The resolution of aneuploid DNA stem lines by flow cytometry: limitations imposed by the coefficient of variation and the percentage of aneuploid nuclei

Factors important in the resolution of cell sub-populations with differing DNA contents were investigated using an EPICS C flow cytometer. Software is available for the EPICS C which permits data from any two histograms to be superimposed or added together before display. Samples of fresh and archival thyroid tissue, stained with propidium iodide, were analysed on the flow cytometer and the peak channel number noted. The photomultiplier (PMT) voltage was increased and the sample analysed again producing a second histogram with a higher peak channel number. The two histograms were added together to simulate a cell suspension with two sub-populations with a different DNA content. By systematically altering the PMT voltage and the number of nuclei included in each analysis, it was possible to examine the importance of DNA index and the percentage of tumor cells with an aneuploid DNA content for both fresh and paraffin-embedded thyroid nuclei. The crucial importance of achieving a low coefficient of variation (CV) was demonstrated and consequently the reservations that pertain when archival material is studied, particularly in tumours where DNA aneuploidy is frequently expressed with a low DNA index.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Environmental,geographical and time-related impacts on avian malaria infections in native and introduced populations of house sparrows (Passer domesticus), a globally invasive species

Aim

The increasing spread of vector-borne diseases has resulted in severe health concerns for humans, domestic animals and wildlife, with changes in land use and the introduction of invasive species being among the main possible causes for this increase. We explored several ecological drivers potentially affecting the local prevalence and richness of avian malaria parasite lineages in native and introduced house sparrows (Passer domesticus) populations.

Location

Global.

Time period

2002–2019.

Major taxa studied

Avian Plasmodium parasites in house sparrows.

Methods

We analysed data from 2,220 samples from 69 localities across all continents, except Antarctica. The influence of environment (urbanization index and human density), geography (altitude, latitude, hemisphere) and time (bird breeding season and years since introduction) were analysed using generalized additive mixed models (GAMMs) and random forests.

Results

Overall, 670 sparrows (30.2%) were infected with 22 Plasmodium lineages. In native populations, parasite prevalence was positively related to urbanization index, with the highest prevalence values in areas with intermediate urbanization levels. Likewise, in introduced populations, prevalence was positively associated with urbanization index; however, higher infection occurred in areas with either extreme high or low levels of urbanization. In introduced populations, the number of parasite lineages increased with altitude and with the years elapsed since the establishment of sparrows in a new locality. Here, after a decline in the number of parasite lineages in the first 30 years, an increase from 40 years onwards was detected.

Main conclusions

Urbanization was related to parasite prevalence in both native and introduced bird populations. In invaded areas, altitude and time since bird introduction were related to the number of Plasmodium lineages found to be infecting sparrows.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Evaluation of Nosema locustae (Microsporida) as a control agent of grasshopper populations in Saskatchewan

A quantitative study of the effect of a microsporidan, Nosema locustae, as a control agent against grasshopper populations in Saskatchewan, Canada, revealed that 50% of the populations of Melanoplus sanguinipes, M. packardii, and Camnula pellucida were infected between 4 and 5 weeks (400 to 424 degree-days) after application of the pathogen. Maxima of 95–100% infection were evident between 9 and 12 weeks (600–700 degree-days) after application. The percentage reduction in surviving populations of M. sanguinipes, i.e., those that did not die from natural causes, reached about 20% by the 4th week (400 degree-days) after inoculation, about 50% by the 9th week (600 + degree-days), and a maximum of about 60% by the 12th week (700 degree-days). An exponential relationship was obtained between percentage reduction and percentage infection in all three species. However, a similar percentage infection resulted in different percentage reduction in the populations. Results also revealed that the rate of reduction in populations reached its peak by about 40–80 degree-days before the maximum rate of infection was attained for each species. Egg production in the two Melanoplus species in treated plots was significantly lower (P < 0.05) than in the control plot.     

Emerging aspects of oesophageal and gastro-oesophageal junction cancer histopathology - an update for the surgical oncologist

Adenocarcinoma of the oesophagus and gastro-oesophageal junction are rapidly increasing in incidence and have a well described sequence of carcinogenesis: the Barrett's metaplasia-dysplasia-adenocarcinoma sequence. During recent years there have been changes in the knowledge surrounding disease progression, cancer management and histopathology specimen reporting. Tumours around the gastro-oesophageal junction (GOJ) pose several specific challenges. Numerous difficulties arise when the existing TNM staging systems for gastric and oesophageal cancers are applied to GOJ tumours. The issues facing the current TNM staging and GOJ tumour classification systems are reviewed in this article. Recent evidence regarding the importance of several histopathologically derived prognostic factors, such as circumferential resection margin status and lymph node metastases, have implications for specimen reporting. With the rising use of multimodal treatments for oesophageal cancer it is important that the response of the tumour to this therapy is carefully documented pathologically. In addition, several controversial and novel areas such as endoscopic mucosal resection, lymph node micrometastases and the sentinel node concept are being studied. We aim to review these aspects, with special relevance to oesophageal and gastro-oesophageal cancer specimen reporting, to update the surgical oncologist with an interest in upper gastrointestinal cancer.     

The molecular machinery of germ line specification

Germ cells occupy a unique position in animal reproduction, development, and evolution. In sexually reproducing animals, only they can produce gametes and contribute genetically to subsequent generations. Nonetheless, germ line specification during embryogenesis is conceptually the same as the specification of any somatic cell type: germ cells must activate a specific gene regulatory network in order to differentiate and go through gametogenesis. While many genes with critical roles in the germ line have been characterized with respect to expression pattern and genetic interactions, it is the molecular interactions of the relevant gene products that are ultimately responsible for germ cell differentiation. This review summarizes the current state of knowledge on the molecular functions and biochemical connections between germ line gene products. We find that homologous genes often interact physically with the same conserved molecular partners across the metazoans. We also point out cases of nonhomologous genes from different species whose gene products play analogous biological roles in the germ line. We suggest a preliminary molecular definition of an ancestral “pluripotency module” that could have been modified during metazoan evolution to become specific to the germ line. Mol. Reprod. Dev. 77: 3–18, 2010. © 2009 Wiley‐Liss, Inc.