PhyTB: Phylogenetic tree visualisation and sample positioning for M. tuberculosis

Background

Phylogenetic-based classification of M. tuberculosis and other bacterial genomes is a core analysis for studying evolutionary hypotheses, disease outbreaks and transmission events. Whole genome sequencing is providing new insights into the genomic variation underlying intra- and inter-strain diversity, thereby assisting with the classification and molecular barcoding of the bacteria. One roadblock to strain investigation is the lack of user-interactive solutions to interrogate and visualise variation within a phylogenetic tree setting.

Results

We have developed a web-based tool called PhyTB (http://pathogenseq.lshtm.ac.uk/phytblive/index.php) to assist phylogenetic tree visualisation and identification of M. tuberculosis clade-informative polymorphism. Variant Call Format files can be uploaded to determine a sample position within the tree. A map view summarises the geographical distribution of alleles and strain-types. The utility of the PhyTB is demonstrated on sequence data from 1,601 M. tuberculosis isolates.

Conclusion

PhyTB contextualises M. tuberculosis genomic variation within epidemiological, geographical and phylogenic settings. Further tool utility is possible by incorporating large variants and phenotypic data (e.g. drug-resistance profiles), and an assessment of genotype-phenotype associations. Source code is available to develop similar websites for other organisms (http://sourceforge.net/projects/phylotrack).     

BEEtag: A Low-Cost,Image-Based Tracking System for the Study of Animal Behavior and Locomotion

A fundamental challenge common to studies of animal movement, behavior, and ecology is the collection of high-quality datasets on spatial positions of animals as they change through space and time. Recent innovations in tracking technology have allowed researchers to collect large and highly accurate datasets on animal spatiotemporal position while vastly decreasing the time and cost of collecting such data. One technique that is of particular relevance to the study of behavioral ecology involves tracking visual tags that can be uniquely identified in separate images or movie frames. These tags can be located within images that are visually complex, making them particularly well suited for longitudinal studies of animal behavior and movement in naturalistic environments. While several software packages have been developed that use computer vision to identify visual tags, these software packages are either (a) not optimized for identification of single tags, which is generally of the most interest for biologists, or (b) suffer from licensing issues, and therefore their use in the study of animal behavior has been limited. Here, we present BEEtag, an open-source, image-based tracking system in Matlab that allows for unique identification of individual animals or anatomical markers. The primary advantages of this system are that it (a) independently identifies animals or marked points in each frame of a video, limiting error propagation, (b) performs well in images with complex backgrounds, and (c) is low-cost. To validate the use of this tracking system in animal behavior, we mark and track individual bumblebees (Bombus impatiens) and recover individual patterns of space use and activity within the nest. Finally, we discuss the advantages and limitations of this software package and its application to the study of animal movement, behavior, and ecology.     

Characteristics and N-terminal amino acid sequence of a manganese peroxidase purified from Lentinula edodes cultures grown on a commercial wood substrate

Summary Extracellular culture filtrates from ligninolytic cultures of the lignin-degrading basidiomycete Lentinula (syn. Lentinus) edodes (Berk.) Pegler contained one major peroxidase when grown on a commercial oak-wood substrate. The peroxidase was purified by polyethylenimine clarification, anion-exchange chromatography, and hydrophobic-interaction HPLC. The enzyme (MnP1) was a heme-iron protein with an apparent molecular weight of 44 600 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and an isoelectric point of pH 3.2. The native enzyme had an absorption maximum at 407 nm, which shifted to 420 nm upon H₂O₂ addition. The pyridine-hemochrome-absorption spectrum indicated that one heme group was present per enzyme as protoporphyrin IX. N-terminal amino acid sequencing showed that MnP1 had higher sequence homology with manganese peroxidases than with lignin peroxidases reported from Phanerochaete chrysosporium. L. edodes MnP1 was capable of oxidizing lignin and lignin-model compounds in the presence of manganese and H₂O₂.On leave from the Department of Biochemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand.Research carried out while a visiting scientist at the USDA Forest Products Laboratory from the National Chemistry Laboratory, Pune, India 41 1008 Offprint requests to: I. T. Forrester     

The biological effects of syngeneic and allogeneic cytokine-expressing prophylactic whole cell vaccines and the influence of irradiation in a murine melanoma model

Allogeneic whole tumour cell vaccines are inherently practical compared with autologous vaccines. Cell lines are derived from allogeneic tumour, grown in bulk and then administered as a vaccine to the patient, following irradiation, which not only prevents any replication but also enhances antigen presentation. Protection is believed to occur through the presentation of antigens shared between the syngeneic and allogeneic tumours. Although cytokine-transfected tumour whole cell vaccines have been used clinically, little data is available comparing the effects of immunomodulatory cytokine-transfection directly on the same cells when used as both an allogeneic and autologous vaccine. To address this, weakly immunogenic B16-F10 (H-2^b) murine melanoma was transfected to secrete either GM-CSF, IL-4 or IL-7. Prophylactic vaccination of both syngeneic C57/BL6 (H-2^b) (B6) and allogeneic C3H/Hej (H-2^k) (C3H) mice showed the effects of transfected cytokine varied between models. Both GM-CSF and IL-7 significantly (P<0.05) increased the levels of protection within syngeneic B6 mice, but had a diminished effect (P>0.05) within C3H allogeneic mice. Allogeneic B16-F10 cells and syngeneic K1735 cells generated CTL against K1735 suggesting cross-reactive immunity. Using cells labeled with fluorescent dye we demonstrate that irradiated vaccines, of either syngeneic or allogeneic origin, appear to generate potent immune responses and fragments of either vaccine remain at the injection site for up to 9 days. This study shows that protection can be enhanced in vivo by using transfected cytokine, but suggests that irradiated whole cell vaccines, of either tissue-type, are rapidly processed. This leads to the conclusion that the cytokine effects are transient and thus transfection with cytokine may be of limited long-term use in situ.     

Social inequalities and mortality in europe - results from a large multi-national cohort

Background

Socio-economic inequalities in mortality are observed at the country level in both North America and Europe. The purpose of this work is to investigate the contribution of specific risk factors to social inequalities in cause-specific mortality using a large multi-country cohort of Europeans.

Methods

A total of 3,456,689 person/years follow-up of the European Prospective Investigation into Cancer and Nutrition (EPIC) was analysed. Educational level of subjects coming from 9 European countries was recorded as proxy for socio-economic status (SES). Cox proportional hazard model''s with a step-wise inclusion of explanatory variables were used to explore the association between SES and mortality; a Relative Index of Inequality (RII) was calculated as measure of relative inequality.

Results

Total mortality among men with the highest education level is reduced by 43% compared to men with the lowest (HR 0.57, 95% C.I. 0.52–0.61); among women by 29% (HR 0.71, 95% C.I. 0.64–0.78). The risk reduction was attenuated by 7% in men and 3% in women by the introduction of smoking and to a lesser extent (2% in men and 3% in women) by introducing body mass index and additional explanatory variables (alcohol consumption, leisure physical activity, fruit and vegetable intake) (3% in men and 5% in women). Social inequalities were highly statistically significant for all causes of death examined in men. In women, social inequalities were less strong, but statistically significant for all causes of death except for cancer-related mortality and injuries.

Discussion

In this European study, substantial social inequalities in mortality among European men and women which cannot be fully explained away by accounting for known common risk factors for chronic diseases are reported.     

Processing of Chlamydia abortus Polymorphic Membrane Protein 18D during the Chlamydial Developmental Cycle

Background

Chlamydia possess a unique family of autotransporter proteins known as the Polymorphic membrane proteins (Pmps). While the total number of pmp genes varies between Chlamydia species, all encode a single pmpD gene. In both Chlamydia trachomatis (C. trachomatis) and C. pneumoniae, the PmpD protein is proteolytically cleaved on the cell surface. The current study was carried out to determine the cleavage patterns of the PmpD protein in the animal pathogen C. abortus (termed Pmp18D).

Methodology/Principal Findings

Using antibodies directed against different regions of Pmp18D, proteomic techniques revealed that the mature protein was cleaved on the cell surface, resulting in a100 kDa N-terminal product and a 60 kDa carboxy-terminal protein. The N-terminal protein was further processed into 84, 76 and 73 kDa products. Clustering analysis resolved PmpD proteins into three distinct clades with C. abortus Pmp18D, being most similar to those originating from C. psittaci, C. felis and C. caviae.

Conclusions/Significance

This study indicates that C. abortus Pmp18D is proteolytically processed at the cell surface similar to the proteins of C. trachomatis and C. pneumoniae. However, patterns of cleavage are species-specific, with low sequence conservation of PmpD across the genus. The absence of conserved domains indicates that the function of the PmpD molecule in chlamydia remains to be elucidated.     

Brm Inhibits the Proliferative Response of Keratinocytes and Corneal Epithelial Cells to Ultraviolet Radiation-Induced Damage

Ultraviolet radiation (UV) from sunlight is the primary cause of skin and ocular neoplasia. Brahma (BRM) is part of the SWI/SNF chromatin remodeling complex. It provides energy for rearrangement of chromatin structure. Previously we have found that human skin tumours have a hotspot mutation in BRM and that protein levels are substantially reduced. Brm−/− mice have enhanced susceptibility to photocarcinogenesis. In these experiments, Brm−/− mice, with both or a single Trp53 allele were exposed to UV for 2 or 25 weeks. In wild type mice the central cornea and stroma became atrophic with increasing time of exposure while the peripheral regions became hyperplastic, presumably as a reparative process. Brm−/−, Trp53+/−, and particularly the Brm−/− Trp53+/− mice had an exaggerated hyperplastic regeneration response in the corneal epithelium and stroma so that the central epithelial atrophy or stromal loss was reduced. UV induced hyperplasia of the epidermis and corneal epithelium, with an increase in the number of dividing cells as determined by Ki-67 expression. This response was considerably greater in both the Brm−/− Trp53+/+ and Brm−/− Trp53+/− mice indicating that Brm protects from UV-induced enhancement of cell division, even with loss of one Trp53 allele. Cell division was disorganized in Brm−/− mice. Rather than being restricted to the basement membrane region, dividing cells were also present in the suprabasal regions of both tissues. Brm appears to be a tumour suppressor gene that protects from skin and ocular photocarcinogenesis. These studies indicate that Brm protects from UV-induced hyperplastic growth in both cutaneous and corneal keratinocytes, which may contribute to the ability of Brm to protect from photocarcinogenesis.     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).