A focus on fixation

Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation–retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed (post-fixation). This was accepted as the “gold standard” for a long time. Post-fixation, however, may have serious consequences for preservation of small peptides leaking from the cut open cells, whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as “gold standard” no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.     

Association between pygmy right whales (Caperea marginata) and areas of high marine productivity off Australia and New Zealand

Abstract

Opportunistic sightings and strandings of Caperea marginata (n=196) from the vicinity of Australia and New Zealand (1884 to early 2007) were used to relate geographic and temporal patterns to oceanographic and broad-scale climatic variability. Records were not uniformly distributed along the coast and more (69%) were from Australia than New Zealand. Seven coastal whale ‘hotspots’ were identified which accounted for 61% of records with locality data. Half of the hotspot records were from southeast (37) and northwest (20) Tasmania—others each had 9–15 events. Upwelling and/or high zooplankton abundance has been documented near all whale hotspots. Records of C. marginata occurred in all months, with 75% in spring and summer. Inter-annual variability showed broad agreement between increased whale records (usually in spring/summer) and strongly positive ‘Niño 3.4’ during 1980–1995 but not thereafter. Coastal upwelling and productivity increase during climatic phenomena such as El Niño and are likely to be quickly beneficial to plankton-feeding whales such as C. marginata.     

Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection

Background

Host genetics has been shown to play a role in porcine reproductive and respiratory syndrome (PRRS), which is the most economically important disease in the swine industry. A region on Sus scrofa chromosome (SSC) 4 has been previously reported to have a strong association with serum viremia and weight gain in pigs experimentally infected with the PRRS virus (PRRSV). The objective here was to identify haplotypes associated with the favorable phenotype, investigate additional genomic regions associated with host response to PRRSV, and to determine the predictive ability of genomic estimated breeding values (GEBV) based on the SSC4 region and based on the rest of the genome. Phenotypic data and 60 K SNP genotypes from eight trials of ~200 pigs from different commercial crosses were used to address these objectives.

Results

Across the eight trials, heritability estimates were 0.44 and 0.29 for viral load (VL, area under the curve of log-transformed serum viremia from 0 to 21 days post infection) and weight gain to 42 days post infection (WG), respectively. Genomic regions associated with VL were identified on chromosomes 4, X, and 1. Genomic regions associated with WG were identified on chromosomes 4, 5, and 7. Apart from the SSC4 region, the regions associated with these two traits each explained less than 3% of the genetic variance. Due to the strong linkage disequilibrium in the SSC4 region, only 19 unique haplotypes were identified across all populations, of which four were associated with the favorable phenotype. Through cross-validation, accuracies of EBV based on the SSC4 region were high (0.55), while the rest of the genome had little predictive ability across populations (0.09).

Conclusions

Traits associated with response to PRRSV infection in growing pigs are largely controlled by genomic regions with relatively small effects, with the exception of SSC4. Accuracies of EBV based on the SSC4 region were high compared to the rest of the genome. These results show that selection for the SSC4 region could potentially reduce the effects of PRRS in growing pigs, ultimately reducing the economic impact of this disease.     

Archamamelis,hamamelidalean flowers from the Upper Cretaceous of Sweden

Lignite fossil flowers (including pollen) and isolated stamens of probable hamamelidalean (possible hamamelidaceous) affinities from the upper Cretaceous (Late Santonian or Early Campanian) of Sweden are described. The flowers are 6–7-merous with probably a double perianth, one whorl of stamens and (2-?)3 carpels. The stamens are disporangiate; each theca opens by a valve towards the centre of the flower. Pollen is tricolpate, tectate-columellate and reticulate; the endexine is lamellated in the apertural region. The gynoecium has free styles and a syncarpous ovary. In the one flower that was serially sectioned the ovary is either non-functional or development of the few (2?) ovules is retarded.     

The ancestor of the <Emphasis Type="Italic">Paulinella</Emphasis> chromatophore obtained a carboxysomal operon by horizontal gene transfer from a <Emphasis Type="Italic">Nitrococcus</Emphasis>-like γ-proteobacterium

Background  

Paulinella chromatophora is a freshwater filose amoeba with photosynthetic endosymbionts (chromatophores) of cyanobacterial origin that are closely related to free-living Prochlorococcus and Synechococcus species (PS-clade). Members of the PS-clade of cyanobacteria contain a proteobacterial form 1A RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) that was acquired by horizontal gene transfer (HGT) of a carboxysomal operon. In rDNA-phylogenies, the Paulinella chromatophore diverged basal to the PS-clade, raising the question whether the HGT occurred before or after the split of the chromatophore ancestor.     

Expression of anti-sense mRNA in H-ras transfected NIH/3T3 cells does not suppress the transformed phenotype

We have attempted to reverse the transformed phenotype of cells expressing the H-ras oncogene. A plasmid in which the first exon of the H-ras oncogene was coupled to the SV40 early promoter in an anti-sense orientation was constructed. This construct was introduced into a clone of H-ras-transformed NIH/3T3 cells. Simultaneous expression of both the SV40 anti-sense construct and H-ras was observed. Anti-sense RNA was present in a 10-20-fold excess over sense H-ras RNA. Only a small fraction of the cytoplasmic RNA was present in a sense: anti-sense duplexed form. The expression of anti-sense H-ras RNA was not accompanied by a phenotypic reversion of transformed cells. The only phenotypic reversion we observed was accompanied by a loss of transfected H-ras sequences. The loss of transfected H-ras sequences occurs with a high frequency in cells supertransfected with the SV40 anti-sense construct.     

Appomattoxia ancistrophora gen. et sp. nov., a new Early Cretaceous plant with similarities to Circaeaster and extant Magnoliidae

A new genus and species of fossil angiosperm (Appomattoxia ancistrophora) is established based on well-preserved fruiting units and associated pollen from the Early Cretaceous (Early or Middle Albian) Puddledock locality in the Potomac Group sequence of Virginia, eastern North America. Fruiting units are small, unilocular, and with a single, pendulous, orthotropous seed. The fruit surface is characterized by densely spaced unicellular spines with hooklike tips, which probably functioned in biotic dispersal. Pollen grains adhering to the stigmatic area of many specimens are monocolpate and tectate with granular to columellate infratectal structure, and are similar to dispersed grains assigned to Tucanopollis and Transitoripollis. Comparison of fossil Appomattoxia ancistrophora with extant plants reveals an unusual combination of characters that includes similarities with some magnoliid taxa, particularly Piperales (Piperaceae, Saururaceae) and Laurales (Chloranthaceae), as well as the monotypic ranunculid family Circaeasteraceae. Appomattoxia ancistrophora differs from extant Piperales in having a pendulous rather than erect ovule, and differs from extant Circaeaster in details of the fruit wall, as well as the presence of monosulcate rather than tricolpate pollen.     

Persistence of accuracy of genomic estimated breeding values over generations in layer chickens

Background

The predictive ability of genomic estimated breeding values (GEBV) originates both from associations between high-density markers and QTL (Quantitative Trait Loci) and from pedigree information. Thus, GEBV are expected to provide more persistent accuracy over successive generations than breeding values estimated using pedigree-based methods. The objective of this study was to evaluate the accuracy of GEBV in a closed population of layer chickens and to quantify their persistence over five successive generations using marker or pedigree information.

Methods

The training data consisted of 16 traits and 777 genotyped animals from two generations of a brown-egg layer breeding line, 295 of which had individual phenotype records, while others had phenotypes on 2,738 non-genotyped relatives, or similar data accumulated over up to five generations. Validation data included phenotyped and genotyped birds from five subsequent generations (on average 306 birds/generation). Birds were genotyped for 23,356 segregating SNP. Animal models using genomic or pedigree relationship matrices and Bayesian model averaging methods were used for training analyses. Accuracy was evaluated as the correlation between EBV and phenotype in validation divided by the square root of trait heritability.

Results

Pedigree relationships in outbred populations are reduced by 50% at each meiosis, therefore accuracy is expected to decrease by the square root of 0.5 every generation, as observed for pedigree-based EBV (Estimated Breeding Values). In contrast the GEBV accuracy was more persistent, although the drop in accuracy was substantial in the first generation. Traits that were considered to be influenced by fewer QTL and to have a higher heritability maintained a higher GEBV accuracy over generations. In conclusion, GEBV capture information beyond pedigree relationships, but retraining every generation is recommended for genomic selection in closed breeding populations.     

Potomacanthus lobatus gen. et sp. nov., a new flower of probable Lauraceae from the Early Cretaceous (Early to Middle Albian) of eastern North America

A charcoalified fossil flower, Potomacanthus lobatus gen. et sp. nov., is described from the Early Cretaceous (Early to Middle Albian) Puddledock locality, Virginia, USA. Internal floral structure was studied using nondestructive synchrotron-radiation x-ray tomographic microscopy (SRXTM). The flower is bisexual and trimerous. The perianth consists of two whorls of tepals. The androecium has two whorls of fertile stamens. Anthers open by two distally hinged valves. The gynoecium consists of a single carpel that is plicate in the style and ascidiate in the ovary and contains a single pendant ovule. The fossil flower shares many similarities with flowers of extant Lauraceae and is unlike flowers of other families of Laurales. However, the fossil flower also differs in detail from all extant or fossil Lauraceae, particularly in configuration of the androecium. The new taxon, together with previously described but more fragmentary material from the Puddledock locality, provides the earliest fossil record of plants more closely related to Lauraceae than to any other extant family. It reveals several derived morphological characters that are potential synapomorphies among extant representatives of the family Lauraceae and contributes to the growing evidence for an early diversification of Laurales before the end of the Early Cretaceous.