The genus Atheris (Serpentes: Viperidae) in East Africa: phylogeny and the role of rifting and climate in shaping the current pattern of species diversity.

Past climatic and tectonic events are believed to have strongly influenced species diversity in the Eastern Afromontane Biodiversity Hotspot. We investigated the phylogenetic relationships and historical biogeography of the East African genus Atheris (Serpentes: Viperidae), and explored temporal and spatial relationships between Atheris species across Africa, and the impact of palaeoclimatic fluctuations and tectonic movements on cladogenesis of the genus. Using mitochondrial sequence data, the phylogeny of East African species of Atheris shows congruent temporal patterns that link diversification to major tectonic and aridification events within East Africa over the last 15million years (my). Our results are consistent with a scenario of a delayed direct west-east colonisation of the Eastern Arc Mountains of Atheris by the formation of the western rift. Based on the phylogenetic patterns, this terrestrial, forest-associated genus has dispersed into East Africa across a divided route, on both west-southeasterly and west-northeasterly directions (a C-shaped route). Cladogenesis in the Eastern Arc Mountains and Southern Highlands of Tanzania corresponds to late Miocene and Plio-Pleistocene climatic shifts. Taxonomically, our data confirmed the monophyly of Atheris as currently defined, and reveal four major East African clades, three of which occur in discrete mountain ranges. Possible cryptic taxa are identified in the Atheris rungweensis and A. ceratophora clades.

Interleukin 7-dependent B lymphocyte precursor cells are ultrasensitive to apoptosis.

We have compared the sensitivity of clonogenic interleukin 7 (IL-7)-dependent murine B cell precursors with that of clonogenic mature B cells and myeloid precursors to alpha-particles from plutonium-238 and X radiation. All three populations are relatively sensitive, but B cell precursors are ultrasensitive. This differential sensitivity is also observed with corticosteroid, etoposide, and cisplatin, all apoptosis-inducing drugs used in the treatment of leukemia and other cancers. Further, we show that x-rays and drugs induce the bulk of the B cell precursor population to undergo rapid apoptosis, despite the continued presence of IL-7. B cell precursors were found to express very low levels of BCL-2 protein compared with mature splenic B cells and their resistance to x-rays and corticosteroid could be enhanced by expression of a BCL-2 transgene. These data have important implications for normal lymphopoiesis and for the behavior of leukemic lymphoid precursor cells.

Blinded comparison of faecal loading on plain radiography versus radio-opaque marker transit studies in the assessment of constipation.

AIM: To compare faecal loading on plain radiography versus radio-opaque marker transit studies in the assessment of constipation. METHODS: The study group was a convenience sample of patients attending the Durham Constipation Clinic. All patients underwent transit studies according to an established protocol, and severity of constipation was assessed contemporaneously using a validated questionnaire (PAC-SYM). Transit studies were performed using radio-opaque markers that were ingested over 3 consecutive days, with a radiograph taken on the fourth day. Digital images of the radiograph were digitally altered to remove all traces of the transit markers without affecting the underlying pattern of faecal loading. Four observers assessed faecal loading independently; two clinicians (C1 and C2) and two radiologists (R1 and R2). C1 and R1 used a previously described formal scoring method of assessing faecal loading, whereas C2 and R2 assessed the images as if they were in a clinic or reporting session, grading the faecal loading as mild, moderate, or severe. RESULTS: One hundred patients were recruited out of 186 presenting in a 2-year period. All patients completed assessments. The correlation between observers was only fair to moderate (r ranging from 0.34-0.51). There were some surprisingly marked disagreements in 10-18% of assessments. The correlation between faecal loading and transit was weak for all observers (r ranging from 0.261-0.311). Symptom severity did not correlate with faecal loading. CONCLUSION: These results suggest that there is considerable inter-observer variation in the radiological assessment of faecal loading, irrespective of the training or method used by the observer, and that there is very poor correlation with colonic transit. The diagnosis of constipation, and the assessment of severity, is best performed clinically.

A novel approach to the prediction of musculotendon paths.

One of the most important aspects of the modelling of musculoskeletal systems is the determination of muscle moment arms which are dependent upon the paths of the muscles. These paths are often required to wrap around passive structures that can be modelled as simple geometric shapes. A novel technique for the prediction of the paths of muscles modelled as strings when wrapping around smooth analytical surfaces is presented. The theory of geodesics is used to calculate the shortest path of the string on the surface and a smoothness constraint is used to determine the correct solutions for the string path between insertions. The application of the technique to tapered cylinders and ellipsoids is presented as an extension of previous work on right-circular cylinders and spheres. The technique is assessed with reference to a particular biomechanical scenario; string lengths and moment arms are calculated and compared with alternative approximate methods. This illustrates the potential of the technique to provide more accurate muscle moment arm predictions.

Algorithms for exact multi-object muscle wrapping and application to the deltoid muscle wrapping around the humerus.

Lines of action of muscle forces imply the function and performance of muscles acting around joints. It is not always possible to determine muscle force lines of action in vivo, and so computational techniques are often used to predict them. It is common to model a muscle as a taut elastic string that follows the shortest geodesic path between attachments over the wrapping geometry. A number of studies have been concerned with wrapping paths over single wrapping objects, and those that have considered more objects have applied the single-object solutions with iterative approaches to the search for a solution. This study presents a more efficient methodology for finding the exact solutions to a certain class of wrapping problems in which the path is constrained by multiple surfaces. It also introduces a more general wrapping technique based on the idea of energy minimization, which has been successfully validated against the exact solution. These methods are applied to the case of an element of the deltoid wrapping around the humerus modelled as a composite sphere-cylinder. Comparison of results with those obtained from approximated single-object solutions demonstrates the need to include correct multi-object wrapping algorithms in biomechanical models.

In-house development of a dedicated data acquisition and monitoring system for intracranial pressure, patient posture and patient symptoms in a regional neurosciences centre.

Management of traumatic brain injury and cerebrospinal fluid (CSF) flow disorders can be aided by measurement and monitoring of intracranial pressure (ICP). In addition to pressure measurement, knowledge of patient symptoms and posture during monitoring are also valuable, particularly in the management of CSF flow disorders. ICP monitoring systems have been developed in this centre to meet clinical needs in the absence of commercially available solutions. An early system (mark I) was developed and the technical challenges in its design are described, along with limitations to this system that motivated the development of a new mark II system. The mark II system is then described.

Bound PCNA in nuclei of primary rat tracheal epithelial cells after exposure to very low doses of plutonium-238 alpha particles.

Bystander effects from ionizing radiation have been detailed for a number of cell systems and a number of end points. We wished to use a cell culture/ex vivo rat model of respiratory tissue to determine whether a bystander effect detected in culture could also be shown in a tissue. Examination by immunofluorescence techniques of tracheal cell cultures after exposure to very low doses of alpha particles revealed a large proportion of cells with proliferating cell nuclear antigen (PCNA) bound in their nuclei. PCNA was selected as an end point because it is involved in both DNA repair and the changes in cell cycle that are typical of many reported bystander effects. Maximum response can be detected in up to 28% of the cells in sub-confluent cultures with a dose of only 2 mGy. At this dose less than 2% of the cell nuclei have experienced a particle traversal and less than 6% of the cells have experienced an alpha-particle traversal through either their nucleus or some part of their cytoplasm. The hypothesis that this bystander response in nontargeted cells is mediated through secreted factor(s) is presented, and supporting evidence was found using partial irradiation and co-culture experiments. Examination of the effect with excised pieces of trachea demonstrated a response similar to that seen in culture.

The effect of dimethyl sulfoxide on the induction of DNA double-strand breaks in V79-4 mammalian cells by alpha particles.

The present study was undertaken to assess the protective effect of dimethyl sulfoxide (DMSO) against the induction and rejoining of DNA double-strand breaks (DSBs) and inactivation of V79-4 Chinese hamster cells by both high- and low-linear energy transfer (LET) radiations. The cells were exposed under aerobic conditions as monolayers to either low-LET photons (60Co gamma rays) or high-LET alpha particles (238Pu) at 277 K. The initial yield of DSBs, determined by elution under nondenaturing conditions, is linearly dependent on dose. When the irradiation was carried out in the presence of DMSO (0-0.6 mol dm-3), the initial yields of DSBs induced by both gamma and alpha-particle irradiation decrease. With gamma irradiation at [DMSO] > 0.6 mol dm-3, a further decrease in the yield of DSBs occurs. DMSO (0.5 mol dm-3) reduces the initial yield of DSBs by 50 +/- 5% and 32 +/- 4% for photons and alpha particles, respectively. DMSO protects more effectively against cellular inactivation and DSB induction at low LET compared with alpha-particle irradiation with protection factors of 1.7 and 1.4, respectively, for survival and 2.0 and 1.5, respectively, for DSBs. After incubation of the irradiated cells for 3 h at 310 K after high-LET irradiation, the residual yield of DSBs is reduced by < 13% when the irradiations were carried out in the presence of 0.5 mol dm-3 DMSO. With gamma irradiation in the presence of 0.5 mol dm-3 DMSO, 90% of the DSBs are rejoined by 3 h incubation at 310 K. Therefore, the nonscavengeable DSBs induced by alpha particles are not significantly rejoined within 3 h, in contrast to rejoining of the majority of the nonscavengeable DSBs induced by gamma irradiation. From comparison of the data on DSBs and survival for alpha-particle irradiation, it is inferred that the severity of damage is reduced by DMSO through minimizing the formation of OH-induced sugar/base modifications in the vicinity of nonscavengeable DSBs.

Long-term genomic instability in human lymphocytes induced by single-particle irradiation.

Recent evidence suggests that genomic instability, which is an important step in carcinogenesis, may be important in the effectiveness of radiation as a carcinogen, particularly for high-LET radiations. Understanding the biological effects underpinning the risks associated with low doses of densely ionizing radiations is complicated in experimental systems by the Poisson distribution of particles that can be delivered. In this study, we report an approach to determine the effect of the lowest possible cellular radiation dose of densely ionizing alpha particles, that of a single particle traversal. Using microbeam technology and an approach for immobilizing human T-lymphocytes, we have measured the effects of single alpha-particle traversals on the surviving progeny of cells. A significant increase in the proportion of aberrant cells is observed 12-13 population doublings after exposure, with a high level of chromatid-type aberrations, indicative of an instability phenotype. These data suggest that instability may be important in situations where even a single particle traverses human cells.

Nuclear area measurement on viable cells, using confocal microscopy.

We describe a rapid procedure for the accurate measurements of nuclear areas on unperturbed living cells as used in radiobiological experiments, using the confocal laser scanning microscope. The microdosimetric interpretation of radiobiological data requires precise information on the nuclear area of cells as irradiated with high-LET radiation.

Radiation-induced chromosomal instability in human fibroblasts: temporal effects and the influence of radiation quality.

PURPOSE: To determine whether chromosomal instability is induced in human diploid fibroblasts by ionizing radiation and to investigate the effects of radiation quality by comparing X-rays, neutrons and alpha-particles. MATERIALS AND METHODS: Cells from two human diploid fibroblast lines, HF12 and HF19, were irradiated and analysed cytogenetically at 3, 20 and 35 population doublings post-irradiation. RESULTS: Exposure of HF19 cells to neutrons and alpha-particles resulted in a consistently increased frequency of unstable aberrations, particularly chromatid-type aberrations, compared to control cultures. Aberration frequency after X-irradiation was not significantly greater than controls at 20 population doublings but was significantly increased after 35 population doublings, although not to the same level as that following neutron or alpha-irradiation. No chromosomal instability was demonstrated in the progeny of HF12 cells after X-, neutron or alpha-particle irradiation. CONCLUSIONS: The data are consistent with the progeny of irradiated HF19 cells expressing chromosomal instability. All three radiations are effective in inducing instability, but the expression of the phenotype is influenced by radiation quality. The absence of radiation-induced chromosomal instability in HF12 cells may reflect the influence of genetic factors.

Lethality and mutagenesis of B lymphocyte progenitor cells following exposure to alpha-particles and X-rays.

B lymphocyte precursor cells are the target cells for the major subtype of paediatric cancer, acute lymphoblastic leukaemia. Using a murine IL-7-dependent clonogenic assay for normal B cell precursors as a model, we have investigated the sensitivity of these cells versus other normal and leukaemic haemopoietic cells to alpha-particle radiation. We find that B cell precursors are remarkably susceptible to the lethal effects of alpha-particles and have a very low probability of surviving a single alpha-track. B cell precursors are also very sensitive to the lethal effects of low LET X-rays. The mutation frequency in a marker gene (HPRT) does not, however, appear to be greater in B cell precursors that survive X-radiation than in other haemopoietic cells.

Frequencies of complex chromosome exchange aberrations induced by 238Pu alpha-particles and detected by fluorescence in situ hybridization using single chromosome-specific probes.

We undertook an analysis of chromosome-type exchange aberrations induced by alpha-particles using fluorescence in situ hybridization (FISH) with whole chromosome-specific probes for human chromosomes 1 or 4, together with a pan-centromeric probe. Contact-inhibited primary human fibroblasts (in G1) were irradiated with 0.41-1.00 Gy 238Pu alpha-particles and aberrations were analysed at the next mitosis following a single chromosome paint. Exchange and aberration painting patterns were classified according to Savage and Simpson (1994a). Of exchange aberrations, 38-47% were found to be complex derived, i.e. resulting from three or more breaks in two or more chromosomes, and the variation with dose was minimal. The class of complex aberrations most frequently observed were insertions, derived from a minimum of three breaks in two chromosomes. There was also an elevated frequency of rings. The high level of complex aberrations observed after alpha-particle irradiation indicates that, when chromosome domains are traversed by high linear energy transfer alpha-particle tracks, there is an enhanced probability of production of multiple localized double-strand breaks leading to more complicated interactions.

Complex chromosome aberrations in peripheral blood lymphocytes as a potential biomarker of exposure to high-LET alpha-particles.

PURPOSE: To determine the induction and transmission, to second and third division cells, of complex chromosome aberrations in peripheral blood lymphocytes after exposure to high-LET alpha-particles in vitro. MATERIALS AND METHODS: Separated peripheral blood lymphocytes collected from four healthy donors were irradiated in vitro with either high-LET alpha-particles (121 keV/microm; 0.5 Gy) or low-LET X-rays (250kV constant potential; 3 Gy). Cells were harvested in first, second and third division post-irradiation and chromosome aberrations observed at each cell division were analysed by combining the techniques of FISH and DAPI/Hoechst 33258 harlequin staining. Whole chromosome probes were used for chromosomes 1, 2 and 5, together with a pan-centromeric probe and the resulting chromosome 'painting' patterns were classified according to the Savage and Simpson (S & S) scheme (Savage and Simpson 1994a, Savage and Tucker 1996). RESULTS: A greater proportion of complex chromosome aberrations was observed, defined as involving three or more breaks in two or more chromosomes, relative to total exchanges, after exposure to 0.5 Gy alpha-particles (mean 1 track/cell) than after the high reference dose of 3 Gy X-rays (49-56% and 20-22%, respectively). Qualitatively, alpha-particles induced chromosome aberrations of far greater complexity than those observed after X-rays. This was reflected by both the rapid reduction in the percentage of damaged cells between first and second division indicative of cell death, and the spectrum of aberration types observed in second and third division cells post-irradiation. Regardless of this complexity, 15% of the complexes induced by alpha-particles at first division were potentially transmissible and by third division, transmissible-type complexes, specifically insertions, represented the predominant complex type (65%). CONCLUSION: Transmissible-type complexes were observed, specifically insertions, in both second and third division cells after exposure to high-LET alpha-particles (0.5 Gy) in vitro. The authors predict these cells to be stable and to be capable of persisting through many cell generations. Considering that the induction of complex chromosome aberrations by low-LET radiation is strongly dependent on dose, so that they are expected to be undetectable at environmental exposures, insertions are much more likely to be a characteristic feature of high-LET radiation at all doses. From this the usefulness of insertions as biomarkers of past exposure to environmentally relevant doses of high-LET alpha-particles is supported.

Dose- and time-response relationships for lethal mutations and chromosomal instability induced by ionizing radiation in an immortalized human keratinocyte cell line.

PURPOSE: To investigate the relationship between two well-established delayed effects of ionizing radiation, experiments were conducted to determine the induction and expression of lethal mutations (delayed reproductive death) and chromosomal instability with respect to dose and time in a human immortalized keratinocyte cell line. METHODS: HPV-G cells were gamma- or alpha-irradiated and maintained in culture for up to 72 population doublings. At intervals, measurements were made of cloning efficiency and the cells examined for apoptosis and cytogenetic aberrations. RESULTS: The descendants of cells surviving 1 or 3 Gy gamma-irradiation, but not 0.5 Gy gamma-irradiation, exhibited a reduced colony-forming efficiency. The reduction persisted at a constant rate of 15-20% clonogenic cell loss per population doubling for up to 72 population doublings. Apoptosis was demonstrated in all colonies in the 1 and 3 Gy groups at 30 and 72 population doublings post-irradiation but not in the 0.5 Gy group. A significant persistent reduction in colony-forming ability (approximately 80%) was demonstrated in the progeny of cells irradiated with 0.5 Gy alpha-particles. After 30 population doublings, the proportion of chromosomally aberrant cells was significantly greater than control values for all doses of both high- and low-LET radiations. The major cytogenetic aberrations (chromatid breaks, chromosome fragments and minutes) were consistent with the transmission of chromosomal instability. The expression of instability declined between 30 and 72 population doublings in the 0.5 Gy and 3 Gy gamma-irradiation groups, but persisted up to 72 population doublings in the 1 Gy group. The expression of chromosomal instability was greater in the descendants of alpha-irradiated cells and showed little evidence of reduction with time. CONCLUSIONS: Unstable aberrations characteristic of radiation-induced chromosomal instability may commonly result in apoptosis and account for a component of the delayed reproductive death/lethal mutation phenotype in HPV-G cells. However, the absence of lethal mutations in the descendants of 0.5 Gy gamma-irradiated cells indicates a low-LET threshold effect for this particular endpoint. Overall, and particularly at low doses, there is no direct correlation between the two endpoints, indicating the absence of a simple relationship between these manifestations of radiation-induced genomic instability.

Is the increased relative biological effectiveness of high LET particles due to spatial or temporal effects? Characterization and OER in V79-4 cells.

The efficiency of producing biological damage varies with radiation quality. Conventional explanations rely on spatial differences in the radiation track structure; generally however there are also very large temporal differences in delivery of the radiation at the cellular level. High-LET radiation normally deposits substantial amounts of energy by individual heavily ionizing tracks on a timescale of the order of picoseconds. By contrast each low-LET radiation track deposits a small amount of energy. Many of these tracks, distributed over the whole cell, are required to deliver an equivalent dose to a high-LET track and they are usually delivered over much longer timescales (typically seconds) during which chemical, biochemical and biological processes are occurring. In this paper the design, characterization and initial application of a high-brightness, laser-plasma ultrasoft x-ray source is described. This has been used to investigate the importance of the temporal differences by irradiating mammalian cells with an energy deposition with spatial properties of low-LET radiation and temporal properties similar to high-LET radiation. The present system delivers a typical dose, to the incident surface of the cells, of 0.12 Gy per pulse delivered in <10 ps. The capabilities of the x-ray source were tested by determining the survival of V79-4 hamster cells irradiated with picosecond pulses of ultrasoft x-rays under aerobic and anaerobic conditions, which were found to be consistent with previously published non pulsed data with x-rays of similar energy. These results support the expectation that the disappearance of an oxygen effect for high-LET radiation particles is due to their spatial properties rather than the very short timescale of each particle traversal. For other effects, particularly non-targeted phenomena such as induced genomic instability, expectations may be less clear cut.

Occurrence of toxigenic Clostridium botulinum type C in the soil of wetlands in Saskatchewan.

Mouse-lethal toxin identified as that of Clostridium botulinum type C by antitoxin neutralization was present in cultures of 38.0% of 326 soil samples collected from 28 wetlands in Saskatchewan. There was no difference in prevalence of toxicity between samples collected in spring and summer, and no relationship was evident between the occurrence of toxicity and water salinity, marsh type or water depth. There was a strong association between the prior occurrence of avian botulism in a marsh and the presence of toxin in cultures from soil; 59.2% of soil samples from marshes with a known history of botulism produced toxin, whereas only 6.2% of soil samples from marshes with no history of the disease produced toxin. Eight of the 10 soil samples collected from a marsh that had been dry for several years, and from another marsh that had not had a recognized outbreak of botulism for 11 yr produced toxin, indicating a long residual effect after a botulism outbreak. The results suggest that any wetland with a history of botulism is likely to suffer repeated occurrences because of heavy contamination of the soil with spores, and should be managed to control the disease.

A request for an abortion.

PIP: How to manage to abortion request by a hypothetical 30-year old married woman who states the she fears a deformed child because of taking an antibiotic combination, cotrimoxazole, containing trimethoprim is discussed by 3 physicians. The 1st doctor would confirm pregnancy with an exam and a laboratory test, schedule another consultation for counseling, and schedule a pelvic ultrasound if she decides to carry the pregnancy. If she wants an abortion, the physician would counsel her at length about her marriage and the emotional consequences of abortion. The 2nd physician would advise her that fetal abnormality from trimethoprim has not been reported in women. Since this doctor is personally opposed to abortion, she would refer the patient to another doctor to make the arrangements, and counsel her again afterward. The 3rd physician added the advice that 1-2% of all U.K. births are abnormal in some way. He would take steps to establish the precise gestational date, recommend an ultrasound scan at 18 weeks to cover himself legally and suggest that the patient's husband join in the counseling session to help bring out feelings about the marriage and the pregnancy.^ieng