Genetic parentage in the squat lobsters Munida rugosa and M. sarsi (Crustacea, Anomura, Galatheidae)

Munida is the most diverse and cosmopolitan genus of the galatheid squat lobsters. The group has attracted much attention in recent years from both systematic and evolutionary perspectives, yet information on the biology, ecology, and evolution of this genus is very limited. We investigated the genetic parentage of two North Atlantic species; Munida rugosa and M. sarsi sampled from the Clyde Sea on the west coast of Scotland. Microsatellite markers were used to establish the parental contribution from embryos of berried females (M. rugosa, n=25 and M. sarsi, n=5). The frequency of multiple paternity observed in both species (86% for M. rugosa and 100% for M. sarsi) is the highest ever reported for any marine crustaceans. Invariably more than two sires were involved in each case (minimum of two to three for M. rugosa and four for M. sarsi). Findings indicate that multiple paternity is likely to be the norm in both species. Within most multiply sired broods, sire contribution was highly skewed towards a single male (66% of broods for M. rugosa and 100% for M. sarsi). Furthermore, embryos from different sires were randomly distributed across the female's brood patch. This is the first report of multiple paternity in galatheids. While a number of theories can account for the high incidence of multiple paternity in these species (e.g. convenience polyandry as a result of cryptic female choice, forced copulations, the influence of fishing pressures), at present it is not possible to disentangle their individual and/or combined effects.

Landscape effects on extremely fragmented populations of a rare solitary bee, Colletes floralis

Globally there is concern over the decline of bees, an ecologically important group of pollinating insects. Genetic studies provide insights into population structure that are crucial for conservation management but that would be impossible to obtain by conventional ecological methods. Yet conservation genetic studies of bees have primarily focussed on social species rather than the more species-rich solitary bees. Here we investigate the population structure of Colletes floralis, a rare and threatened solitary mining bee, in Ireland and Scotland using nine microsatellite loci. Genetic diversity was surprisingly as high in Scottish (Hebridean island) populations at the extreme northwestern edge of the species range as in mainland Irish populations further south. Extremely high genetic differentiation among populations was detected; multilocus FST was up to 0.53, and G’ST and Dest were even higher (maximum: 0.85 and 1.00 respectively). A pattern of isolation by distance was evident for sites separated by land. Water appears to act as a substantial barrier to gene flow yet sites separated by sea did not exhibit isolation by distance. Colletes floralis populations are extremely isolated and probably not in regional migration-drift equilibrium. GIS-based landscape genetic analysis reveals urban areas as a potential and substantial barrier to gene flow. Our results highlight the need for urgent site-specific management action to halt the decline of this and potentially other rare solitary bees.

Genetic differentiation across the social transition in a socially polymorphic sweat bee, Halictus rubicundus

Eusociality is widely considered a major evolutionary transition. The socially polymorphic sweat bee Halictus rubicundus, solitary in cooler regions of its holarctic range and eusocial in warmer parts, is an excellent model organism to address this transition, and specifically the question of whether sociality is associated with a strong barrier to gene flow between phenotypically divergent populations. Mitochondrial DNA (COI) from specimens collected across the British Isles, where both solitary and social phenotypes are represented, displayed limited variation, but placed all specimens in the same European lineage; haplotype network analysis failed to differentiate solitary and social lineages. Microsatellite genetic variability was high and enabled us to quantify genetic differentiation among populations and social phenotypes across Great Britain and Ireland. Results from conceptually different analyses consistently showed greater genetic differentiation between geographically distant populations, independently of their social phenotype, suggesting that the two social forms are not reproductively isolated. A landscape genetic approach revealed significant isolation by distance (Mantel test r = 0.622, p

Evidence for a modifier of onset age in Huntington disease linked to the HD gene in 4p16

Huntington disease (HD) is a neurodegenerative disorder caused by the abnormal expansion of CAG repeats in the HD gene on chromosome 4p16.3. A recent genome scan for genetic modifiers of age at onset of motor symptoms (AO) in HD suggests that one modifier may reside in the region close to the HD gene itself. We used data from 535 HD participants of the New England Huntington cohort and the HD MAPS cohort to assess whether AO was influenced by any of the three markers in the 4p16 region: MSX1 (Drosophila homeo box homologue 1, formerly known as homeo box 7, HOX7), Delta2642 (within the HD coding sequence), and BJ56 (D4S127). Suggestive evidence for an association was seen between MSX1 alleles and AO, after adjustment for normal CAG repeat, expanded repeat, and their product term (model P value 0.079). Of the variance of AO that was not accounted for by HD and normal CAG repeats, 0.8% could be attributed to the MSX1 genotype. Individuals with MSX1 genotype 3/3 tended to have younger AO. No association was found between Delta2642 (P=0.44) and BJ56 (P=0.73) and AO. This study supports previous studies suggesting that there may be a significant genetic modifier for AO in HD in the 4p16 region. Furthermore, the modifier may be present on both HD and normal chromosomes bearing the 3 allele of the MSX1 marker.

Interaction of normal and expanded CAG repeat sizes influences age at onset of Huntington disease

Huntington disease (HD) is a neurodegenerative disorder caused by the abnormal expansion of CAG repeats in the HD gene on chromosome 4p16.3. Past studies have shown that the size of expanded CAG repeat is inversely associated with age at onset (AO) of HD. It is not known whether the normal Huntington allele size influences the relation between the expanded repeat and AO of HD. Data collected from two independent cohorts were used to test the hypothesis that the unexpanded CAG repeat interacts with the expanded CAG repeat to influence AO of HD. In the New England Huntington Disease Center Without Walls (NEHD) cohort of 221 HD affected persons and in the HD-MAPS cohort of 533 HD affected persons, we found evidence supporting an interaction between the expanded and unexpanded CAG repeat sizes which influences AO of HD (P = 0.08 and 0.07, respectively). The association was statistically significant when both cohorts were combined (P=0.012). The estimated heritability of the AO residual was 0.56 after adjustment for normal and expanded repeats and their interaction. An analysis of tertiles of repeats sizes revealed that the effect of the normal allele is seen among persons with large HD repeat sizes (47-83). These findings suggest that an increase in the size of the normal repeat may mitigate the expression of the disease among HD affected persons with large expanded CAG repeats. (C) 2003 Wiley-Liss, Inc.

Polymorphisms in SLC6A4, PAH, GABRB3, and MAOB and modification of psychotic disorder features

We tested four genes [phenylalanine hydroxylase (PAH), the serotonin transporter (SLC6A4), monoamine oxidase B (MAOB), and the gamma-aminobutyric acid A receptor beta-3 subunit (GABRB3)] for their impact on five schizophrenia symptom factors: delusions, hallucinations, mania, depression, and negative symptoms. In a 90 family subset of the Irish Study of High Density Schizophrenia Families, the PAH 232 bp microsatellite allele demonstrated significant association with the delusions factor using both QTDT (F = 8.0, p = .031) and QPDTPHASE (chi-square = 12.54, p = .028). Also, a significant association between the GABRB3 191 bp allele and the hallucinations factor was detected using QPDTPHASE (chi-square 15.51, p = .030), but not QTDT (chi-square = 2.07, p = .560). (C) 2009 Elsevier B.V. All rights reserved.

Phylogeographic analysis of North American populations of the parasitic herbaceous plant Monotropa hypopitys reveals a complex history of range expansion from multiple late glacial refugia

Aim We carried out a phylogeographic study across the range of the herbaceous plant species Monotropa hypopitys L. in North America to determine whether its current disjunct distribution is due to recolonization from separate eastern and western refugia after the Last Glacial Maximum (LGM). Location North America: Pacific Northwest and north-eastern USA/south-eastern Canada. Methods Palaeodistribution modelling was carried out to determine suitable climatic regions for M. hypopitys at the LGM. We analysed between 155 and 176 individuals from 39 locations spanning the species' entire range in North America. Sequence data were obtained for the chloroplast rps2 gene (n=168) and for the nuclear ITS region (n=158). Individuals were also genotyped for eight microsatellite loci (n=176). Interpolation of diversity values was used to visualize the range-wide distribution of genetic diversity for each of the three marker classes. Minimum spanning networks were constructed showing the relationships between the rps2 and ITS haplotypes, and the geographical distributions of these haplotypes were plotted. The numbers of genetic clusters based on the microsatellite data were estimated using Bayesian clustering approaches. Results The palaeodistribution modelling indicated suitable climate envelopes for M. hypopitys at the LGM in both the Pacific Northwest and south-eastern USA. High levels of genetic diversity and endemic haplotypes were found in Oregon, the Alexander Archipelago, Wisconsin, and in the south-eastern part of the species' distribution range. Main conclusions Our results suggest a complex recolonization history for M. hypopitys in North America, involving persistence in separate eastern and western refugia. A generally high degree of congruence between the different marker classes analysed indicated the presence of multiple refugia, with at least two refugia in each area. In the west, putative refugia were identified in Oregon and the Alexander Archipelago, whereas eastern refugia may have been located in the southern part of the species' current distribution, as well as in the 'Driftless Area'. These findings are in contrast to a previous study on the related species Orthilia secunda, which has a similar disjunct distribution to M. hypopitys, but which appears to have recolonized solely from western refugia. © 2011 Blackwell Publishing Ltd.

Metastatic testicular carcinoma from the colon with clinical, immunophenotypical, and molecular characterization: Report of a case

We report a case of testicular metastasis from a colonic adenocarcinoma. The presentation of testicular metastasis, diagnosis, management, and possible modes of spread are reported. In addition to conventional investigations and histopathologic techniques, a molecular study of the testicular metastasis and colon primary, using microsatellite analysis, was performed to confirm the primary origin. Its potential uses are discussed.

Immunocytochemistry versus molecular fingerprinting of metastases

Examination of cytological samples of cancer to suggest a possible primary site of origin is one of the commonest and most difficult tasks of diagnostic cytopathologists. Currently, both cytomorphology and immunocytochemistry are the main approaches to this diagnostic dilemma. We report the application of microsatellite analysis in cytological samples in a patient with a primary colonic tumour and two subsequent lung nodules, which were suspected on CT scans of the chest, and compared the findings with those obtained with conventional immunocytochemistry. The molecular results were in agreement with the radiological impression and conflicted with the immunocytochemistry. We conclude that immunocytochemical and molecular biology approaches to the diagnosis of tumours may give rise to contradictory results.

Unique genetic variation at a species' rear edge is under threat from global climate change

Global climate change is having a significant effect on the distributions of a wide variety of species, causing both range shifts and population extinctions. To date, however, no consensus has emerged on how these processes will affect the range-wide genetic diversity of impacted species. It has been suggested that species that recolonized from low-latitude refugia might harbour high levels of genetic variation in rear-edge populations, and that loss of these populations could cause a disproportionately large reduction in overall genetic diversity in such taxa. In the present study, we have examined the distribution of genetic diversity across the range of the seaweed Chondrus crispus, a species that has exhibited a northward shift in its southern limit in Europe over the last 40 years. Analysis of 19 populations from both sides of the North Atlantic using mitochondrial single nucleotide polymorphisms (SNPs), sequence data from two singlecopy nuclear regions and allelic variation at eight microsatellite loci revealed unique genetic variation for all marker classes in the rear-edge populations in Iberia, but not in the rear-edge populations in North America. Palaeodistribution modelling and statistical testing of alternative phylogeographic scenarios indicate that the unique genetic diversity in Iberian populations is a result not only of persistence in the region during the last glacial maximum, but also because this refugium did not contribute substantially to the recolonization of Europe after the retreat of the ice. Consequently, loss of these rear-edge populations as a result of ongoing climate change will have a major effect on the overall genetic diversity of the species, particularly in Europe, and this could compromise the adaptive potential of the species as a whole in the face of future global warming.

A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex

DNA telomeric repeats in mammalian cells are transcribed to guanine-rich RNA sequences, which adopt parallel-stranded G-quadruplexes with a propeller-like fold. The successful crystallization and structure analysis of a bimolecular human telomeric RNA G-quadruplex, folded into the same crystalline environment as an equivalent DNA oligonucleotide sequence, is reported here. The structural basis of the increased stability of RNA telomeric quadruplexes over DNA ones and their preference for parallel topologies is described here. Our findings suggest that the 2'-OH hydroxyl groups in the RNA quadruplex play a significant role in redefining hydration structure in the grooves and the hydrogen bonding networks. The preference for specific nucleotides to populate the C3'-endo sugar pucker domain is accommodated by alterations in the phosphate backbone, which leads to greater stability through enhanced hydrogen bonding networks. Molecular dynamics simulations on the DNA and RNA quadruplexes are consistent with these findings. The computations, based on the native crystal structure, provide an explanation for RNA G-quadruplex ligand binding selectivity for a group of naphthalene diimide ligands as compared to the DNA G-quadruplex.

Molecular dynamics and principal components analysis of human telomeric quadruplex multimers.

Guanine-rich DNA repeat sequences located at the terminal ends of chromosomal DNA can fold in a sequence-dependent manner into G-quadruplex structures, notably the terminal 150–200 nucleotides at the 3' end, which occur as a single-stranded DNA overhang. The crystal structures of quadruplexes with two and four human telomeric repeats show an all-parallel-stranded topology that is readily capable of forming extended stacks of such quadruplex structures, with external TTA loops positioned to potentially interact with other macromolecules. This study reports on possible arrangements for these quadruplex dimers and tetramers, which can be formed from 8 or 16 telomeric DNA repeats, and on a methodology for modeling their interactions with small molecules. A series of computational methods including molecular dynamics, free energy calculations, and principal components analysis have been used to characterize the properties of these higher-order G-quadruplex dimers and tetramers with parallel-stranded topology. The results confirm the stability of the central G-tetrads, the individual quadruplexes, and the resulting multimers. Principal components analysis has been carried out to highlight the dominant motions in these G-quadruplex dimer and multimer structures. The TTA loop is the most flexible part of the model and the overall multimer quadruplex becoming more stable with the addition of further G-tetrads. The addition of a ligand to the model confirms the hypothesis that flat planar chromophores stabilize G-quadruplex structures by making them less flexible.

A Robust Curve-Fitting Procedure for the Analysis of Plasmid DNA Strand Break Data from Gel Electrophoresis

A robust method for fitting to the results of gel electrophoresis assays of damage to plasmid DNA caused by radiation is presented. This method makes use of nonlinear regression to fit analytically derived dose response curves to observations of the supercoiled, open circular and linear plasmid forms simultaneously, allowing for more accurate results than fitting to individual forms. Comparisons with a commonly used analysis method show that while there is a relatively small benefit between the methods for data sets with small errors, the parameters generated by this method remain much more closely distributed around the true value in the face of increasing measurement uncertainties. This allows for parameters to be specified with greater confidence, reflected in a reduction of errors on fitted parameters. On test data sets, fitted uncertainties were reduced by 30%, similar to the improvement that would be offered by moving from triplicate to fivefold repeats (assuming standard errors). This method has been implemented in a popular spreadsheet package and made available online to improve its accessibility. (C) 2011 by Radiation Research Society

Phenotypic and genotypic variations within a single bacteriophage species.

Background: Although horizontal gene transfer plays a pivotal role in bacteriophage evolution, many lytic phage genomes are clearly shaped by vertical evolution. We investigated the influence of minor genomic deletions and insertions on various phage-related phenotypic and serological properties. Findings. We collected ten different isolates of Pseudomonas aeruginosa bacteriophage KMV. All sequenced genomes (42-43 kb, long direct terminal repeats) are nearly identical, which intuitively implied strongly similar infections cycles. However, their latent periods vary between 21 and 28 minutes and they are able to lyse between 5 and 58% of a collection of 107 clinical P. aeruginosa strains. We also noted that phages with identical tail structures displayed profound differences in host spectra. Moreover, point mutations in tail and spike proteins were sufficient to evade neutralization by two phage-specific antisera, isolated from rabbits. Conclusion: Although all analyzed phages are 83-97% identical at the genome level, they display a surprisingly large variation in various phenotypic properties. The small overlap in host spectrum and their ability to readily escape immune defences against a nearly identical phage are promising elements for the application of these phages in phage therapy. © 2011 Ceyssens et al; licensee BioMed Central Ltd.


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Tissue biomarker development in a multicentre trial context: a feasibility study on the PETACC3 stage II and III colon cancer adjuvant treatment trial.

Purpose: We evaluated the feasibility of biomarker development in the context of multicenter clinical trials.

Experimental Design: Formalin-fixed, paraffin-embedded (FFPE) tissue samples were collected from a prospective adjuvant colon cancer trial (PETACC3). DNA was isolated from tumor as well as normal tissue and used for analysis of microsatellite instability, KRAS and BRAF genotyping, UGT1A1 genotyping, and loss of heterozygosity of 18 q loci. Immunohistochemistry was used to test expression of TERT, SMAD4, p53, and TYMS. Messenger RNA was retrieved and tested for use in expression profiling experiments.

Results: Of the 3,278 patients entered in the study, FFPE blocks were obtained from 1,564 patients coming from 368 different centers in 31 countries. In over 95% of the samples, genomic DNA tests yielded a reliable result. Of the immmunohistochemical tests, p53 and SMAD4 staining did best with reliable results in over 85% of the cases. TERT was the most problematic test with 46% of failures, mostly due to insufficient tissue processing quality. Good quality mRNA was obtained, usable in expression profiling experiments.

Conclusions: Prospective clinical trials can be used as framework for biomarker development using routinely processed FFPE tissues. Our results support the notion that as a rule, translational studies based on FFPE should be included in prospective clinical trials.