New embedding medium for sectioning undecalcified bone

Methylmethacrylate (MMA) is the most commonly used embedding medium for sectioning undecalcified bone; however, a number of problems exist with its use in a research laboratory. MMA requires a long infiltration time and temperature control, and it reacts with many polymers. We used Kleer Set resin? as an alternative embedding medium for sectioning undecalcified bone specimens. Fluorochrome labeled bone specimens were sectioned transversely using a ground section technique and longitudinally on a sledge macrotome. The slides were viewed using both transmitted light and epifluorescence microscopy. High quality sections were obtained using Kleer Set resin? for both sectioning techniques. We have shown that this new embedding medium is simpler, safer, quicker to use and does not interfere with visualization of fluorochromes.     

A 16-Channel Receive,Forced Current Excitation Dual-Transmit Coil for Breast Imaging at 7T

Purpose

To enable high spatial and temporal breast imaging resolution via combined use of high field MRI, array coils, and forced current excitation (FCE) multi channel transmit.

Materials and Methods

A unilateral 16-channel receive array insert was designed for use in a transmit volume coil optimized for quadrature operation with dual-transmit RF shimming at 7T. Signal-to-noise ratio (SNR) maps, g-factor maps, and high spatial and temporal resolution in vivo images were acquired to demonstrate the utility of the coil architecture.

Results

The dual-transmit FCE coil provided homogeneous excitation and the array provided an increase in average SNR of 3.3 times (max 10.8, min 1.5) compared to the volume coil in transmit/receive mode. High resolution accelerated in vivo breast imaging demonstrated the ability to achieve isotropic spatial resolution of 0.5 mm within clinically relevant 90 s scan times, as well as the ability to perform 1.0 mm isotropic resolution imaging, 7 s per dynamics, with the use of bidirectional SENSE acceleration of up to R = 9.

Conclusion

The FCE design of the transmit coil easily accommodates the addition of a sixteen channel array coil. The improved spatial and temporal resolution provided by the high-field array coil with FCE dual-channel transmit will ultimately be beneficial in lesion detection and characterization.     

Digestibility of Duddingtonia flagrans chlamydospores in ruminants: in vitro and in vivo studies

Background

Rabies is a widespread disease in African domestic dogs and a serious public health problem in developing countries. Canine rabies became established in Africa during the 20th century, coinciding with ecologic changes that favored its emergence in canids. This paper reports the results of a cross-sectional study of dog ecology in the Antananarivo urban community in Madagascar. A questionnaire survey of 1541 households was conducted in Antananarivo from October 2007 to January 2008. The study addressed both owned and unowned dogs. Various aspects of dog ecology were determined, including size of dog population, relationship between dogs and humans, rabies vaccination.

Results

Dog ownership was common, with 79.6 to 94.1% (mean 88.9%) of households in the six arrondissements owning dogs. The mean owned dog to person ratio was 1 dog per 4.5 persons and differed between arrondissements (administrative districts), with ratios of 1:6.0 in the first arrondissement, 1:3.2 persons in the 2^nd, 1:4.8 in the 3^rd, 1:5.2 in the 4^th, 1:5.6 in the 5^th and 1:4.4 in the 6^th arrondissement. Overall, there were more male dogs (61.3%) and the male/female sex ratio was estimated to be 1.52; however, mature females were more likely than males to be unowned (OR: 1.93, CI 95%; 1.39Conclusion Antananarivo has a higher density of dogs than many other urban areas in Africa. The dog population is unrestricted and inadequately vaccinated against rabies. This analysis of the dog population will enable targeted planning of rabies control efforts.     

Seed characteristics and fatty acid composition of castor (<i>Ricinus communis</i> L.) varieties in Northeast China

Oil content and fatty acid composition were investigated on 12 castor varieties and strains by using the soxhlet extraction method and capillary gas chromatography. This was made to provide a reference and theoretical basis for castorbean breeding with high oil content, determine variability of seed compounds for breeding purposes, and broaden chemical material choices. Results revealed that crude fat percentage in seeds ranged from 18.91 to 35.84% with an average of 25.91%; the absolute content of ricinoleic acid varied between 171.65 g/kg and 314.03 g/kg with an average of 222.43 g/kg, and kernel crude fat percentage was between 24.28 and 46.97% with an average of 34.30%. All these study variables were highest in the 2129 strain. The percentage of ricinoleic acid in crude fat was between 83.85 to 87.62%, and the highest value was found in the zhebi4 accession. The other fatty acids appeared in small concentrations, and showed small amplitude: 1.12 to 1.61%, 1.21 to 1.61%, 3.53 to 4.80%, 5.35 to 6.38%, 0.52 to 0.79%, 0.05 to 0.08% and 0.43 to 0.55%, for palmitic, stearic, oleic, linolic, linolenic, arachidic, and arachidonic acids, respectively. One hundred seed weight was determined for each accession. One hundred seed weight ranged from 25.7 g to 34.0 g with an average of 29.9 g. There was a significant correlation between seed weight and oil content, but the correlation value was low (r=0.51). Cluster analysis by SSPS based on the content of fatty acid composition revealed that the accessions were divided into three independent clusters. These findings will clearly provide useful information for further research in breeding and utilization of castor oil.     

Dynamics of intracellular calcium in hair cells isolated from the semicircular canal of the frog.

Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]i) were monitored optically in hair cells mechanically isolated from frog semicircular canals using the membrane-impermeant form of the Ca(2+)-selective dye Oregon Green 488 BAPTA-1 (OG, 100 microM). Cells stimulated by depolarization under whole-cell voltage clamp conditions revealed Ca(2+) entry at selected sites (hotspots) located mostly in the lower (synaptic) half of the cell body. [Ca(2+)]i at individual hotspots rose with a time constant tau1 approximately 70 ms and decayed with a bi-exponential time-course (tau2 approximately 160, tau3 approximately 2500 ms) following a 160 ms depolarization to -20 mV. With repeated stimulation [Ca(2+)]i underwent independent amplitude changes at distinct hotspots, suggesting that the underlying Ca(2+) channel clusters can be regulated differentially by intracellular signalling pathways. Block by nifedipine indicated that the L-type Ca(2+)channels are distributed at different densities in distinct hotspots. No diffusion barrier other than the nuclear region was found in the cytosol, so that, during a prolonged depolarization (lasting up to 1s), Ca(2+) was able to reach the cell apical ciliated pole. The effective Ca(2+) diffusion constant, measured from the progression of Ca(2+) wavefronts in the cytosol, was approximately 57 microm(2)/s. Our results indicate that in these hair cells, buffered diffusion of Ca(2+) proceeds evenly from the source point to the cell interior and is dominated by the diffusion constant of the endogenous mobile buffers.     

Cryopreservation of seabream (Sparus aurata) spermatozoa

The aim of this research was to optimize protocols for freezing spermatozoa of seabream (Sparus aurata). All the phases of the cryopreservation procedure (sampling, choosing the cryoprotective extender, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa under examination, so as to be able to restore on thawing the morphological and physiological characteristics of fresh semen. Seabream spermatozoa were collected by stripping and transported to the laboratory chilled (0-2 degrees C). Five cryoprotectants, dimethyl sulfoxide (Me(2)SO), ethylene glycol (EG), 1,2-propylene glycol (PG), glycerol, and methanol, were tested at concentrations between 5 and 15% by volume to evaluate their effect on the motility of semen exposed for up to 30 min at 26 degrees C. The less toxic cryoprotectants, 10% EG, 10% PG, and 5% Me(2)SO, respectively, were added to 1% NaCl to formulate the extenders for freezing. The semen was diluted 1:6 with the extender, inserted into 0.25-ml plastic straws by Pasteur pipette, and frozen using a cooling rate of either 10 or 15 degrees C/min to -150 degrees C followed by transfer and storage in liquid nitrogen (-196 degrees C). The straws were thawed at 15 degrees C/s. On thawing, the best motility was obtained with 5% Me(2)SO, although both 10% PG and EG showed good results; no differences were found between the two freezing gradients, although semen frozen with the 10 degrees C/min gradient showed a slightly higher and more prolonged motility.     

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

Background

CCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively.

Results

Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure.

Conclusion

Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.     

Ca<Superscript>2+</Superscript> current of frog vestibular hair cells is modulated by intracellular ATP but not by long-lasting depolarisation

Some aspects of Ca²⁺ channel modulation in hair cells isolated from semicircular canals of the frog (Rana esculenta) have been investigated using the whole-cell technique and intra and extracellular solutions designed to modify the basic properties of the Ca²⁺ macrocurrent. With 1 mM ATP in the pipette solution, about 60% of the recorded cells displayed a Ca²⁺ current constituted by a mix of an L and a drug-resistant (R2) component; the remaining 40% exhibited an additional drug-resistant fraction (R1), which inactivated in a Ca-dependent manner. If the pipette ATP was raised to 10 mM, cells exhibiting the R1 current fraction displayed an increase of both the R1 and L components by ∼280 and ∼70%, respectively, while cells initially lacking R1 showed a similar increase in the L component with R1 becoming apparent and raising up to a mean amplitude of ∼44 pA. In both cell types the R2 current fraction was negligibly affect by ATP. The current run-up was unaffected by cyclic nucleotides, and was not triggered by 10 mM ATPγS, ADP, AMP or GTP. Long-lasting depolarisations (>5 s) produced a progressive, reversible decay in the inward current despite the presence of intracellular ATP. Ca²⁺ channel blockade by Cd²⁺ unmasked a slowly activating outward Cs⁺ current flowing through a non-Ca²⁺ channel type, which became progressively unblocked by prolonged depolarisation even though Cs⁺ and TEA⁺ were present on both sides of the channel. The outward current waveform could be erroneously ascribed to a Ca- and/or voltage dependence of the Ca²⁺ macrocurrent. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.