Effects of fixation on routine,special and immunohistochemical stains: introduction to a symposium for the Biological Stain Commission

Orcein was first proposed as an elastic fiber stain by Taenzer in 1890, and has proved very useful for the purpose. It is an important constituent of the author's “Quad” stain. Unfortunately not all orcein samples have proved equally satisfactory, whether they are derived from the lichens from which the dye was originally prepared or have been manufactured by a synthetic process. At the present time several brands are available, and two of the brands of the synthetic product investigated have not proved to be the same thing; one of the latter proves only fair as an elastin stain, the other is one of the best samples the author has ever tried.     

News from the Biological Stain Commission No. 5

In this fifth issue of News from the Biological Stain Commission (BSC), under the heading of Regulatory Affairs, the BSC's International Affairs Committee provides more information from the meeting of the International Standards Organization ISO/TC 212 Committee that took place on June 2–4, 2008 at Vancouver, Canada. In addition, we give an update on the current situation regarding the supplies of hematoxylin.     

Benzidine-based dyes: effects of industrial practices,regulations, and world trade on the biological stains market

One of the most sweeping changes in the dye industry since the advent of synthetic dyes grew out of the health risks associated with benzidine. Dyes made from benzidine and its derivatives were used around the world until adverse health effects become incontrovertible. Workers and family members of workers involved in production and use of benzidine-based dyes had a high incidence of bladder cancer. Following publication of several reports documenting this health hazard, dye makers in the USA, Europe, and Japan phased these dyes out of production in the 1970s. Government regulations lent legal support for these voluntary initiatives. Two strategies subsequently evolved to compensate: developed nations brought alternative substances to market while emerging countries increased production of carcinogenic dyes and sold them at discount prices around the world. Nearly all dye manufacturing now has moved away from nations whose costs of production and compliance rendered them unable to compete. The purpose of this brief review is to publicize the health risks associated with dyes made from benzidine and its congeners, and to alert all companies and end users handling these dyes for biomedical applications that composition of the product and lot-to-lot variability may be problematic because of the manufacturing and distribution practices of the countries where they are produced.     

Arginine protects muscle cells from wasting in vitro in an mTORC1-dependent and NO-independent manner

Amino acids are potent regulators of muscle protein synthesis and breakdown and have received considerable attention for the treatment of muscle wasting conditions. Arginine is critically involved in numerous physiological functions including providing substrate for the production of creatine, urea and nitric oxide (NO) and in the synthesis of new proteins. However, little is known about the direct effects of arginine on skeletal muscle protein synthesis during catabolic conditions. The aims of this study were to determine whether exogenous arginine could protect skeletal muscle cells from wasting directly and whether this effect was dependent on production of NO and/or activation of the rapamycin-sensitive mechanistic target of rapamycin complex 1 (mTORC1) signalling pathway. To explore these aims, we deprived mature C2C12 myotubes from nutrients and growth factors by incubating them in HEPES buffered saline with arginine or equimolar concentrations of alanine (control). Our results show that arginine: increased the ratio of phosphorylated to total mTOR (146 %), S6 (40 %) and 4EBP1 (69 %); increased protein synthesis (69 %) during the first hour of treatment; and increased myotube diameter by ~15 %. Experiments using the NO synthase inhibitor l-NG-Nitroarginine Methyl Ester showed a NO-independent protection from muscle wasting. On the other hand, the mTORC1 inhibitor rapamycin prevented increases in phosphorylated S6, protein synthesis and myotube diameter. The activation of mTORC1 and protein synthesis by arginine was not associated with changes in the phosphorylation status of Akt, but rather increased the expression of the amino acid-sensitive type III PI3-kinase Vps34 signalling protein. These data support a direct role for arginine in the regulation of mTORC1 in skeletal muscle.     

A cross-sectional study to determine the seroprevalence of bluetongue virus serotype 8 in sheep and goats in 2006 and 2007 in the Netherlands

Background

In August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe. In the course of 2007 it became evident that BTV8 had survived the winter in North-West Europe, re-emerged and spread exponentially. Recently, the European Union decided to start vaccination against BTV8. In order to improve the understanding of the epidemiological situation, it was necessary to execute a cross-sectional serological study at the end of the BT vector season. Cattle were the target species for cross-sectional serological studies in Europe at the end of 2006 and 2007. However, there was no information on the BTV8-seroprevalence in sheep and goats.

Results

On the basis of our cross-sectional study, the estimated seroprevalence of BTV8-exposed locations in the Netherlands in 2006 was 0% for goats (95% confidence interval: 0 – 5.6%) and 7.0% for sheep (95% confidence interval: 3.5 – 12.9%). The estimated seroprevalence of BTV-8 exposed locations in 2007 was 47% for goats (95% confidence interval: 36 – 58%) and 70% for sheep (95% confidence interval: 63 – 76%). There was a wide range in within-location seroprevalence in locations with goats and sheep (1 – 100%). A gradient in seroprevalence was seen, with the highest level of seroprevalence in the southern Netherlands, the area where the epidemic started in 2006, and a decreasing seroprevalence when going in a northern direction.

Conclusion

There is a much higher estimated seroprevalence of locations with goats exposed to BTV8 than can be inferred from the rather low number of reported clinical outbreaks in goats. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep. The wide range in within-location seroprevalence observed means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between flocks. This should be taken into account when vaccinating animals.

     

Local spread of classical swine fever upon virus introduction into The Netherlands: Mapping of areas at high risk

Background

In the recent past, the introduction of Classical Swine Fever Virus (CSFV) followed by between-herd spread has given rise to a number of large epidemics in The Netherlands and Belgium. Both these countries are pork-exporting countries. Particularly important in these epidemics has been the occurrence of substantial "neighborhood transmission" from herd to herd in the presence of base-line control measures prescribed by EU legislation. Here we propose a calculation procedure to map out "high-risk areas" for local between-herd spread of CSFV as a tool to support decision making on prevention and control of CSFV outbreaks. In this procedure the identification of such areas is based on an estimated inter-herd distance dependent probability of neighborhood transmission or "local transmission". Using this distance-dependent probability, we derive a threshold value for the local density of herds. In areas with local herd density above threshold, local transmission alone can already lead to epidemic spread, whereas in below-threshold areas this is not the case. The first type of area is termed 'high-risk' for spread of CSFV, while the latter type is termed 'low-risk'.

Results

As we show for the case of The Netherlands, once the distance-dependent probability of local transmission has been estimated from CSFV outbreak data, it is possible to produce a map of the country in which areas of high-risk herds and of low-risk herds are identified. We made these maps even more informative by estimating border zones between the two types of areas. In these border zones the risk of local transmission of infection to a nearby high-risk area exceeds a certain level.

Conclusion

The risk maps provide an easily understandable visualization of the spatial heterogeneities in transmission risk. They serve as a tool for area-specific designs of control strategies, and possibly also for spatial planning of areas where livestock farming is allowed. Similar risk maps can in principle be constructed for other highly-transmissible livestock infections that spread via neighborhood transmission.

     

Benefits to shorebirds from invasion of a non-native shellfish

Introductions of non-native species are seen as major threats to ecosystem function and biodiversity. However, invasions of aquatic habitats by non-native species are known to benefit generalist consumers that exhibit dietary switches and prey upon the exotic species in addition to or in preference to native ones. There is, however, little knowledge concerning the population-level implications of such dietary changes. Here, we show that the introduction of the Manila clam Tapes philippinarum into European coastal waters has presented the Eurasian oystercatcher Haematopus ostralegus ostralegus with a new food resource and resulted in a previously unknown predator-prey interaction between these species. We demonstrate, with an individuals-based simulation model, that the presence of this non-native shellfish, even at the current low density, has reduced the predicted over-winter mortality of oystercatchers at one recently invaded site. Further increases in clam population density are predicted to have even more pronounced effects on the density dependence of oystercatcher over-winter mortality. These results suggest that if the Manila clam were to spread around European coastal waters, a process which is likely to be facilitated by global warming, this could have considerable benefits for many shellfish-eating shorebird populations.     

Intersection tests for single marker QTL analysis can be more powerful than two marker QTL analysis

Background

It has been reported in the quantitative trait locus (QTL) literature that when testing for QTL location and effect, the statistical power supporting methodologies based on two markers and their estimated genetic map is higher than for the genetic map independent methodologies known as single marker analyses. Close examination of these reports reveals that the two marker approaches are more powerful than single marker analyses only in certain cases.Simulation studies are a commonly used tool to determine the behavior of test statistics under known conditions. We conducted a simulation study to assess the general behavior of an intersection test and a two marker test under a variety of conditions. The study was designed to reveal whether two marker tests are always more powerful than intersection tests, or whether there are cases when an intersection test may outperform the two marker approach.We present a reanalysis of a data set from a QTL study of ovariole number in Drosophila melanogaster.

Results

Our simulation study results show that there are situations where the single marker intersection test equals or outperforms the two marker test. The intersection test and the two marker test identify overlapping regions in the reanalysis of the Drosophila melanogaster data. The region identified is consistent with a regression based interval mapping analysis.

Conclusion

We find that the intersection test is appropriate for analysis of QTL data. This approach has the advantage of simplicity and for certain situations supplies equivalent or more powerful results than a comparable two marker test.

     

Lidocaine block of cardiac sodium channels

Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 μM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 μM, at a negative holding potential where inactivation was completely removed, to approximately 10 μM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (τless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (τ approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 μM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 μM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the “modulated-receptor hypothesis” of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.