Refugial persistence and postglacial recolonization of North America by the cold-tolerant herbaceous plant Orthilia secunda

Previous phylogeographical and palaeontological studies on the biota of northern North America have revealed a complex scenario of glacial survival in multiple refugia and differing patterns of postglacial recolonization. Many putative refugial regions have been proposed both north and south of the ice sheets for species during the Last Glacial Maximum, but the locations of many of these refugia remain a topic of great debate. In this study, we used a phylogeographical approach to elucidate the refugial and recolonization history of the herbaceous plant species Orthilia secunda in North America, which is found in disjunct areas in the west and east of the continent, most of which were either glaciated or lay close to the limits of the ice sheets. Analysis of 596-bp of the chloroplast trnS-trnG intergenic spacer and five microsatellite loci in 84 populations spanning the species' range in North America suggests that O.secunda persisted through the Last Glacial Maximum (LGM) in western refugia, even though palaeodistribution modelling indicated a suitable climate envelope across the entire south of the continent. The present distribution of the species has resulted from recolonization from refugia north and south of the ice sheets, most likely in Beringia or coastal regions of Alaska and British Columbia, the Washington/Oregon region in the northwest USA, and possibly from the region associated with the putative 'ice-free corridor' between the Laurentide and Cordilleran ice sheets. Our findings also highlight the importance of the Pacific Northwest as an important centre of intraspecific genetic diversity, owing to a combination of refugial persistence in the area and recolonization from other refugia.

Genomic organization, complex splicing pattern and expression of a human septin gene on chromosome 17q25.3

The Ov/Br septin gene, which is also a fusion partner of MLL in acute myeloid leukaemia, is a member of a family of novel GTP binding proteins that have been implicated in cytokinesis and exocytosis. In this study, we describe the genomic and transcriptional organization of this gene, detailing seventeen exons distributed over 240 kb of sequence. Extensive database analyses identified orthologous rodent cDNAs that corresponded to new, unidentified 5' splice variants of the Ov/Br septin gene, increasing the total number of such variants to six. We report that splicing events, occurring at non-canonical sites within the body of the 3' terminal exon, remove either 1801 bp or 1849 bp of non-coding sequence and facilitate access to a secondary open reading frame of 44 amino acids maintained near the end of the 3' UTR. These events constitute a novel coding arrangement and represent the first report of such a design being implemented by a eukaryotic gene. The various Ov/Br proteins either differ minimally at their amino and carboxy termini or are equivalent to truncated versions of larger isoforms. Northern analysis with an Ov/Br septin 3' UTR probe reveals three transcripts of 4.4, 4 and 3 kb, the latter being restricted to a sub-set of the tissues tested. Investigation of the identified Ov/Br septin isoforms by RT-PCR confirms a complex transcriptional pattern, with several isoforms showing tissue-specific distribution. To date, none of the other human septins have demonstrated such transcriptional complexity.

Distribution of the common lizard (Zootoca vivipara) and landscape favourability for the species in Northern Ireland

The common lizard (Zootoca vivipara) is Ireland’s only native reptile, forming a key part of the island’s biodiversity. However, there is a general paucity of distributional and abundance data for the species. In this study, we collated incidental records for common lizard sightings to define the distribution of the species in Northern Ireland. Maximum entropy modelling was employed to describe species-habitat associations. The resulting predicted landscape favourability was used to evaluate the current status of the species based on the distribution of its maximum potential range in relation to the degree of fragmentation of remaining suitable habitat. In common with previous studies in the Republic of Ireland, sightings were highly clustered indicating under-recording, observer bias, and fragmentation of suitable habitat. A total of 98 records were collated from 1905 to 2009. The species was recorded in 63 (ca. 34%) of 186 × 10 km Northern Irish grid squares. Lizard occurrence was strongly and positively associated with landscapes dominated by heathland, bog and coastal habitats. The single best approximating model correctly classified the presence of lizards in 84.2% of cases. Upland heath, lowland raised bog and sand dune systems are all subject to Habitat Action Plans in Northern Ireland and are threatened by conversion to agriculture, afforestation, invasive species encroachment and infrastructural development. Consequently, remaining common lizard populations are likely to be small, isolated and highly fragmented. Establishment of an ecological network to preserve connectivity of remaining heath and bog will not only benefit remaining common lizard populations but biodiversity in general.

Cytopathogenesis of Sendai virus in well-differentiated primary pediatric bronchial epithelial cells

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.

MicroRNA 203 Expression in Keratinocytes Is Dependent on Regulation of p53 Levels by E6.

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i). We show that the induction of miR-203 observed during calcium-induced differentiation of HFKs is significantly reduced in HFKs expressing E6 and in p53i HFKs. Induction of miR-203 in response to DNA damage is also reduced in the absence of p53. We report that proliferation of HFKs is dependent on the level of miR-203 expression and that overexpression of miR-203 can reduce overproliferation in E6/E7-expressing and p53i HFKs. In summary, these results indicate that expression of miR-203 is dependent on p53, which may explain how expression of HPV16 E6 can disrupt the balance between proliferation and differentiation, as well as the response to DNA damage, in keratinocytes.

Replication of association between schizophrenia and ZNF804A in the Irish Case-Control Study of Schizophrenia sample

A recent genome-wide association study reported association between schizophrenia and the ZNF804A gene on chromosome 2q32.1. We attempted to replicate these findings in our Irish Case-Control Study of Schizophrenia (ICCSS) sample (N=1021 cases, 626 controls). Following consultation with the original investigators, we genotyped three of the most promising single-nucleotide polymorphisms (SNPs) from the Cardiff study. We replicate association with rs1344706 (trend test one-tailed P=0.0113 with the previously associated A allele) in ZNF804A. We detect no evidence of association with rs6490121 in NOS1 (one-tailed P=0.21), and only a trend with rs9922369 in RGRIP1L (one-tailed P=0.0515). On the basis of these results, we completed genotyping of 11 additional linkage disequilibrium-tagging SNPs in ZNF804A. Of 12 SNPs genotyped, 11 pass quality control criteria and 4 are nominally associated, with our most significant evidence of association at rs7597593 (P=0.0013) followed by rs1344706. We observe no evidence of differential association in ZNF804A on the basis of family history or sex of case. The associated SNP rs1344706 lies in approximately 30 bp of conserved mammalian sequence, and the associated A allele is predicted to maintain binding sites for the brain-expressed transcription factors MYT1l and POU3F1/OCT-6. In controls, expression is significantly increased from the A allele of rs1344706 compared with the C allele. Expression is increased in schizophrenic cases compared with controls, but this difference does not achieve statistical significance. This study replicates the original reported association of ZNF804A with schizophrenia and suggests that there is a consistent link between the A allele of rs1344706, increased expression of ZNF804A and risk for schizophrenia.

Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6 and E7-Expressing Keratinocytes

Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G(2)/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G(2) damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle. Little is known about the mechanism that allows these cells to gain entry into and exit from mitosis. Here, we show that in the presence of DNA damage, E6/E7 cells have elevated levels of cyclin B, which would allow entry into mitosis. Also, as required for exit from mitosis, cyclin B is degraded in these cells, permitting initiation of the next round of DNA synthesis and cell cycle progression. Proteasomal degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) is, in part, due to elevated levels of the E2-conjugating enzyme, Ubch10, and the substrate recognition protein, Cdc20, of APC/C. Also, in E6/E7 cells with DNA damage, while Cdc20 is complexed with BubR1, indicating an active checkpoint, it is also present in complexes free of BubR1, presumably allowing APC/C activity and slippage through the checkpoint.

Conventional 3D staging PET/CT in CT simulation for lung cancer: impact of rigid and deformable target volume alignments for radiotherapy treatment planning

Objective: Positron emission tomography (PET)/CT scans can improve target definition in radiotherapy for non-small cell lung cancer (NSCLC). As staging PET/CT scans are increasingly available, we evaluated different methods for co-registration of staging PET/CT data to radiotherapy simulation (RTP) scans.

Methods: 10 patients underwent staging PET/CT followed by RTP PET/CT. On both scans, gross tumour volumes (GTVs) were delineated using CT (GTVCT) and PET display settings. Four PET-based contours (manual delineation, two threshold methods and a source-to-background ratio method) were delineated. The CT component of the staging scan was co-registered using both rigid and deformable techniques to the CT component of RTP PET/CT. Subsequently rigid registration and deformation warps were used to transfer PET and CT contours from the staging scan to the RTP scan. Dice’s similarity coefficient (DSC) was used to assess the registration accuracy of staging-based GTVs following both registration methods with the GTVs delineated on the RTP PET/CT scan.

Results: When the GTVCT delineated on the staging scan after both rigid registration and deformation was compared with the GTVCT on the RTP scan, a significant improvement in overlap (registration) using deformation was observed (mean DSC 0.66 for rigid registration and 0.82 for deformable registration, p50.008). A similar comparison for PET contours revealed no significant improvement in overlap with the use of deformable registration.

Conclusions: No consistent improvements in similarity measures were observed when deformable registration was used for transferring PET-based contours from a staging PET/CT. This suggests that currently the use of rigid registration remains the most appropriate method for RTP in NSCLC.

Assessing the image quality of pelvic MR images acquired with a flat couch for radiotherapy treatment planning

Objectives: To improve the integration of MRI with radiotherapy treatment planning, our department fabricated a flat couch top for our MR scanner. Setting up using this couch top meant that the patients were physically higher up in the scanner and, posteriorly, a gap was introduced between the patient and radiofrequency coil.

Assessing the daily consistency of bladder filling using an ultrasonic Bladderscan device in men receiving radical conformal radiotherapy for prostate cancer

Consistency in target organ and organ at risk position from planning to treatment is an important basic principle of radiotherapy. This study evaluates the effectiveness of bladder-filling instructions in achieving a consistent and reproducible bladder volume at the time of planning CT and daily during the course of radical radiotherapy for prostate cancer. It also assessed the rate of bladder filling before and at the end of radiotherapy.

In vivo tropism of attenuated and pathogenic measles virus expressing green fluorescent protein in macaques

The global increase in measles vaccination has resulted in a significant reduction of measles mortality. The standard route of administration for the live-attenuated measles virus (MV) vaccine is subcutaneous injection, although alternative needle-free routes, including aerosol delivery, are under investigation. In vitro, attenuated MV has a much wider tropism than clinical isolates, as it can use both CD46 and CD150 as cellular receptors. To compare the in vivo tropism of attenuated and pathogenic MV, we infected cynomolgus macaques with pathogenic or attenuated recombinant MV expressing enhanced green fluorescent protein (GFP) (strains IC323 and Edmonston, respectively) via the intratracheal or aerosol route. Surprisingly, viral loads and cellular tropism in the lungs were similar for the two viruses regardless of the route of administration, and CD11c-positive cells were identified as the major target population. However, only the pathogenic MV caused significant viremia, which resulted in massive virus replication in B and T lymphocytes in lymphoid tissues and viral dissemination to the skin and the submucosa of respiratory epithelia. Attenuated MV was rarely detected in lymphoid tissues, and when it was, only in isolated infected cells. Following aerosol inhalation, attenuated MV was detected at early time points in the upper respiratory tract, suggesting local virus replication. This contrasts with pathogenic MV, which invaded the upper respiratory tract only after the onset of viremia. This study shows that despite in vitro differences, attenuated and pathogenic MV show highly similar in vivo tropism in the lungs. However, systemic spread of attenuated MV is restricted.

Direct evidence for a bystander effect of ionizing radiation in primary human fibroblasts

Bystander responses underlie some of the current efforts to develop gene therapy approaches for cancer treatment. Similarly, they may have a role in strategies to treat tumours with targeted radioisotopes. In this study we show direct evidence for the production of a radiation-induced bystander response in primary human fibroblasts, We utilize a novel approach of using a charged-particle microbeam, which allows individual cells within a population to be selected and targeted with counted charged particles. Individual primary human fibroblasts within a population of 600-800 cells were targeted with between 1 and 15 helium ions (effectively, alpha -particles). The charged particles were delivered through the centre of the nucleus with an accuracy of +/- 2 mum and a detection and counting efficiency of greater than 99%. When scored 3 days later, even though only a single cell had been targeted, typically an additional 80-100 damaged cells were observed in the surviving population of about 5000 cells. The yield of damaged cells was independent of the number of charged particles delivered to the targeted cell, Similar results of a 2-3-fold increase in the background level of damage present in the population were observed whether 1 or 4 cells were targeted within the dish. Also, when 200 cells within one quadrant of the dish were exposed to radiation, there was a 2-3-fold increase in the damage level in an unexposed quadrant of the dish, This effect was independent of the presence of serum in the culture medium and was only observed when a cell was targeted, but not when only the medium was exposed, confirming that a cell-mediated response is involved. (C) 2001 Cancer Research Campaign.

Chemoenzymatic synthesis of a mixed phosphine-phosphine oxide catalyst and its application to asymmetric allylation of aldehydes and hydrogenation of alkenes

The chemoenzymatic synthesis of a Lewis basic phosphine-phosphine oxide organocatalyst from a cis-dihydrodiol metabolite of bromobenzene proceeds via a palladium-catalysed carbon-phosphorus bond coupling and a novel room temperature Arbuzov [2,3]-sigmatropic rearrangement of an allylic diphenylphosphinite. Allylation of aromatic aldehydes were catalysed by the Lewis basic organocatalyst giving homoallylic alcohols in up to 57% ee. This compound also functioned as a ligand for rhodium-catalysed asymmetric hydrogenation of acetamidoacrylate giving reduction products with ee values of up to 84%.