Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20 degrees C or cooled down to 5 degrees C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/Pl. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V. and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5 degrees C than at 20 degrees C. which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature. (C) 2011 Elsevier B.V. All rights reserved.

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na2SO4, (NH4)(2)SO4, MgSO4, LiSO4, Na2HPO4, sodium phosphate buffer (pH 7.0), NaH2PO4, K2HPO4, potassium phosphate buffer (pH 7.0), KH2PO4, C6H8O7, Na3C6HSO7, KCHO2, NaCHO2, NaCO3, NaHCO3, C2H4O2, sodium acetate buffer (pH 4.5), and NaC2H3O2] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents. (C) 2011 Elsevier Inc. All rights reserved.

Partitioning and extraction of collagenase from Penicillium aurantiogriseum in poly(ethylene glycol)/phosphate aqueous two-phase system

Purification of collagenase produced by Penicillium aurantiogriseum URM4622 was carried using a PEG/phosphate aqueous two-phase system (ATPS). A 2(3)-full experimental design was used to investigate the influence of PEG molar mass, PEG concentration and phosphate concentration on the selected responses, namely partition coefficient, activity yield and purification factor. The ATPS was composed of PEG (molar mass of 550, 1500 and 4000 g/mol) at concentrations of 15.0, 17.5 and 20.0% (w/w) and phosphate at concentrations of 12.5, 15.0 and 17.5% (w/w). The best results of one-step extraction of collagenase from the fermentation broth (partition coefficient of 1.01, activity yield of 242% and purification factor of 23.5) were obtained at pH 6.0 using 20.0% (w/w) PEG 550 and 17.5% (w/w) phosphate. The results of this preliminary study demonstrate that the selected ATPS is satisfactorily selective for the extraction of such a collagenase. (C) 2012 Elsevier B.V. All rights reserved.

Two-phase flow patterns and pressure drop inside horizontal tubes containing twisted-tape inserts

This paper presents an experimental study on two-phase flow patterns and pressure drop of R134a inside a 15.9 mm ID tube containing twisted-tape inserts. Experimental results were obtained in a horizontal test section for twisted-tape ratios of 3, 4, 9 and 14, mass velocities ranging from 75 to 250 kg/m(2) s and saturation temperatures of 5 and 15 degrees C. An unprecedented discussion on two-phase flow patterns inside tubes containing twisted-tape inserts is presented and the flow pattern effects on the frictional pressure drop are carefully discussed. Additionally, a new method to predict the frictional pressure drop during two-phase flow inside tubes containing twisted-tape inserts is proposed. (C) 2012 Elsevier Ltd. All rights reserved.

Two-Phase Frictional Pressure Drop and Flow Boiling Heat Transfer for R245fa in a 2.32-mm Tube

Experimental two-phase frictional pressure drop and flow boiling heat transfer results are presented for a horizontal 2.32-mm ID stainless-steel tube using R245fa as working fluid. The frictional pressure drop data was obtained under adiabatic and diabatic conditions. Experiments were performed for mass velocities ranging from 100 to 700 kg m−2 s−1 , heat flux from 0 to 55 kW m−2 , exit saturation temperatures of 31 and 41◦C, and vapor qualities from 0.10 to 0.99. Pressures drop gradients and heat transfer coefficients ranging from 1 to 70 kPa m−1 and from 1 to 7 kW m−2 K−1 were measured. It was found that the heat transfer coefficient is a strong function of the heat flux, mass velocity, and vapor quality. Five frictional pressure drop predictive methods were compared against the experimental database. The Cioncolini et al. (2009) method was found to work the best. Six flow boiling heat transfer predictive methods were also compared against the present database. Liu and Winterton (1991), Zhang et al. (2004), and Saitoh et al. (2007) were ranked as the best methods. They predicted the experimental flow boiling heat transfer data with an average error around 19%.

CFD modeling of two-phase boiling flows in the slug flow regime with an interface capturing technique

The objective of this thesis was to improve the commercial CFD software Ansys Fluent to obtain a tool able to perform accurate simulations of flow boiling in the slug flow regime. The achievement of a reliable numerical framework allows a better understanding of the bubble and flow dynamics induced by the evaporation and makes possible the prediction of the wall heat transfer trends. In order to save computational time, the flow is modeled with an axisymmetrical formulation. Vapor and liquid phases are treated as incompressible and in laminar flow. By means of a single fluid approach, the flow equations are written as for a single phase flow, but discontinuities at the interface and interfacial effects need to be accounted for and discretized properly. Ansys Fluent provides a Volume Of Fluid technique to advect the interface and to map the discontinuous fluid properties throughout the flow domain. The interfacial effects are dominant in the boiling slug flow and the accuracy of their estimation is fundamental for the reliability of the solver. Self-implemented functions, developed ad-hoc, are introduced within the numerical code to compute the surface tension force and the rates of mass and energy exchange at the interface related to the evaporation. Several validation benchmarks assess the better performances of the improved software. Various adiabatic configurations are simulated in order to test the capability of the numerical framework in modeling actual flows and the comparison with experimental results is very positive. The simulation of a single evaporating bubble underlines the dominant effect on the global heat transfer rate of the local transient heat convection in the liquid after the bubble transit. The simulation of multiple evaporating bubbles flowing in sequence shows that their mutual influence can strongly enhance the heat transfer coefficient, up to twice the single phase flow value.

Large Sample Theory for Semiparametric Regression Models with Two-Phase, Outcome Dependent Sampling

Outcome-dependent, two-phase sampling designs can dramatically reduce the costs of observational studies by judicious selection of the most informative subjects for purposes of detailed covariate measurement. Here we derive asymptotic information bounds and the form of the efficient score and influence functions for the semiparametric regression models studied by Lawless, Kalbfleisch, and Wild (1999) under two-phase sampling designs. We show that the maximum likelihood estimators for both the parametric and nonparametric parts of the model are asymptotically normal and efficient. The efficient influence function for the parametric part aggress with the more general information bound calculations of Robins, Hsieh, and Newey (1995). By verifying the conditions of Murphy and Van der Vaart (2000) for a least favorable parametric submodel, we provide asymptotic justification for statistical inference based on profile likelihood.

Two-Phase Flow In Microchannels: Morphology And Interface Phenomena

The existence and morphology, as well as the dynamics of micro-scale gas-liquid interfaces is investigated numerically and experimentally. These studies can be used to assess liquid management issues in microsystems such as PEMFC gas flow channels, and are meant to open new research perspectives in two-phase flow, particularly in film deposition on non-wetting surfaces. For example the critical plug volume data can be used to deliver desired length plugs, or to determine the plug formation frequency. The dynamics of gas-liquid interfaces, of interest for applications involving small passages (e.g. heat exchangers, phase separators and filtration systems), was investigated using high-speed microscopy - a method that also proved useful for the study of film deposition processes. The existence limit for a liquid plug forming in a mixed wetting channel is determined by numerical simulations using Surface Evolver. The plug model simulate actual conditions in the gas flow channels of PEM fuel cells, the wetting of the gas diffusion layer (GDL) side of the channel being different from the wetting of the bipolar plate walls. The minimum plug volume, denoted as critical volume is computed for a series of GDL and bipolar plate wetting properties. Critical volume data is meant to assist in the water management of PEMFC, when corroborated with experimental data. The effect of cross section geometry is assessed by computing the critical volume in square and trapezoidal channels. Droplet simulations show that water can be passively removed from the GDL surface towards the bipolar plate if we take advantage on differing wetting properties between the two surfaces, to possibly avoid the gas transport blockage through the GDL. High speed microscopy was employed in two-phase and film deposition experiments with water in round and square capillary tubes. Periodic interface destabilization was observed and the existence of compression waves in the gas phase is discussed by taking into consideration a naturally occurring convergent-divergent nozzle formed by the flowing liquid phase. The effect of channel geometry and wetting properties was investigated through two-phase water-air flow in square and round microchannels, having three static contact angles of 20, 80 and 105 degrees. Four different flow regimes are observed for a fixed flow rate, this being thought to be caused by the wetting behavior of liquid flowing in the corners as well as the liquid film stability. Film deposition experiments in wetting and non-wetting round microchannels show that a thicker film is deposited for wetting conditions departing from the ideal 0 degrees contact angle. A film thickness dependence with the contact angle theta as well as the Capillary number, in the form h_R ~ Ca^(2/3)/ cos(theta) is inferred from scaling arguments, for contact angles smaller than 36 degrees. Non-wetting film deposition experiments reveal that a film significantly thicker than the wetting Bretherton film is deposited. A hydraulic jump occurs if critical conditions are met, as given by a proposed nondimensional parameter similar to the Froude number. Film thickness correlations are also found by matching the measured and the proposed velocity derived in the shock theory. The surface wetting as well as the presence of the shock cause morphological changes in the Taylor bubble flow.

DEVELOPMENT OF CONFOCAL IMAGING TECHNIQUES FOR PROBING INTERFACIAL DYNAMICS IN MICROSCALE, GAS-LIQUID, TWO-PHASE FLOW

Micro-scale, two-phase flow is found in a variety of devices such as Lab-on-a-chip, bio-chips, micro-heat exchangers, and fuel cells. Knowledge of the fluid behavior near the dynamic gas-liquid interface is required for developing accurate predictive models. Light is distorted near a curved gas-liquid interface preventing accurate measurement of interfacial shape and internal liquid velocities. This research focused on the development of experimental methods designed to isolate and probe dynamic liquid films and measure velocity fields near a moving gas-liquid interface. A high-speed, reflectance, swept-field confocal (RSFC) imaging system was developed for imaging near curved surfaces. Experimental studies of dynamic gas-liquid interface of micro-scale, two-phase flow were conducted in three phases. Dynamic liquid film thicknesses of segmented, two-phase flow were measured using the RSFC and compared to a classic film thickness deposition model. Flow fields near a steadily moving meniscus were measured using RSFC and particle tracking velocimetry. The RSFC provided high speed imaging near the menisci without distortion caused the gas-liquid interface. Finally, interfacial morphology for internal two-phase flow and droplet evaporation were measured using interferograms produced by the RSFC imaging technique. Each technique can be used independently or simultaneously when.

Two-phase change in CO2, Antarctic temperature and global climate during Termination II

The end of the Last Glacial Maximum (Termination I), roughly 20 thousand years ago (ka), was marked by cooling in the Northern Hemisphere, a weakening of the Asian monsoon, a rise in atmospheric CO2 concentrations and warming over Antarctica. The sequence of events associated with the previous glacial–interglacial transition (Termination II), roughly 136 ka, is less well constrained. Here we present high-resolution records of atmospheric CO2 concentrations and isotopic composition of N2—an atmospheric temperature proxy—from air bubbles in the EPICA Dome C ice core that span Termination II. We find that atmospheric CO2 concentrations and Antarctic temperature started increasing in phase around 136 ka, but in a second phase of Termination II, from 130.5 to 129 ka, the rise in atmospheric CO2 concentrations lagged that of Antarctic temperature unequivocally. We suggest that during this second phase, the intensification of the low-latitude hydrological cycle resulted in the development of a CO2 sink, which counteracted the CO2 outgassing from the Southern Hemisphere oceans over this period.

Design of a Two-Phase buck converter with fourth-order output filter for envelope amplifiers of limited bandwidth

The use of techniques such as envelope tracking (ET) and envelope elimination and restoration (EER) can improve the efficiency of radio frequency power amplifiers (RFPA). In both cases, high-bandwidth DC/DC converters called envelope amplifiers (EA) are used to modulate the supply voltage of the RFPA. This paper addresses the analysis and design of a modified two-phase Buck converter optimized to operate as EA. The effects of multiphase operation on the tracking capabilities are analyzed. The use of a fourth-order output filter is proposed to increase the attenuation of the harmonics generated by the PWM operation, thus allowing a reduction of the ratio between the switching frequency and the converter bandwidth. The design of the output filter is addressed considering envelope tracking accuracy and distortion caused by the side bands arising from the nonlinear modulation process. Finally, the proposed analysis and design methods are supported by simulation results, as well as demonstrated by experiments obtained using two 100-W, 10-MHz, two-phase Buck EAs capable of accurately tracking a 1.5-MHz bandwidth OFDM signal.

Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots

Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a two-phase integration mechanism. In the “preintegration” phase, transforming plasmid molecules (either intact or partial) are spliced together. This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences. Subsequently, integration of transgenic DNA into the host genome is initiated. Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus. The resulting transgenic locus may have plant DNA separating the transgenic sequences. Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism. Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically. Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through Agrobacterium, the qualitative and/or quantitative efficiency of transformation events is not compromised. Our results cast doubt on the role of Agrobacterium vir genes in the integration process.