Effects of gamma-aminobutyric acid, progesterone and ionophore A23187 on acrosome reaction of tree shrew sperm in vitro: examination of acrosome reaction with an improved fluorescence microscopy

A number of acrosome reaction (AR) initiators have been found to be effective in inducing AR of human, laboratory and domestic animal sperm. Using an improved simple fluorescence microscopy, effects of gamma-aminobutyric acid (GABA), progesterone and ionophore A23187 on sperm AR of tree shrew, a useful animal model in biomedical research, have been investigated. Spontaneous AR in 4.92-7.53% of viable sperm was observed. Complete AR in 10.31-18.25% of viable tree shrew sperm was obviously induced by 5 mu M and 10 mu M calcium ionophore A23187, 1 mM GABA, and 5 mu M progesterone, and there were no significant differences between their abilities to initiate complete AR. No significant differences of AR percentages between 1- and 2-h treatments with A23187, progesterone and/or GABA were observed. These results suggested that the responses of tree shrew sperm to these AR initiators are similar to that of human and other mammalian sperm. (C) 1997 Elsevier Science B.V.

An improved microtiter assay for evaluating anti-HIV-I neutralizing antibodies from sera or plasma

Background: The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay. Methods: In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor( s), the tested sera or plasma was removed by a washout procedure after initial 3 - 6 h culture in the assay. Result: This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method. Conclusion: The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma.

In vitro anti-HIV and -HSV activity and safety of sodium rutin sulfate as a microbicide candidate

Sodium rutin sulfate (SRS) is a sulfated rutin modified from the natural flavonol glycoside rutin. Here, we investigated its in vitro anti-HIV and -HSV activities and its cytotoxic profile. Fifty percent inhibitory concentration (IC50) values of SRS against HIV-1 X4 virus IIIB, HIV-1 R5 isolates Ada-M and Ba-L were 2.3 +/- 0.2, 4.5 +/- 2.0 and 8.5 +/- 3.8 mu M with a selectivity index (SI) of 563, 575 and 329, respectively. Its IC50 against primary R5 HIV-1 isolate from Yunnan province in China was 13.1 +/- 5.5 mu M, with a Sl of 197. In contrast, unsulfated rutin had no activity against any of the HIV-1 isolates tested. Further study indicated that SRS blocked viral entry and virus-cell fusion likely through interacting with the HIV- I envelope glycoprotein. SRS also demonstrated some activity against human herpes simplex virus (HSV) with an IC50 of 88.3 +/- 0.1 mu M and a Sl of 30. The 50% cytotoxicity concentration (CC50) of SRS was >3.0 mM, as determined in human genital ME 180, HeLa and primary human foreskin fibroblast cells. Minimum inhibitory concentration of SRS for vaginal lactobacilli was >3.0 mM. These results collectively indicate that SRS represents a novel candidate for anti-HIV-1/HSV microbicide development. (C) 2007 Elsevier B.V. All rights reserved.

Energy stable and momentum conserving hybrid finite element method for the incompressible Navier-Stokes equations

A hybrid method for the incompressible Navier-Stokes equations is presented. The method inherits the attractive stabilizing mechanism of upwinded discontinuous Galerkin methods when momentum advection becomes significant, equal-order interpolations can be used for the velocity and pressure fields, and mass can be conserved locally. Using continuous Lagrange multiplier spaces to enforce flux continuity across cell facets, the number of global degrees of freedom is the same as for a continuous Galerkin method on the same mesh. Different from our earlier investigations on the approach for the Navier-Stokes equations, the pressure field in this work is discontinuous across cell boundaries. It is shown that this leads to very good local mass conservation and, for an appropriate choice of finite element spaces, momentum conservation. Also, a new form of the momentum transport terms for the method is constructed such that global energy stability is guaranteed, even in the absence of a pointwise solenoidal velocity field. Mass conservation, momentum conservation, and global energy stability are proved for the time-continuous case and for a fully discrete scheme. The presented analysis results are supported by a range of numerical simulations. © 2012 Society for Industrial and Applied Mathematics.

Anti-counterfeiting and supply chain security

Counterfeit trade developed into a severe problem for many industries. While established security features such as holograms, micro printings or chemical markers do not seem to efficiently avert trade in illicit imitation products, RFID technology, with its potential to automate product authentications, may become a powerful tool to enhance brand and product protection. The following contribution contains an overview on the implication of product counterfeiting on affected companies, provides a starting point for a structured requirements definition for RFID-based anti-counterfeiting systems, and outlines several principal solution approaches that are discussed in greater detail in the subsequent chapters. © 2008 Springer-Verlag Berlin Heidelberg.

CFD investigation of turbulent premixed flame response to transverse forcing

Screech is a high frequency oscillation that is usually characterized by instabilities caused by large-scale coherent flow structures in the wake of bluff-body flameholders and shear layers. Such oscillations can lead to changes in flame surface area which can cause the flame to burn unsteadily, but also couple with the acoustic modes and inherent fluid-mechanical instabilities that are present in the system. In this study, the flame response to hydrodynamic oscillations is analyzed in a controlled manner using high-fidelity Computational Fluid Dynamics (CFD) with an unsteady Reynolds-averaged Navier-Stokes approach. The response of a premixed flame with and without transverse velocity forcing is analyzed. When unforced, the flame is shown to exhibit a self-excitation that is attributed to the anti-symmetric shedding of vortices in the wake of the flameholder. The flame is also forced using two different kinds of low-amplitude out-of-phase inlet velocity forcing signals. The first forcing method is harmonic forcing with a single characteristic frequency, while the second forcing method involves a broadband forcing signal with frequencies in the range of 500 - 1000 Hz. For the harmonic forcing method, the flame is perturbed only lightly about its mean position and exhibits a limit cycle oscillation that is characteristic of the forcing frequency. For the broadband forcing method, larger changes in the flame surface area and detachment of the flame sheet can be seen. Transition to a complicated trajectory in the phase space is observed. When analyzed systematically with system identification methods, the CFD results, expressed in the form of the Flame Transfer Function (FTF) are capable of elucidating the flame response to the imposed perturbation. The FTF also serves to identify, both spatially and temporally, regions where the flame responds linearly and nonlinearly. Locking-in between the flame's natural self-excited frequency and the subharmonic frequencies of the broadband forcing signal is found to alter the dynamical behaviour of the flame. Copyright © 2013 by ASME.

EFFECT OF ELEVATED BICARBONATE CONCENTRATION ON GROWTH, CHLOROPHYLL A FLUORESCENCE AND ULTRA-STRUCTURE OF Microcystis aeruginosa (CYANOBACTERIUM)

The influence of bicarbonate (HCO3-) on Microcystis aeruginosa FACHB 905 was assessed in this study. Growth curves, chlorophyll a fluorescence and ultrastructure were measured at two HCO3- concentrations, 2.3 mM and 12.4 mM. A treatment of sodium chloride (NaCl) was also conducted alongside to establish the influence level of sodium. It was found that upon treatment with elevated HCO3- concentrations of 2.3 mM and 12.4 mM, cell densities were 13% and 27% (respectively) higher than controls. In photosynthetic performance, elevated HCO3- concentration initially stimulated Fv/Fm at the prophase of culture and then subsequently inhibited it. The inhibition of 2.3mM was higher than that of 12.4mM HCO3-. The maximum relative electron transport rate (ETRmax) exhibited inhibition at elevated HCO3- concentrations. DI0/CS was decreased at 2.3 mM and increased at 12.4mM. In the case of both treatments. ABS/CSI TR0/CS, ET0/CS, RC/CS0 and RC/CSm were decreased by elevated HCO3- concentrations, which indicated damage to photosynthetic apparati and an inactivation of a fraction of reaction centers. This point was also proven by ultrastructural photos. High HCO3--exposed cells lost the characteristic photosynthetic membrane arrangement compared with the control and high salinity treated samples. At the 2.3mM concentration of HCO3-. damage to photosynthetic apparati caused decreased photosynthetic activity. These findings suggested that elevated HCO3- concentration stimulated the growth and photosynthesis of M. aeruginosa FACHB 905 in a short time. Exposure to high HCO3- concentrations for a longer period of time will damage photosynthetic apparatus. In addition, the ultrastructure indicated that elevated HCO3--concentration lead to photosynthetic apparati damage. In our experiment, it was observed that the inhibition effect of 2.3mM HCO3- was higher than that of 12.4mM HCO3-. We hypothesized that M. aeruginosa FACHB 905 induced a protective mechanism under high concentrations of HCO3-.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.

Effects of sand burial on biomass, chlorophyll fluorescence and extracellular polysaccharides of man-made cyanobacterial crusts under experimental conditions

Soil cyanobacterial crusts occur throughout the world, especially in the semiarid and arid regions. It always encounters sand burial, which is an important feature of mobile sand dunes. A greenhouse 41 study was conducted to determine the effects of sand burial on biomass, chlorophyll fluorescence and extracellular polysaccharides of man-made cyanobacterial crusts in six periods of time (0, 5, 10, 15, 20 and 30 d after burying) and at five depths (0, 0.2, 0.5, 1 and 2cm). The results indicated that with the increase of the burial time and burial depth extracellular polysaccharides content and Fv/Fm decreased correspondingly and there were no significant differences between 20 and 30 burial days under different burial depths. The degradation of chlorophyll a content appeared only at 20 and 30 burial days and there was also no significant difference between them under different burial depths. It was also observed a simultaneous decrease of the values of the Fv/Fm and the content of extracellular polysaccharides happened in the crusted cyanobacterium Microcoleus vaginatus Gom. It may suggest that there exists a relationship between extracellular polysaccharides and recovery of the activity of photosystem II (PS II) after rehydration.

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Effects of anti-bursin monoclonal antibody on immunosuppression in the duck (Cherry Valley duck)

To study the immunologic function of bursin, we analyzed the effects of anti-bursin monoclonal antibody (mAb) on the immunosuppression in ducks (Cherry Valley duck) by injecting various doses of the anti-bursin mAb into 13-d duck embryos. After hatch, cell-mediated immune activity and humoral responses were studied using lymphocyte proliferation test, tube agglutination test, and indirect enzyme-linked immuno-sorbent assay to detect anti-Escherichia coli antibodies and antibodies to Riemerella anatipester, respectively. Simultaneously, relative weights (BW-adjusted) of bursa of Fabricius (BF), spleen, and thymus were determined. Additionally, the morphology of BF, spleen, and thymus was examined at various ages using conventional histology. Follicle morphology of BF was analyzed by image analysis. The results indicated that anti-bursin mAb markedly decreased duck lymphocyte proliferation, the antibody-producing ability to bacteria, as well as the relative BF weight. Moreover, the anti-bursin mAb hindered the development of BF follicles.

Study on fluorescence emission and synchronous-scan fluorescence spectrea of Nitzschia hantzschiana solution with FE(III)

The characterization of the algal Nitzschia hantzschiana solution with (or without) Fe(III) was carried out using fluorescence emission and synchronous-scan spectroscopy. An emission peak (excited at 440 nm) was observed at 675 nm for Nitzschia hantzschiana solution. The effective characterization method used was synchronous-scan fluorescence spectroscopy (SFS). A wavelength difference (Delta lambda) of 90 nm was maintained between excitation and emission wavelengths. The peak was observed at about 236(ex) nm (326(em) nm) for synchronous fluorescence spectroscopy. Fe(III) was an effective quencher. The relationship between I-0/I (quenching efficiency) and c (concentration of Fe (III) added) was a linear correlation for the algal solution with Fe(III). Effects of pH on synchronous-scan fluorescence intensity were evident.

Modification of two-photon excited fluorescence from quantum dots on SiN photonic crystals

We investigate two-photon excited fluorescence from CdSe quantum dots with a center-emitting wavelength of 655 nm on SiN photonic crystals. We find that two-photon excited fluorescence is enhanced by more than 1 order of magnitude in the vertical direction when a photonic crystal is used compared to the fluorescence spectra in the absence of photonic crystals. The spectrum of two-photon excited fluorescence from quantum dots on SiN photonic crystal is observed to shift to blue compared to that from quantum dots on SiN without photonic crystals. (C) 2010 Optical Society of America