Sperm DNA damage has a negative association with live birth rates after IVF.

Sperm DNA damage has a negative impact on pregnancy rates following assisted reproduction treatment (ART). The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage. Following IVF, couples with <25% sperm DNA fragmentation had a live-birth rate of 33%; in contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13%. Following ICSI, no significant differences in sperm DNA damage were found between any groups of patients. Sperm DNA damage was also associated with low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men with idiopathic infertility have high sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF.

Sperm DNA damage has a negative impact on assisted reproduction treatment outcome, in particular, on pregnancy rates. The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage and treatment outcome. Following IVF, couples with <25% sperm DNA fragmentation had a live birth rate of 33%. In contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13% following IVF. Following ICSI, there were no significant differences in levels of sperm DNA damage between any groups of patients. Sperm DNA damage was also associated with the very low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men have high level of sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF.

A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles

The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacterium Bacteroides fragilis were examined. Outer membrane vesicles are released from the surface of B. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide of B. fragilis confirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations of B. fragilis strains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL); non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence of B. fragilis.

Aggregation Limits Surface Expression of Homomeric GluA3 Receptors

AMPA receptors are glutamate-gated cation channels assembled from GluA1-4 subunits and have properties that are strongly dependent on the subunit composition. The subunits have different propensities to form homomeric or various heteromeric receptors expressed on cell surface, but the underlying mechanisms are still poorly understood. Here, we examined the biochemical basis for the poor ability of GluA3 subunits to form homomeric receptors, linked previously to two amino acid residues, Y454 and R461, in its ligand-binding domain (LBD). Surface expression of GluA3 was improved by co-assembly with GluA2 but not with stargazin, a trafficking chaperone and modulator of AMPA receptors. The secretion efficiency of GluA2 and GluA3 LBDs paralleled the transport difference between the respective full-length receptors and was similarly dependent on Y454/R461, but not on LBD stability. In comparison to GluA2, GluA3 homomeric receptors showed a strong and Y454/R461-dependent tendency to aggregate both in the macroscopic scale measured as lower solubility in nonionic detergent and in the microscopic scale evident as the preponderance of hydrodynamically large structures in density gradient centrifugation and native gel electrophoresis. We conclude that the impaired surface expression of homomeric GluA3 receptors is caused by nonproductive assembly and aggregation to which LBD residues Y454 and R461 strongly contribute. This aggregation inhibits the entry of newly synthesized GluA3 receptors to the secretory pathway.

Structural effects of neutral lipids on the divalent cation-induced interactions of phosphatidylserine - containing bilayers

As Ca2+ and phosphatidylserine (PS) are known to induce the adhesion of bilayer vesicles and form collapsed multibilayer structures in vitro, it was the aim of this study to examine how that interaction and the resultant structures might be modified by neutral lipid species. X-ray diffraction data from multilamellar systems suggest that phosphatidylcholine (PC) and diacylglycerol (DG) might be in the collapsed phase up to a concentration of -30 mole % and that above this concentration these neutral lipids may modify Ca2+-induced bilayer interactions. Using large unilamellar vesicles and long incubations in excess Ca2+ to ensure equilibration, similar preliminary results were again obtained with PC, and also with phosphatidylethanolamine (PE). A combination of X-ray diffraction, thin-layer chromatography, density gradient centrifugation and freeze-fracture electron microscopy, used in conjunction with an osmotic stress technique, showed that (i) -30 mole % PC can be accomodated in the Ca(DOPS)2 phase; and (ii) higher PC levels modify Ca2+-induced bilayer interactions resulting in single lamellar phases of larger dimension and reduced tendency for REV collapse. Importantly, the data suggest that PC is dehydrated during the rapid collapse process leading. to Ca(DOPS)2 formation and exists with this dehydrated phase. Similar results were obtained using PS isolated from bovine brain. Preliminary studies using two different phosphatidylethanolamine (PE) species indicated accomodation by Ca(DOPS)2 of -25-30 mole 0/0 PE and bulk phase separation, of species favouring a non-bilayer phase, at higher levels. Significantly, all PS/PE vesicles appear to undergo a complete Ca2+-induced collapse, even with contents of up to 90 mole % PE. These data suggest that PE may have an important role in fusion mechanisms in vivo. In sum the data lend both structural and stoichiometric evidence for th~ existence of laterally segregated neutral lipid molecules within the same bilayers as PS domains exposed to Ca2+.

Detecting uterine cervical cancer cells using molecular biomarkers

Arrière-plan: les cellules tumorales circulantes (CTC) sont détectables dans de nombreux cancers et peuvent être utiles cliniquement pour le pronostic de la maladie, pour mesurer la récidive et pour prédire la sensibilité aux medicaments chimiothérapeutiques. Au cours des dernières années, l’études des CTC dans de nombreux cancers tels que le cancer du sein, du poumon, du côlon et de la prostate a grandement évolué. Alternativement, il y peu d'études à ce sujet concernant le cancer du col de l’utérus (CCU). Objectifs: Notre objectif est d’optimiser le processus d'enrichissement des CTC dans le CCU et la détection moléculaire des biomarqueurs E6 et E7. Matériel et Méthodes: Dans l’optique de mimer la présence de CTC dans le sang, nous avons dilué des cellules cancéreuses CaSki VPH16-positif provenant d’un CCU dans du sang humain prélevé sur des volontaires sains. Les CaSki ont été collectées suite à une centrifugation par densité avec le Ficoll, la lyse des globules rouges (RBC) et la lyse des RBC combinée avec un enrichissement positif et négatif à l’aide de marqueurs de surface cellulaire. Les CTC ont été détectées par la mesure d’expression des oncogènes E6 et E7 du virus du papillome humain (VPH), de la cytokératine 19 (CK19) et de la cycline p16INK4 en utilisant la technique quantitative en temps réel de Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). Pour valider notre méthode de détection des CTC in vivo, nous avons recruté dix patientes atteintes d’un CCU VPH16 positif et six contrôles sains. Résultats: Dans le modèle de dilutions de cellules CaSki, la lyse des RBC seule ou combinée avec l'enrichissement négatif ou positif suggèrent des limites de détection de 1 CTC par mL de sang pour tous les biomarqueurs moléculaires utilisés. La sensibilité de détection est accrue lors de l'utilisation de l’enrichissement positif et négatif en réduisant le bruit de fond causé par les monocytes sanguins. Contrairement aux oncogènes E6 et E7, les marqueurs CK19 et p16INK4A ont été détectés chez des individus sains, les niveaux d'expression de base appropriés doivent donc être déterminés avec précision par rapport aux patientes CCU. Le gradient de densité par Ficoll a une limite de détection de seulement environ 1000 cellules par mL de sang. Enfin, les CTC ont été détectées dans 2/10 patientes en utilisant le marqueur CK19. Cependant, ces patientes étaient négatives pour les oncogènes E6/E7. Le marqueur p16INK4A était exprimé au même niveau dans tous les échantillons (CCU et normaux). Conclusion: Notre étude suggère que les oncogènes E6 et E7 du VPH16 sont les marqueurs biologiques les plus sensibles et spécifiques en qRT-PCR pour détecter les CTC dans le modèle de dilution de cellules de CCU dans le sang. Chez les patientes atteintes d’un CCU de stade précoce, seulement CK19 a révélé la présence potentielle de CTC, ce qui suggère que ces cellules sont rares à ce stade de la maladie. Mots clés: cancer du col de l’utérus, cellules tumorales circulantes, RT-qPCR, E6 et E7, CK19, p16INK4A, enrichissement immunomagnétique, détection moléculaire.

Ribosome modulation factor protects Escherichia coli during heat stress, but this may not be dependent on ribosome dimerisation

The role of ribosome modulation factor (RMF) in protecting heat-stressed Escherichia coli cells was identified by the observation that cultures of a mutant strain lacking functional RMF (HMY15) were highly heat sensitive in stationary phase compared to those of the parent strain (W3110). No difference in heat sensitivity was observed between these strains in exponential phase, during which RMF is not synthesised. Studies by differential scanning calorimetry demonstrated that the ribosomes of stationary-phase cultures of the mutant strain had lower thermal stability than those of the parent strain in stationary phase, or exponential-phase ribosomes. More rapid breakdown of ribosomes in the mutant strain during heating was confirmed by rRNA analysis and sucrose density gradient centrifugation. Analyses of ribosome composition showed that the 100S dimers dissociated more rapidly during heating than 70S particles. While ribosome dimerisation is a consequence of the conformational changes caused by RMF binding, it may not therefore be essential for RMF-mediated ribosome stabilisation.

The key role of the western boundary in linking the AMOC strength to the north-south pressure gradient

A key idea in the study of the Atlantic meridional overturning circulation (AMOC) is that its strength is proportional to the meridional density gradient, or more precisely, to the strength of the meridional pressure gradient. A physical basis that would tell us how to estimate the relevant meridional pressure gradient locally from the density distribution in numerical ocean models to test such an idea, has been lacking however. Recently, studies of ocean energetics have suggested that the AMOC is driven by the release of available potential energy (APE) into kinetic energy (KE), and that such a conversion takes place primarily in the deep western boundary currents. In this paper, we develop an analytical description linking the western boundary current circulation below the interface separating the North Atlantic Deep Water (NADW) and Antarctic Intermediate Water (AAIW) to the shape of this interface. The simple analytical model also shows how available potential energy is converted into kinetic energy at each location, and that the strength of the transport within the western boundary current is proportional to the local meridional pressure gradient at low latitudes. The present results suggest, therefore, that the conversion rate of potential energy may provide the necessary physical basis for linking the strength of the AMOC to the meridional pressure gradient, and that this could be achieved by a detailed study of the APE to KE conversion in the western boundary current.

The importance of wind and buoyancy forcing for the boundary density variations and the geostrophic component of the AMOC at 26°N

It is widely thought that changes in both the surface buoyancy fluxes and wind stress drive variability in the Atlantic meridional overturning circulation (AMOC), but that they drive variability on different time scales. For example, wind forcing dominates short-term variability through its effects on Ekman currents and coastal upwelling, whereas buoyancy forcing is important for longer time scales (multiannual and decadal). However, the role of the wind forcing on multiannual to decadal time scales is less clear. Here the authors present an analysis of simulations with the Nucleus for European Modelling of the Ocean (NEMO) ocean model with the aim of explaining the important drivers of the zonal density gradient at 26°N, which is directly related to the AMOC. In the experiments, only one of either the wind stress or the buoyancy forcing is allowed to vary in time, whereas the other remains at its seasonally varying climatology. On subannual time scales, variations in the density gradient, and in the AMOC minus Ekman, are driven largely by local wind-forced coastal upwelling at both the western and eastern boundaries. On decadal time scales, buoyancy forcing related to the North Atlantic Oscillation dominates variability in the AMOC. Interestingly, however, it is found that wind forcing also plays a role at longer time scales, primarily impacting the interannual variability through the excitation of Rossby waves in the central Atlantic, which propagate westward to interact with the western boundary, but also by modulating the decadal time-scale response to buoyancy forcing.

Investigation of the Europium Emission Spectra of the Europium-Oxytetracycline Complex in the Presence of Human Low-Density Lipoproteins

Low-Density Lipoprotein (LDL), often known as ""bad cholesterol"" is one of the responsible to increase the risk of coronary arterial diseases. For this reason, the cholesterol present in the LDL particle has become one of the main parameters to be quantified in routine clinical diagnosis. A number of tools are available to assess LDL particles and estimate the cholesterol concentration in the blood. The most common methods to quantify the LDL in the plasma are the density gradient ultracentrifugation and nuclear magnetic resonance (NMR). However, these techniques require special equipments and can take a long time to provide the results. In this paper, we report on the increase of the Europium emission in Europium-oxytetracycline complex aqueous solutions in the presence of LDL. This increase is proportional to the LDL concentration in the solution. This phenomenum can be used to develop a method to quantify the number of LDL particles in a sample. A comparison between the performances of the oxytetracycline and the tetracycline in the complexes is also made.

Occurrence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in Crotalus durissus terrificus (Serpentes, Viperidae) in Brazil

The objective of the present study was to investigate the prevalence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in the snake Crotalus durissus terrificus (Serpentes, Viperidae). Fifty animals were evaluated for the presence of oocysts of Cryptosporidium sp. at the time of arrival and 30 and 60 days later. Intestinal washings with saline solution (1% body weight), fecal samples, and organ scrapings were collected during the study. Oocysts were concentrated by an ether-phosphate-buffered saline sedimentation technique and then separated by a density gradient centrifugation technique. Smears were made with the sediment and submitted to modified acid-fast and auramine-rhodamine staining. Cryptosporidium-positive smears were used as controls for the experimental findings. The overall prevalence of Cryptosporidium sp. oocysts was 14%. Among the positive snakes, oocysts were detected only in the intestinal washing in two specimens, only in the feces in four specimens, and in both materials at least once in one specimen. The positive snakes were predominantly from Santa Maria da Serra city State of São Paulo (57.1%). We also observed that all of the examinations that presented positive results were obtained at least 27 days after the capture of the animals.

Sexagem de espermatozoides bovinos por centrifugação em gradiente descontínuo de densidade de Percoll

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Simulation and economic analysis of bovine sex selection

A simulation model implemented in the programming software Delphi XE® was applied to evaluate sex selection in bovine. The hypothesis under investigation was that a dynamic model with stochastic and deterministic elements could detect the sexed semen technique to minimize pregnancy cost and to determine the adequate number of recipients required for in vivo (ET) and in vitro embryo production (IVP) in the proposed scenarios. Sex selection was compared through semen sexed using flow cytometry (C1) and density gradient centrifugation techniques (C2) in ET and IVP. Sensibility analyses were used to identify the adequate number of recipients for each scenario. This number was reinserted into the model to determine the biological and financial values that maximized ET and IVP using sexed semen (C1M and C2M). New scenarios showed that the density gradient technique minimized pregnancy cost based on the proposed scenarios. In addition, the adequate number of recipients (ET - C1M - 115 and C2M - 105)/(IVP - C1M - 145 and C2M - 140) per donor used was determined to minimize the pregnancy cost in all scenarios.

Embryos refrozen-thawed by vitrification lead to live births: Case report

Objective: To describe two successful cases of utilizing refrozen blastocysts by vitrification derived from supernumerary embryos. Design: Case report. Setting: Private fertility clinic. Subjects: Two infertility couple. Interventions: Refrozen blastocysts by vitrification derived from supernumerary embryos. Main outcome measures: Obstetric and pediatric results. Results: Two pregnancies obtained from refrozen-warmed blastocysts led to two healthy babies being born without clinical or genetic problems. Conclusion: These case reports support the notion of safely repeating cryopreservation. However, despite these favorable results, there is still a need for prospective controlled studies on the obstetric and neonatal repercussions of refreezing and of vitrification in particular. © 2010 Middle East Fertility Society.

Avaliação de novas propostas em arcabouços tridimencionais (3D) para cultura de células-tronco mesenquinas e condogênese

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influência do método de separação dos espermatozóides viáveis (swim-up) na eficiência de seleção do sexo de bovinos por gradiente descontínuo de densidade e o impacto no melhoramento genético animal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)