Gross anatomy of the muscle systems of Fasciola hepatica as visualized by phalloidin-fluorescence and confocal microscopy

Neuropeptides, biogenic amines and acetylcholine are expressed abundantly within the nervous systems of parasitic flatworms, and are particularly evident in the innervation of the musculature. Such associations have implicated the nervous system in locomotion, host attachment and reproductive co-ordination. Information on the muscle systems of parasitic flatworms is generally sparse, in particular those muscles associated with the reproductive system, intestinal tract and attachment apparatus. Also, the use of sectioned material has left description of the 3-dimensional organization of the musculature largely unrecorded. Using fluorescein isothiocyanate (FITC)-labelled phalloidin as a site-specific probe for filamentous actin, applied to whole-mount preparations of adult Fasciola hepatica and examined by confocal scanning laser microscopy, the present work reports on the organization of the major muscle systems in this trematode parasite. A highly regular array of outer circular, intermediate longitudinal and inner diagonal fibres distinguishes the body wall musculature, which is also involved in the development of both ventral and oral suckers. Circular fibres dominate the duct walls of the male and female reproductive systems, whereas the muscles of the intestinal tract have a somewhat diffuse arrangement of fibres. An understanding of the structural complexity of the muscle systems of parasitic flatworms is considered as fundamental to the interpretation of results from physiological experiments.

RP-LC with fluorescence detection of amino acids in rat brain synaptosomes

For the first time, a simple and validated reversed-phase liquid chromatography (RP-LC) with fluorescence detection has been developed for the simultaneous analysis of glutamate (Glu), ?-aminobutyric acid (GABA), glycine (Gly) and taurine (Tau) in Wistar and tremor rats brain synaptosomes. The samples were separated on a C18 analytical column with gradient elution of methanol and 0.1 mol L-1 potassium acetate at a flow rate of 1 mL min-1. Total run time was approximately 25 min. All calibration curves exhibited good linearity (r 2 > 0.999) within test ranges. The reproducibility was estimated by intra-and inter-day assays and RSD values were less than 2.48%. The recoveries were between 96.32 and 105.21%. The method was successfully applied to the quantification of amino acids in Wistar and tremor rats brain synaptosomes. Through this developed protocol, the levels of Glu in hippocampal and prefrontal cortical synaptosomes of tremor rats were both significantly elevated than those of adult Wistar rats whereas significantly decreased concentrations of GABA and Gly were observed in the hippocampal region of tremor rats without evident difference in the prefrontal cortex between experimental and control groups. In addition, our studies also showed a marked elevation of Tau in tremor rats hippocampal synaptosomes although there was no pronounced difference in the prefrontal cortical region of Wistar and tremor rats.

A novel dual-fluorescence strategy for functionally validating microRNA targets in 3-prime untranslated regions: regulation of the inward rectifier potassium channel Kir2.1 by miR-212.

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 (KCNJ2) which is dysregulated in cardiac and vascular disorders. The 3'UTR was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known downregulator of Kir2.1 expression, and was used to investigate targeting of the Kir2.1 3'UTR by miR-212. Red/green ratio was lower in miR-212-expressing cells compared to non-targeting controls, an effect that was attenuated by mutating the predicted target site. MiR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

Excitation of the 3p^4(4s,3d,4p) Ar^+ states during Ar photoionization:Intensity, alignment, and orientation

Ar photoionization is studied using the R-matrix formalism with emphasis on the simultaneous excitation of the residual A^r+ ion. Cross sections have been obtained for excitation of the 3p^4(3d,4s,4p) states. A comparison with experiments having a resolution of 70 meV shows reasonable agreement for the position and shape of resonance structures. The relative magnitude of the resonances proves to be more elusive. The partial cross section for excitation of the 3p^4(3Pe)4p(2P_3/2^o) and (2D_3/2^o) levels is treated in more detail. A comparison of LS-coupling calculations with high-resolution experimental results shows good agreement for both the excitation cross sections and the polarization of the fluorescence. We also predict the orientation for both levels. We demonstrate that the polarization of the fluorescence originating from the (2D_3/2^o) level can be employed to study spin-orbit effects in Ar photoionization.

Fluorescence yields for 3lnl′ Rydberg series in ions belonging to the He isoelectronic sequence

Fluorescence yields are reported for 3lnl' Rydberg series members in He-like ions of N, O and Ne. Results are presented for singlet series members with n values between 3 and 9, i.e. up to the region of overlap with the states belonging to the 4l4l' manifold in these atoms. This data is required, for example, for the interpretation of charge-exchange experiments involving bare N, O and Ne nuclei. Fluorescence yields, averaged over all 3lnl' singlet states, larger than 50% are obtained from about n = 7.

INFLUENCE OF CORE POLARIZATION ON THE ELECTRON-AFFINITY OF CA

We have developed a method, based on the use of B-spline basis sets and model potentials, for determining properties of systems with two or three electrons outside a polarizable closed-shell core. It is applied to the calculation of the electron affinity of Ca and the resulting value of 17.7 meV is in excellent agreement with the most recent experiments. It is found that the dielectronic core-valence interaction reduces the electron affinity by 39.5 meV.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples

Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.

Detection of Paralytic Shellfish Toxins by a Solid-Phase Inhibition Immunoassay Using a Microsphere-Flow Cytometry System

Paralytic shellfish poisoning is a toxic syndrome described in humans following the ingestion of seafood contaminated with saxitoxin and/or its derivatives. The presence of these toxins in shellfish is considered an important health threat and their levels in seafood destined to human consumption are regulated in many countries, as well as the levels of other chemically unrelated toxins. We studied the feasibility of immunodetection of saxitoxin and its analogs using a solid-phase microsphere assay coupled to flow cytometry detection in a Luminex 200 system. The technique consists of a competition assay where the toxins in solution compete with bead-bound saxitoxin for binding to an antigonyautoxin 2/3 monoclonal antibody (GT-13A). The assay allowed the detection of saxitoxin both in buffer and mussel extracts in the range of 2.2-19.7 ng/mL (IC(20)-IC(80)). Moreover, the assay cross-reactivity with other toxins of the group is similar to previously published immunoassays, with adequate detection of most analogs except N-1 hydroxy analogs. The recovery rate of the assay for saxitoxin was close to 100%. This microsphere-based immunoassay is suitable to be used as a screening method, detecting saxitoxin from 260 to 2360 µg/kg. This microsphere/flow cytometry system provided similar sensitivities to previously published immunoassays and provides a solid background for the development of easy, flexible multiplexing of toxin detection in one sample.

Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 µg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 µg/kg, respectively.

Development and validation of an ultrasensitive fluorescence planar waveguide biosensor for the detection of paralytic shellfish toxins in marine algae

Marine dinoflagellates of the genera Alexandrium are well known producers of the potent neurotoxic paralytic shellfish toxins that can enter the food web and ultimately present a serious risk to public health in addition to causing huge economic losses. Direct coastal monitoring of Alexandrium spp. can provide early warning of potential shellfish contamination and risks to consumers and so a rapid, sensitive, portable and easy-to-use assay has been developed for this purpose using an innovative planar waveguide device. The disposable planar waveguide is comprised of a transparent substrate onto which an array of toxin-protein conjugates is deposited, assembled in a cartridge allowing the introduction of sample, and detection reagents. The competitive assay format uses a high affinity antibody to paralytic shellfish toxins with a detection signal generated via a fluorescently labelled secondary antibody. The waveguide cartridge is analysed by a simple reader device and results are displayed on a laptop computer. Assay speed has been optimised to enable measurement within 15 min. A rapid, portable sample preparation technique was developed for Alexandrium spp. in seawater to ensure analysis was completed within a short period of time. The assay was validated and the LOD and CCß were determined as 12 pg/mL and 20 pg/mL respectively with an intra-assay CV of 11.3% at the CCß and an average recovery of 106%. The highly innovative assay was proven to accurately detect toxin presence in algae sampled from the US and European waters at an unprecedented cell density of 10 cells/L. © 2012 Elsevier B.V. All rights reserved.

Self-potential and spectral induced polarization signals associated with microbial sulphate reduction

Self-potential and spectral induced polarization responses associated with microbial processes involved in sulphate reduction have been monitored in a Perspex Winogradsky column filled with glass beads and growth medium. Salt-bridge is utilized as an electrolytic contact between experiment and control column. Equally spaced SP electrodes are used in combination of Ag-AgCl electrodes to compare electrodic and SP signals associated with the microbial processes involved in sulphate reduction. This study reveals that magnitude of SP varies from 5 to -2 mV and Electrodic potential 0 to -20 mV at the time of domination (day 39) of sulphate reducing bacteria which are very small in comparison to those measured by fixing both measuring and reference Ag-AgCl electrodes in experiment column. We observed that real and imaginary parts of complex conductivities increase with increase in production of H2S and CO in the experiment column. Both real and imaginary parts of surface complex conductivity vary at low frequencies similar to typical growth curve of bacterial population. Sodium lactate as a carbon source, dissolved in Lagan River water was flushed into the column for biostimulation on 144th day. The dissolved oxygen in flushed fluid might have killed the anaerobes in the column and decrease in complex conductivities similar to death phase of bacteria is observed for one week. The results obtained from this experiment should contribute to further understanding the biogeophysical responses involved in complex environments.


Read More: http://library.seg.org/doi/abs/10.1190/segj092009-001.57

Structural effects on the pH-dependent fluorescence of naphthalenic derivatives and consequences for sensing/switching

Naphthalenic compounds are a rich resource for designers of fluorescent sensing/switching/logic systems. The degree of internal charge transfer (ICT) character in the fluorophore excited states can vary from negligible to substantial. Naphthalene-1,8;4,5-diimides (11–13), 1,8-naphthalimides (16) and 4-chloro-1,8-naphthalimides (15) are of the former type. The latter type is represented by the 4-alkylamino-1,8-naphthalimides (1). Whether ICT-based or not, these serve as the fluorophore in ‘fluorophore-spacer-receptor’ switching systems where PET holds sway until the receptor is bound to H+. On the other hand, 4-dialkylamino-1,8-naphthalimides (3–4) show modest H+-induced fluorescence switching unless the 4-dialkylamino group is a part of a small ring (5). Electrostatic destabilization of a non-emissive twisted internal charge transfer (ICT) excited state is the origin of this behaviour. An evolution to the non-emissive twisted ICT excited state is responsible for the weak emission of the model compound 6 (and related structures 7 and 8) across the pH range. Twisted ICT excited states are also implicated in the switch 9 and its model compound 10, which are based on the 6-dialkylamino-3H-benzimidazo[2,1-a]benz[d,e]isoquinolin-3-one fluorophore.