Expression of the male reproduction‐related gene in spermatic ducts of the blue swimming crab,Portunus pelagicus,and transfer of modified protein to the sperm acrosome

Expression of a sex‐specific gene in Macrobrachium rosenbergii (Mr‐Mrr), encoding a male reproduction‐related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp‐Mrr) and Scylla serrata (Ss‐Mrr). We then investigated expression of Pp‐Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp‐Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp‐Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti‐Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp‐Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Measuring error and sampling variation in stereology: comparison of the efficiency of various methods for planar image analysis

An evaluation is made of the relative efficiency (precision of the final estimate per unit time of measurement on a given set of sections) of different methods for planar analysis aimed at estimating aggregate, overall stereological parameters (such as V_v, S_v). The methods tested are point-counting with different densities of test points (4 ≤ P_T ≤ 900 per picture), semiautomatic computer image analysis with MOP and automatic image analysis with Quantimet, for obtaining V_v and S_v estimates. One biological sample as well as three synthetic model structures with known coefficients of variation between sections are used. The standard error of an estimate is mainly determined by the coefficient of variation between sampling units (= sections in the present paper) so that measuring each sample unit with a very high precision is not necessary. Automatic image analysis and point-counting with a 100-point grid were the most efficient methods for reducing the relative standard errors of the V_v and S_v estimates to equivalent levels in the synthetic models. Using a 64-point grid was as precise, and about 11 times faster than using a tracing device for obtaining the estimate of V_v in the biological sample.     

The effect of different cleaning agents and resin cement materials on the bond strength of contaminated zirconia

The purpose of this study was to examine the effect of different cleaning methods and resin cements on the shear bond strength (SBS) of contaminated zirconia. A total of 92 disc-shaped zirconia specimens were contaminated with different procedures. Then, the specimens were grouped according to cleaning methods and resin cements: no cleaning + Variolink Esthetic DC (CN_V), no cleaning + Panavia V5 (CN_P), sandblasted + Variolink Esthetic DC (SB_V), sandblasted + Panavia V5 (SB_P), Ivoclean + Variolink Esthetic DC (IC_V), Ivoclean + Panavia V5 (IC_P), Katana Cleaner + Variolink Esthetic DC (KC_V), and Katana Cleaner + Panavia V5 (KC_P). Following an aging protocol in a 37°C for 1 week, SBS analysis was performed with a universal test machine. For the surface topography and elemental analysis, scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDS) were used. Significance was evaluated as p < .05 and p < .01. The highest SBS results were found in the SB_P group, showing a statistically significant difference from all other groups (p < .05). For the same cleaning method, Panavia V5 showed statistically significantly higher SBS values than Variolink Esthetic DC (p < .01), except the CN_P–CN_V (p = .880) and KC_P–KC_V (p = .082) groups. The most detected surface elements by EDS were Zr, O, C, and N, respectively. The contaminated zirconia surfaces must be cleaned for successful adhesion. The use of phosphate-containing adhesives in combination with sandblasting will increase the adhesion strength, and universal cleaning agents can be a good alternative to sandblasting.     

Analysis of hybrid journal bearing for non‐Newtonian lubricants

The performance characteristics of capillary compensated hole‐entry hybrid journal bearing systems have been studied theoretically. The analysis considers the generalized Reynolds equation governing the flow field of lubricant, having variable viscosity, taking the equation of lubricant flow through a capillary restrictor as constraint. The non‐Newtonian lubricant is assumed to follow the cubic shear stress law. The results obtained from the study suggest that bearing static performance characteristics can be optimized for the particular bearing operating conditions by proper selection of parameters such as bearing land width ratios (ā_b), the restrictor design parameter (C̄_s2), and the non‐linearity factor (K̄). Copyright © 2006 John Wiley & Sons, Ltd.     

Second-order stereology of benign and malignant alterations of the human mammary gland

The purpose of the present study was a quantitative characterization of the three-dimensional arrangement of the epithelial component of benign and malignant alterations of the female breast by combining stereology with stochastic geometry. Twenty cases of fibrous mastopathy and 20 cases of invasive ductal mammary cancer were studied at the light microscopic level. Segmentation of the epithelial tissue component was performed with an image analyser. From the resulting binary images, unbiased estimates of the covariance C(r) and the intensity V_v of the epithelial volume component were obtained automatically by computer. From these data, estimates of the correlation function k(r), of the pair correlation function g(r), of the radial distribution function RDF(r) and of the reduced second moment function K(r) of epithelial volume were determined. The estimates of C(r) and RDF(r) differed between groups, but these functions depend on spatial pattern and V_v. As carcinomas showed a significantly higher epithelial volume density V_v than mastopathies, estimation of C(r) and RDF(r) alone did not permit a safe distinction between possibly different types of spatial arrangement of epithelium in the benign and malignant lesions. Analysis of the estimates of k(r), g(r) and K(r), which are not influenced by V_v, showed definite interaction between epithelial volume elements, with clustering at short distances and repulsion at long distances. In both groups, the null hypothesis of purely random arrangement of epithelium had to be rejected. The clearest distinction between groups was obtained by estimation of g(r), which showed that short-range, tubular pattern as well as long-range, lobular architecture are better preserved in benign than in malignant lesions. The low interindividual scatter of k(r), g(r) and K(r) indicates a high biological significance of spatial pattern, which is presumably under strict genetic control. Potential uses of the method are: (i) the identification of biomathematical models which could contribute to a better understanding of the growth processes involved, (ii) conditional simulation of the underlying three-dimensional structures by computer, and (iii) supporting the diagnosis of mammary lesions with borderline histopathological appearance.     

Three‐dimensional imaging of shear bands in bulk metallic glass composites

The mechanism of the increase in ductility in bulk metallic glass matrix composites over monolithic bulk metallic glasses is to date little understood, primarily because the interplay between dislocations in the crystalline phase and shear bands in the glass could neither be imaged nor modelled in a validated way. To overcome this roadblock, we show that shear bands can be imaged in three dimensions by atom probe tomography from density variations in the reconstructed atomic density, which density‐functional theory suggests being a local‐work function effect. Imaging of near‐interface shear bands in Ti₄₈Zr₂₀V₁₂Cu₅Be₁₅ bulk metallic glass matrix composite permits measurement of their composition, thickness, branching and interactions with the dendrite interface. These results confirm that shear bands here nucleate from stress concentrations in the glass due to intense, localized plastic deformation in the dendrites rather than intrinsic structural inhomogeneities.     

Elimination of Systematic Measurement Inaccuracy in Voltage–Capacitance Spectroscopy of Semiconductor/Insulator Interface

An improved algorithm for measurements of quasi-static voltage–capacitance characteristics (VCC) of metal–insulator–semiconductor (MIS) structures is suggested. This algorithm includes the following stages: high-temperature depolarization of a MIS structure, its cooling at a constant depolarization voltage, measurement of the MIS current I(V _g) upon the linearly time-swept (increasing or decreasing) gate potential, and averaging of the dependences I(V _g) obtained in the modes of sweep rate _v > 0 and _v < 0 ( _v = 0). The method allows the systematic error of the VCC C(V _g) measurement caused by the relaxation polarization of the insulator, leakage current, and shift of the electrometer zero to be eliminated. As a result, the accuracy and reliability of the determination of the interface state (IS) density N _ss(E) are increased, and the energy range of the IS spectra is extended. To test the efficiency of the algorithm developed, it was used for determining the function N _ss(E) in the system SiO₂/(100)Si : P.     

The influence of tissue processing on quantitative histopathology in breast cancer

Objective grading of breast cancer by morphometry has been suggested for improving the precision of the prognostic prediction. However, the tissue components evaluated might be influenced by variations in the processing, reducing the clinical value. In the present study, the impact of the period of fixation, of the acidity of the fixative and of the embedding medium was investigated by allocating tissue samples from 27 surgical breast cancer specimens systematically randomly to different modes of processing. The volume-weighted mean volume of cancer cell nuclei, v?_V(nuc), was estimated using the method of point-sampled intercepts on vertical sections. In addition, estimates of the mean nuclear profile area, ā_H(nuc), the nuclear volume fraction, V_V(nuc), the nuclear profile density, ND, and the mitotic profile frequency, MF, were obtained. The quantitative histopathological estimates were stable with respect to the investigated variables of the tissue processing. No significant differences were found when comparing the estimates obtained in samples from five tumours fixed in formalin at pH 5·0, 6·0, 7·0, 7·4 and 8·0, respectively. Similarly, no significant correlations between the estimates and the period of formalin fixation (24 h, 3 days and 3 months) were found in samples from five other tumours. However, the v?_V(nuc) was 13% larger (2p = 0·004) and the mean ND 17% smaller (2p = 0·04) in hydroxyethyl-methacrylate-embedded samples from 17 tumours as compared to paraffin-embedded samples. Thus, the shrinkage observed in paraffin seems to affect nuclei less than tissue.     

On the estimation of particle number

Two methods are proposed for estimating the number of separated particles within a solid structure per unit volume of structure, N_v. Apart from being arranged with independence of any size parameter, no special assumptions upon the size, shape and orientation of the particles are made. The first method is based on the identity N_V = (N_A)_u ? μ_u^?1, where (N_A)_u is the mean number of particle sections per unit area of a plane probe T_u which is uniform random within the structure and perpendicular to a given direction u, whereas μ_u is the mean particle caliper length along u. The second method uses N_V = A_A?V^?1, where A_A is the mean areal fraction of the particles per unit area of section, whereas v is the mean particle volume. The estimation of (N_A)_u, μ_u, and v requires the examination of parallel serial sections above and below T_u. Particle model reconstructions are not needed, however. Previous approaches to the problem are discussed.     

Stereological length estimation using spherical probes

Lineal structures in biological tissue support a wide variety of physiological functions, including membrane stabilization, vascular perfusion, and cell‐to‐cell communication. In 1953, Smith and Guttman demonstrated a stereological method to estimate the total length density (L_v) of linear objects based on random intersections with a two‐dimensional sampling probe. Several methods have been developed to ensure the required isotropy of object–probe intersections, including isotropic‐uniform‐random (IUR) sections, vertical‐uniform‐random (VUR) slices, and isotropic virtual planes. The disadvantages of these methods are the requirements for inconvenient section orientations (IUR, VUR) or complex counting rules at multiple focal planes (isotropic virtual planes). To overcome these limitations we report a convenient and straightforward approach to estimate L_v and total length, L, for linear objects on tissue sections cut at any arbitrary orientation. The approach presented here uses spherical probes that are inherently isotropic, combined with unbiased fractionator sampling, to demonstrate total L estimation for thin nerve fibres in dorsal hippocampus of the mouse brain.     

Estimation of length and surface of anisotropic capillaries

Surface density (S_V) and length density (L_V) of myocardial capillaries have hitherto been estimated from their profile boundary length (B_A) and their numerical density (Q_A) on transverse sections by the simplifying assumptions of the Krogh model (perfectly anisotropic, straight, unbranched capillaries with constant cross-sectional area). As the capillaries actually are partially anisotropic, curved, branching cylinders with variable cross-sectional area, a geometrical bias arises from the model-reality discrepancies. We have applied and compared two methods to overcome these inconsistencies: (1) estimation of L_V and S_V by a more realistic model (the Dimroth-Watson distribution); (2) estimation of L_V and S_V from isotropic uniform random (IUR) sections. Twelve male Wistar rats were fixed by retrograde vascular perfusion. One pair of longitudinal and transverse sections, and six IUR sections per animal were selected at random from the left ventricular papillary muscles. Ultrathin sections were silver-impregnated and studied by light microscopic morphometry. Nearly identical estimates of L_V and S_V were found by both methods. The model-based estimation provides biologically meaningful anisotropy constants, but it presupposes knowledge of the anisotropy axis. The IUR method provides no measure of anisotropy, but it can be applied in tissues where the anisotropy axis is not known. Both methods are equally efficient and practically unbiased in S_V estimation, but the model-based estimation is far more efficient in L_V estimation.     

Alkyloxy‐s‐triazine derivatives with and without fluorine‐containing substituents as lubricants for steel–steel contact

Three alkyloxy‐s‐triazine derivatives were synthesized and their tribological properties as lubricants for steel–steel contact were evaluated using an Optimol SRV tester at 20°C and 100°C. Their thermal stabilities were also investigated by thermogravimetric analysis. The results show that the three alkyloxy‐s‐triazine lubricants have good thermal stability. Moreover, 2,4,6‐tris(1,1,5‐tri‐H‐octafluoropentyloxy)‐1,3,5‐s‐triazine (FPOT) possesses the best anti‐wear property and good load‐carrying capacity both at 20°C and 100°C. At 20°C the anti‐wear effectiveness of 2,4,6‐tris(n‐pentyloxy)‐1,3,5‐s‐triazine (POT) is the worst, while at 100°C that of the 1,1,5‐tri‐H‐octafluoropentyloxy and/or 1,1,7‐tri‐H‐dodecafluoroheptyloxy tri‐substituted s‐triazine mixture (FMOT) is the worst. In addition, the friction‐reducing properties of the two fluoroalkyloxy‐s‐triazines, FPOT and FMOT, are not as good as those of the non‐fluorine‐containing alkyloxy‐s‐triazine POT. Scanning electron spectroscopy with an energy dispersive analyzer of X‐ray and X‐ray photoelectron spectroscopy analyses of the worn surface indicate that during the rubbing process, tribochemical reactions occur between the lubricants and the metal surface to generate a complex boundary lubrication film comprised of FeF₂, Fe(OH)₂, organofluorine and organonitrogen compounds. Copyright © 2006 John Wiley & Sons, Ltd.     

Accuracy of orientation distribution function determination based on EBSD data-A case study of a recrystallized low alloyed Zr sheet

The question of the statistical accuracy of EBSD data for global texture calculation was re‐explored on the basis of a very large grain population (83 000 grains measured on a recrystallized low‐alloyed Zr sheet). Previous works aimed mainly at identifying and quantifying the main texture components and were based on much smaller data sets. The present work attempts to quantify the accuracy of the complete texture, including low‐density regions of the orientation space. For that purpose, a new statistical parameter, V^Δ, based on the calculation of texture difference functions is proposed. This parameter has two main advantages: it is equally sensitive to both high and low peaks of the orientation density function (ODF), and it has a physical interpretation because it is the material volume fraction corresponding to the difference between a given ODF and a reference ODF (considered, or known to be close to the truth). Two main variables were studied: the number of grains taken into account and the peak width ϕ₀ of Bunge's ‘Gaussian’ model density used as kernel for the actual analysis. The orientation distribution functions were computed by nonparametric kernel density estimation with harmonics up to the order of 34. Minimizing the value of V^Δ serves as the objective function for optimizing the peak width ϕ₀ as a function of the number of grains. The properties of the V^Δ parameter also allows for the definition of a method for estimating the accuracy of a given texture that has been obtained from a limited number of grains, without knowing the true texture of the investigated material.     

Estimating aggregate and overall characteristics from thick sections by transmission microscopy

The random spatial structure considered is the union X of an aggregate of random opaque convex particles in transparent space, the particle centroids being sited at the points of a homogeneous Poisson process. The model is thus the union of an aggregate of non-necessarily-convex disjoint regions. Expressions are derived for the fundamental stereological quantities V_V, S_V, K_V (integrated mean curvature density) and G_V (integrated gaussian curvature density) for X; and for A_A, L_A and C_A (integrated curvature density) for the projection of a slice of thickness t of this model onto a plane parallel to the slice, where t may take any non-negative value. The estimates relevant to transmission microscopy suggested by this theory are presented.     

Reverse current in <Emphasis Type="Italic">p-n</Emphasis> junctions with a field electrode on heteroepitaxial CdHgTe/GaAs structures

Results of measuring the current-voltage characteristics of diodes with a control electrode are presented. The diodes are based on graded energy-gap Cd_0.22Hg_0.78Te (MCT) layers grown by molecular beam epitaxy (MBE) on a semi-insulating GaAs substrate. The diodes are designed for the IR radiation photodetectors with the cutoff wavelength λ_c = 10 μm. It is shown that the surface currents contribute substantially to the reverse currents of MBE MCT photodiodes during enrichment and depletion. A critical built-in charge density value for which the surface leakage level is under 20% of bulk current is obtained.     

Estimation of surface area from vertical sections

‘Vertical’ sections are plane sections longitudinal to a fixed (but arbitrary) axial direction. Examples are sections of a cylinder parallel to the central axis; and sections of a flat slab normal to the plane of the slab. Vertical sections of any object can be generated by placing the object on a table and taking sections perpendicular to the plane of the table. The standard methods of stereology assume isotropic random sections, and are not applicable to this kind of biased sampling. However, by using specially designed test systems, one can obtain an unbiased estimate of surface area. General principles of stereology for vertical sections are outlined. No assumptions are necessary about the shape or orientation distribution of the structure. Vertical section stereology is valid on the same terms as standard stereological methods for isotropic random sections. The range of structural quantities that can be estimated from vertical sections includes V_v, N_v, S_v and the volume-weighted mean particle volume v?_v, but not L_v. There is complete freedom to choose the vertical axis direction, which makes the sampling procedure simple and ‘natural’. Practical sampling procedures for implementation of the ideas are described, and illustrated by examples.     

Beam heating in an asymmetrically illuminated thin circular film

The method of images is used to calculate the temperature increase produced in a circular film when the film is illuminated at any point by an electron beam of circular cross-section and constant current density over its cross-section. Formulae are given in dimensionless form for (i) the temperature increase anywhere along the line joining the centres of the beam and of the film, (ii) the temperature increase at the centre of the beam (v_c), and (iii) the position and value of the maximum temperature increase in the film v_m. The relative sizes of v_c and v_m are discussed and it is shown that (v_m – v_c)/v_m ≤ 0.079. The theory applies best to metallic films where power loss by means of thermal conductivity is dominant. A brief discussion is given of the cases where thermal radiation is important, sample calculations are given for carbon and copper films, and the effect of focusing the electron beam at constant current is considered.     

Determining the Surface Electrostatic Potential Ψ s of a Dielectric-Bordering Semiconductor Using the Method of Ψ s "/Ψ s Diagrams

A quantitative approach to determining the integration constant _s0(V _{g 0}) in an expression relating the semiconductor surface potential _s to the voltage V _g applied to the metal–insulator–semiconductor (MIS) structure and its quasi-static capacitance–voltage characteristic C _v(V _g) (normalized to the dielectric capacitance) is described. The method is based on the analysis of experimental functions _s ^"( _s ), where _s ^" = d _s /dV _g, and the same functions calculated for an ideal MIS structure. The obtained function _s (V _g) is a rather exact and complete characteristic of electron properties of the MIS-structure phase boundary (the integrated interface state density, flat-band voltage V _FB, sign and density of the dielectric fixed charge, and variations of these parameters under the action of various factors). Using the example of a particular n-Si MIS structure, it is shown that the method of _s ^"/ _s diagrams ensures a noticeable (up to 0.93 eV) widening of the Si gap sounding region and observation (by the value of the V _FB shift) of very small ( 1 × 10⁷ cm^–2) variations in the charge density at the Si/SiO₂ phase boundary.     

Effect of different calcium contents on the microstructure and mechanical properties of Mg‐5Al‐1Bi‐0.3Mn magnesium alloy

The effect of different Ca contents on the microstructure and mechanical properties of Mg‐5Al‐1Bi‐0.3Mn (AMB501) magnesium alloys was investigated by conventional melting and casting technique using different Ca contents (1.0, 2.0, and 3.0 wt %). Increasing the Ca content resulted in higher hardness and yield strength, but decreased elongation. The improved tensile properties of the AM50‐1Bi‐xCa alloys were due to the changes in AMB501 alloy microstructure when the Ca content increased, as demonstrated by scanning electron microscope, energy dispersive spectrum, and X‐ray diffractometer. The alloy microstructure indicated that the amount of β‐Mg₁₇Al₁₂ phase on grain boundaries decreased and the morphology of β‐Mg₁₇Al₁₂ phase on grain boundaries changed from quasicontinuous‐net shape to dispersed particles. The Mg₁₇Al₁₂ phase disappeared and a new secondary phase Al₂Ca appeared after a 3.0 wt % Ca addition. Microsc. Res. Tech. 78:65–69, 2015. © 2014 Wiley Periodicals, Inc.     

Ultrastructural aspects of mouse nerve‐muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX‐I and Bbil‐TX: Unknown myotoxic effects

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve‐muscle preparations exposed to Bothrops venoms and their phospholipase A₂ toxins (PLA₂) can exhibit a neurotoxic pattern as increase in frequency of miniature end‐plate potentials (MEPPs) as well as in amplitude of end‐plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA₂ toxins (BthTX‐I and Bbil‐TX) in mouse isolated nerve‐phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX‐I and Bbil‐TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic‐like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.