Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP)

The present work was designed to study the in vitro and in vivo viability, as assessed by blastocyst formation, pregnancy rate and term delivery of bovine embryos produced under completely defined conditions with or without insulin-like growth factor I (IGF-I) following direct transfer after cryopreservation. Slaughterhouse-derived bovine oocytes were matured for 24h, fertilized with frozen-thawed spermatozoa and cultured in vitro under completely defined conditions with or without exposure to IGF-I (5 ng/ml). Only those embryos classified as excellent or good quality blastocysts were frozen. Each blastocyst was individually loaded into a straw, seeded and pre-cooled to -7 degrees C. After 10 min at -7 degrees C straws were frozen further to -30 degrees C at a rate of 0.3 degrees C/min and then plunged into liquid nitrogen. Synchronized recipient cows received one embryo in the horn ipsilateral to the corpus luteum (CL). Pregnancies were diagnosed by ultrasonography 35-45 days after embryo transfer (ET). IGF-I failed to improve cleavage rate, as well as blastocyst production, when added during in vitro culture (IVC). Pregnancy outcome was not significantly improved in cows that received an IGF-I-treated embryo compared with controls (4/10 versus 3/10, respectively). Five out of six calves delivered to date were born alive and healthy. We have shown that it is possible to obtain healthy live offspring from frozen-thawed embryos produced under chemically defined conditions after direct transfer.     

Validation of a biodynamic model of pushing and pulling.

Pushing and pulling during manual material handling can increase the compressive forces on the lumbar disc region while creating high shear forces at the shoe-floor interface. A sagittal plane dynamic model derived from previous biomechanical models was developed to predict L5/S1 compressive force and required coefficients of friction during dynamic cart pushing and pulling. Before these predictions could be interpreted, however, it was necessary to validate model predictions against independently measured values of comparable quantities. This experiment used subjects of disparate stature and body mass, while task factors such as cart resistance and walking speed were varied. Predicted ground reaction forces were compared with those measured by a force platform, with correlations up to 0.67. Predicted erector spinae and rectus abdominus muscle forces were compared with muscle forces derived from RMS-EMGs of the respective muscle groups, using a static force build-up regression relationship to transform the dynamic RMS-EMGs to trunk muscle forces. Although correlations were low, this was attributed in part to the use of surface EMG on subjects of widely varied body mass. The biodynamic model holds promise as a tool for analysis of actual industrial pushing and pulling tasks, when carefully applied.     

Hair pulling: a review

Hair pulling has been reported in humans, six different non-human primate species, mice, guineapigs, rabbits, sheep and muskox, dogs and cats. This behaviour seems to occur only in subjects who are confined in an artificial environment. It has been classified as a mental disorder in humans, as a behavioural pathology in animals. The hair is not only pulled but also, in most species, ingested. Hair pulling can be both self-directed and partner-directed, contains elements of aggression, manifests more often in females than in males, is associated with psychogenic distress, and resists treatment. Research data collected from affected animals are probably not normative, hence scientifically unreliable. The preemptive correction of husbandry deficiencies causing long-term stress may prevent the development of this bizarre behaviour in healthy subjects.     

Measuring two-dimensional receptor-ligand binding kinetics by micropipette.

We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2.     

Comparison of open pulled straw (OPS) vs glass micropipette (GMP) vitrification in mouse blastocysts

The purpose of this study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of mouse blastocysts, and to compare the post-thaw survival of these blastocysts with those cooled in open pulled straws (OPS). The GMP vessel permits higher freezing and warming rates than OPS due to the higher heat conductivity of glass and lower mass of the solution containing the embryos. Groups of 6 mouse blastocysts were sequentially placed into 2 vitrification solutions before being loaded into either the OPS or GMP vessels and immersed into LN2 within 20 to 25 sec. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in holding medium (HM) and modified human tubal fluid medium (mHTF), each for 5 min, and then cultured in mHTF supplemented with 10% FCS for 24 h. The rate of blastocyst re-expansion did not differ significantly for OPS (93.5%) and GMP (95.0%) methods (P<0.05). The hatching rate in OPS (88.7%) was similar to that in GMP (90.0%) but was lower than for the unvitrified control embryos (98.3%, P<0.05). To determine the optimal embryo population per GMP vessel, the pipettes were loaded with 2 to 10 embryos. The rate of blastocyst re-expansion after vitrification was significant for 2 to 4 embryos than for 6 to 10 embryos per vessel. In addition, the rate of blastocyst re-expansion was significantly lower if blastocysts were vitrified in the wide rather than the narrow portion of the micropipette (100 vs 87.5%; P<0.05) even when only 4 blastocysts were loaded per vessel. These results indicate that both vitrification vessels can provide high rates of embryo survival. However, the GMP vessel does not need a cap to protect the vessel from floating after immersion in LN2. The number and location of the embryos (narrow versus wide portion of capillary) were considered to be limiting factors to the viability of mouse embryos.     

Estimation of viscous dissipation inside an erythrocyte during aspirational entry into a micropipette.

Viscous dissipation inside the erythrocyte during its aspirational entry into a micropipette is analyzed. The motion of the intracellular fluid is approximated by a flow into the micropipette orifice from a half space (the portion of the erythrocyte outside the micropipette). The stream function and intracellular pressure (p) in the half space are obtained as a function of radial and axial positions near the orifice. Solution of the boundary value problem for a uniform stream entering a circular hole gives p = 2 eta HQ/pi R3p, where eta H is the intracellular viscosity, Q is the total discharge, and Rp is the pipette radius. The results indicate that the moving erythrocyte membrane helps to drive the intracellular fluid into the orifice. For normal erythrocytes, p is only approximately 0.5% of the total aspiration pressure (delta P). The contribution of p to delta P, however, may become significant when there is a large increase in eta H due to a markedly elevated intracellular hemoglobin concentration or an alteration of the physical state of hemoglobin.     

The application of a homogeneous half-space model in the analysis of endothelial cell micropipette measurements

Experimental studies have shown that endothelial cells which have been exposed to shear stress maintain a flattened and elongated shape after detachment. Their mechanical properties, which are studied using the micropipette experiments, are influenced by the level as well as the duration of the shear stress. In the present paper, we analyze these mechanical properties with the aid of two mathematical models suggested by the micropipette technique and by the geometry peculiar to these cells in their detached post-exposure state. The two models differ in their treatment of the contact zone between the cell and the micropipette. The main results are expressions for an effective Young's modulus for the cells, which are used in conjunction with the micropipette data to determine an effective Young's modulus for bovine endothelial cells, and to discuss the dependence of this modulus upon exposure to shear stress.     

Microapplicator and micropipette for inoculation of the embryonated chicken egg.

Aflatoxin G2 was used to test the speed and accuracy of the microapplicator and the micropipette. The 50% lethal dose of both assay systems was approximately the same, and 4.0 mug of G2 had an 85% lethal effect in both systems. The 50% lethal dose of the microapplicator and the micropipette was lower than that of the syringe but, of these, only the micropipette can combine the accuracy of the microapplicator and the speed of the syringe.     

Microapplicator and micropipette for inoculation of the embryonated chicken egg.

Aflatoxin G2 was used to test the speed and accuracy of the microapplicator and the micropipette. The 50% lethal dose of both assay systems was approximately the same, and 4.0 mug of G2 had an 85% lethal effect in both systems. The 50% lethal dose of the microapplicator and the micropipette was lower than that of the syringe but, of these, only the micropipette can combine the accuracy of the microapplicator and the speed of the syringe.     

Fission and refusion of red blood cells using a micropipette technique

In micropipette experiments with small capillaries and moderate high pressure difference (approximately 1000 Pa) cell fragmentation (fission) of human red blood cells without hemolysis was observed by TV-system for a large number of fresh red blood cells of different donors. After separation, the fragment moves away from the residual cell. In seven cases this process was evaluated quantitatively and was shown that the rate of the fragment was constant in time. Two mechanisms for this phenomenon are discussed. In particular cases a spontaneous re-fusion with the residual cell body in the capillary can be observed. In our opinion probably protein-depleted membrane surfaces arise and membrane fusion is possible simply by mechanical contact without additional electric fields and/or fusion agents.     

Stability analysis of micropipette aspiration of neutrophils

During micropipette aspiration, neutrophil leukocytes exhibit a liquid-drop behavior, i.e., if a neutrophil is aspirated by a pressure larger than a certain threshold pressure, it flows continuously into the pipette. The point of the largest aspiration pressure at which the neutrophil can still be held in a stable equilibrium is called the critical point of aspiration. Here, we present a theoretical analysis of the equilibrium behavior and stability of a neutrophil during micropipette aspiration with the aim to rigorously characterize the critical point. We take the energy minimization approach, in which the critical point is well defined as the point of the stability breakdown. We use the basic liquid-drop model of neutrophil rheology extended by considering also the neutrophil elastic area expansivity. Our analysis predicts that the behavior at large pipette radii or small elastic area expansivity is close to the one predicted by the basic liquid-drop model, where the critical point is attained slightly before the projection length reaches the pipette radius. The effect of elastic area expansivity is qualitatively different at smaller pipette radii, where our analysis predicts that the critical point is attained at the projection lengths that may significantly exceed the pipette radius.     

Optical evaluation of red blood cell geometry using micropipette aspiration.

Although red blood cell (RBC) geometry has been extensively studied by micropipette aspiration, the small size of RBC and pipettes vs. the optical resolution of light microscopy suggests the need to consider potential errors. The present study addressed such difficulties and investigated four specific problems: (1) use of exact equations to calculate RBC membrane area and volume; (2) calibration of the pipette internal diameter (PID); (3) correction for a noncylindrical pipette barrel; (4) diffraction distortion of the RBC image. The observed PID represents a cylinder lens enlargement that can be theoretically derived from the glass/buffer refractive index ratio (1.49/1.33 = 1.12). This enlargement was experimentally confirmed by: (1) studying pipettes bent to allow measurement through the barrel (horizontal) and at the orifice (vertical), with a resulting diameter ratio of 1.12 +/- 0.01; (2) and by replacing the surrounding buffer with immersion oil and hence abolishing the lens phenomenon (ratio = 1.12 +/- 0.02). In addition, use of aspirated oil droplets demonstrated a 3.2 +/- 0.2% error when the PID is focused at a sharp, maximum diameter. The average pipette cone angle was 1.49 +/- 0.09 degrees and varied considerably with pipette pulling procedures; calculated tongue geometry inside the pipette was affected by the noncylindrical pipette barrel. The RBC diffraction error, demonstrated by touching two aspirated cells held by opposing pipettes, was 0.091 +/- 0.002 microns. The PID, cone angle, and diffraction artifacts significantly (p < 0.001) affected calculated RBC geometry (average errors up to 5.4% for area and 9.6% for volume). Two new methods to calculate, rather than directly measure, the PID from images of a single RBC, during either osmotic or pressure manipulation, were evaluated; the osmotic method closely predicted the PID, whereas the pressure method markedly underestimated the PID. Our results thus confirm the need to consider the above-mentioned phenomena when determining RBC geometric parameters via micropipette aspiration.     

The folding mechanics of a knotted protein

An increasing number of proteins are being discovered with a remarkable and somewhat surprising feature, a knot in their native structures. How the polypeptide chain is able to "knot" itself during the folding process to form these highly intricate protein topologies is not known. Here we perform a computational study on the 160-amino-acid homodimeric protein YibK, which, like other proteins in the SpoU family of MTases, contains a deep trefoil knot in its C-terminal region. In this study, we use a coarse-grained C(alpha)-chain representation and Langevin dynamics to study folding kinetics. We find that specific, attractive nonnative interactions are critical for knot formation. In the absence of these interactions, i.e., in an energetics driven entirely by native interactions, knot formation is exceedingly unlikely. Further, we find, in concert with recent experimental data on YibK, two parallel folding pathways that we attribute to an early and a late formation of the trefoil knot, respectively. For both pathways, knot formation occurs before dimerization. A bioinformatics analysis of the SpoU family of proteins reveals further that the critical nonnative interactions may originate from evolutionary conserved hydrophobic segments around the knotted region.     

The mechanics of motility in dissociated cytoplasm.

We stimulate the dynamical behavior of dissociated cytoplasm using the Reactive Flow Model (Dembo, M., and F. Harlow, 1986, Biophys. J., 50:109-121). We find that for the most part the predicted dynamical behavior of the cytoplasm is governed by three nondimensional numbers. Several other nondimensional parameters, the initial conditions, and boundary conditions are found to have lesser effects. Of the three major nondimensional parameters, one (D#) controls the percentage of ectoplasm, the second (C#) controls the sharpness of the endoplasm-ectoplasm boundary, and the third (R#) controls the topological complexity of the endoplasm-ectoplasm distribution. If R# is very small, then the cytoplasm contracts into a single uniform mass, and there is no bulk streaming. If R# is very large, then the cytoplasmic mass breaks up into a number of clumps scattered throughout the available volume. Between these clumps the solution undergoes turbulent or chaotic patterns of streaming. Intermediate values of R# can be found such that the mass of cytoplasm remains connected and yet undergoes coherent modes of motility similar to flares (Taylor, D.L., J.S. Condeelis, P.L. Moore, and R.D. Allen, 1973, J. Cell Biol., 59:378-394) and rosettes (Kuroda, K., 1979, Cell Motility: Molecules and Organization, 347-362).     

Single-molecule pulling experiments: when the stiffness of the pulling device matters

Using Langevin modeling, we investigate the role of the experimental setup on the unbinding forces measured in single-molecule pulling experiments. We demonstrate that the stiffness of the pulling device, K_eff, may influence the unbinding forces through its effect on the barrier heights for both unbinding and rebinding processes. Under realistic conditions the effect of K_eff on the rebinding barrier is shown to play the most important role. This results in a significant increase of the mean unbinding force with the stiffness for a given loading rate. Thus, in contrast to the phenomenological Bell model, we find that the loading rate (the multiplicative value K_effV, V being the pulling velocity) is not the only control parameter that determines the mean unbinding force. If interested in intrinsic properties of a molecular system, we recommend probing the system in the parameter range corresponding to a weak spring and relatively high loading rates where rebinding is negligible.