Multispectral fingerprinting for improved in vivo cell dynamics analysis

Background  

Tracing cell dynamics in the embryo becomes tremendously difficult when cell trajectories cross in space and time and tissue density obscure individual cell borders. Here, we used the chick neural crest (NC) as a model to test multicolor cell labeling and multispectral confocal imaging strategies to overcome these roadblocks.     

Comparison of protein fermentation characteristics in rumen fluid determined with the gas production technique and the nylon bag technique

In this study, a modified version of the gas production technique was used to determine protein fermentation characteristics in rumen fluid of 19 feedstuffs. Performing the incubations in a N-free environment, and with an excess of rapidly fermentable carbohydrates, made N the limiting factor to microbial growth, and so gas production profiles reflected the availability of N from the feed samples. Results showed that fermentation of protein in rumen fluid can be determined with this modified gas production technique, and that there were distinct differences in protein fermentation between the feed samples. Availability of protein for fermentation was highest in wheat, potato pieces and lupin, and lowest in Rumiraap, a formaldehyde treated rapeseed meal, palm kernel expeller and brewery grains. The protein degradation characteristics of the 19 feed ingredients were also determined with the in situ nylon bag technique. With the obtained results, the amount of rumen escape protein (REP) was calculated for each feedstuff. The results showed that the rate of degradation ranged from 0.010/h for Rumiraap to 0.151/h for wheat. The amount of REP ranged from 197 g/kg CP for lupin to 840 g/kg CP for Rumiraap. Comparing the gas production results with the results obtained with the nylon bag technique showed that there was a good relationship between the gas production after 12–25 h of incubation and the calculated amount of REP (r² = 0.83–0.85). The results show that the adapted gas production technique, being depleted of N and using an excess of rapidly fermentable carbohydrates, is suitable to recognize differences in N availability between feed samples and can be used as an alternative to the nylon bag technique and other in vitro techniques.     

A cytochemical method for the demonstration of 5′-nucleotidase in mouse peritoneal macrophages,with cerium ions used as trapping agent

Summary A method was developed for the demonstration of 5-nucleotidase in murine peritoneal resident macrophages. The cells are incubated cytochemically without agitation and cerium chloride is used as a trapping agent. Under these conditions, the great majority of the macrophages in the unstimulated peritoneal cavity show enzyme activity in the plasma membrane. In the presence of AMP-S (an AMP analogue inhibiting 5-nucleotidase, as shown biochemically) there was a decrease in both the number of positive macrophages and the amount of reaction product on the plasma membranes. This indicates that the enzyme activity detected by our cytochemical procedure is attributable to 5-nucleotidase.     

Optimal stimulation settings for CMAP scan registrations

Background

The CMAP (Compound Muscle Action Potential) scan is a non-invasive electrodiagnostic tool, which provides a quick and visual assessment of motor unit potentials as electrophysiological components that together constitute the CMAP. The CMAP scan records the electrical activity of the muscle (CMAP) in response to transcutaneous stimulation of the motor nerve with gradual changes in stimulus intensity. Large MUs, including those that result from collateral reinnervation, appear in the CMAP scan as so-called steps, i.e., clearly visible jumps in CMAP amplitude. The CMAP scan also provides information on nerve excitability. This study aims to evaluate the influence of the stimulation protocol used on the CMAP scan and its quantification.

Methods

The stimulus frequency (1, 2 and 3 Hz), duration (0.05, 0.1 and 0.3 ms), or number (300, 500 and 1000 stimuli) in CMAP scans of 23 subjects was systematically varied while the other two parameters were kept constant. Pain was measured by means of a visual analogue scale (VAS). Non-parametric paired tests were used to assess significant differences in excitability and step variables and VAS scores between the different stimulus parameter settings.

Results

We found no effect of stimulus frequency on CMAP scan variables or VAS scores. Stimulus duration affected excitability variables significantly, with higher stimulus intensity values for shorter stimulus durations. Step variables showed a clear trend towards increasing values with decreasing stimulus number.

Conclusions

A protocol delivering 500 stimuli at a frequency of 2 Hz with a 0.1 ms pulse duration optimized CMAP scan quantification with a minimum of subject discomfort, artefact and duration of the recording. CMAP scan variables were influenced by stimulus duration and number; hence, these need to be standardized in future studies.     

Non-electrolyte permeability as a tool for studying membrane fluidity

1. The reflection coefficient for the permeation of thiourea through bilayers of phosphatidylcholine is a function of the fatty-acid composition of the lipid molecules. By means of these reflection coefficients an index for membrane fluidity has been given to each of those lipids, relative to that of egg phosphatidylcholine. 2. The maximum number of water molecules that can copermeate with each molecule of solute by means of solute-solvent interaction is a function of the packing of the lipid molecules in the bilayer. This parameter has been used in this paper for characterizing the fluidity of cholesterol-containing membranes and for membranes with their lipids in the gel state.