Phylogenomics of the dog and fox family (Canidae,Carnivora) revealed by chromosome painting

Canid species (dogs and foxes) have highly rearranged karyotypes and thus represent a challenge for conventional comparative cytogenetic studies. Among them, the domestic dog is one of the best-mapped species in mammals, constituting an ideal reference genome for comparative genomic study. Here we report the results of genome-wide comparative mapping of dog chromosome-specific probes onto chromosomes of the dhole, fennec fox, and gray fox, as well as the mapping of red fox chromosome-specific probes onto chromosomes of the corsac fox. We also present an integrated comparative chromosome map between the species studied here and all canids studied previously. The integrated map demonstrates an extensive conservation of whole chromosome arms across different canid species. In addition, we have generated a comprehensive genome phylogeny for the Canidae on the basis of the chromosome rearrangements revealed by comparative painting. This genome phylogeny has provided new insights into the karyotypic relationships among the canids. Our results, together with published data, allow the formulation of a likely Canidae ancestral karyotype (CAK, 2n = 82), and reveal that at least 6–24 chromosomal fission/fusion events are needed to convert the CAK karyotype to that of the modern canids. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Complex evolution of X and Y autosomal translocations in the giant mole-rat, Cryptomys mechowi (Bathyergidae)

Cross-species chromosome painting was used to determine homologous chromosomal regions between two species of mole-rat, the naked mole-rat, Heterocephalus glaber (2n = 60), and the giant mole-rat, Cryptomys mechowi (2n = 40), using flow-sorted painting probes representative of all but two of the H. glaber chromosomal complement. In total 43 homologous regions were identified in the C. mechowi genome. Eight H. glaber chromosomes are retained in toto in C. mechowi, and 13 produce two or more signals in this species. The most striking difference in the karyotypes of the two taxa concerns their sex chromosomes. The H. glaber painting probes identified a complex series of translocations that involved the fractionation of four autosomes and the subsequent translocation of segments to the sex chromosomes and to autosomal partners in the C. mechowi genome. An intercalary heterochromatic block (IHB) was detected in sex chromosomes of C. mechowi at the boundary with the translocated autosomal segment. We discuss the likely sequence of evolutionary events that has led to the contemporary composition of the C. mechowi sex chromosomes, and consider these in the light of prevailing views on the genesis of sex chromosomes in mammals.     

Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of SmaI Macrorestriction Fragments: a Multicenter Study

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.The use of electric field pulsing techniques in conjunction with agarose gel electrophoresis for discrimination of large DNA molecules was introduced by Schwarz and Cantor in 1984 (9). During the past decade the methodology has been adapted and improved by various research groups to the point that pulsed-field gel electrophoresis (PFGE) for bacterial strain typing is now utilized with relative ease in a variety of laboratories (1). The combination of contour-clamped homogeneous field electrophoresis and PFGE for the molecular analysis of Staphylococcus aureus has been reported since the late 1980s (7, 19). At present, PFGE is considered to have both the reproducibility and resolving power of a standard technique for the epidemiological typing of bacterial isolates (10, 15).Molecular typing systems can identify different strains within a species, generating data useful for taxonomic or epidemiologic purposes (10, 14). A frequently observed shortcoming of typing systems in general is their lack of reproducibility: most typing systems do not provide a definitive strain identification, which is usually due to the variability of the technique and the lack of large databases containing fragment patterns from a wide variety of organisms to which unknowns can be compared. These problems were recently described in detail for two molecular typing systems. A multicenter study on random amplification of polymorphic DNA for discrimination of S. aureus strains revealed a lack of interlaboratory reproducibility among the banding patterns generated by the participating centers, although the epidemiological interpretation of the data was similar for all the centers involved (16). For PFGE, a similar lack of interlaboratory reproducibility of patterns was observed, although the interpretation of the experimental data also differed per participating center (2). The latter study analyzed 12 different methicillin-resistant S. aureus (MRSA) strains with different techniques optimized in each center and different sources and types of equipment. Since interlaboratory discrepancies with respect to classification of the strains were observed, the study concluded that there is a clear need for standardization of the technique, including the construction of a panel of reference strains to assist the individual researcher in the optimization of the PFGE protocol.The aim of the present study was to compare the fragment patterns of a well-defined collection of MRSA isolates in 12 laboratories using in-house and a standard set of PFGE parameters to determine whether standardization of experimental parameters (DNA preparation and switching protocols) would improve intercenter reproducibility of PFGE analysis.     

Construction of a highly enriched marsupial Y chromosome-specific BAC sub-library using isolated Y chromosomes

The Y chromosome is perhaps the most interesting element of the mammalian genome but comparative analysis of the Y chromosome has been impeded by the difficulty of assembling a shotgun sequence of the Y. BAC-based sequencing has been successful for the human and chimpanzee Y but is difficult to do efficiently for an atypical mammalian model species (Skaletsky et al. 2003, Kuroki et al. 2006). We show how Y-specific sub-libraries can be efficiently constructed using DNA amplified from microdissected or flow-sorted Y chromosomes. A Bacterial Artificial Chromosome (BAC) library was constructed from the model marsupial, the tammar wallaby (Macropus eugenii). We screened this library for Y chromosome-derived BAC clones using DNA from both a microdissected Y chromosome and a flow-sorted Y chromosome in order to create a Y chromosome-specific sub-library. We expected that the tammar wallaby Y chromosome should detect ∼100 clones from the 2.2 times redundant library. The microdissected Y DNA detected 85 clones, 82% of which mapped to the Y chromosome and the flow-sorted Y DNA detected 71 clones, 48% of which mapped to the Y chromosome. Overall, this represented a ∼330-fold enrichment for Y chromosome clones. This presents an ideal method for the creation of highly enriched chromosome-specific sub-libraries suitable for BAC-based sequencing of the Y chromosome of any mammalian species.     

Treatment of malignant gliomas in adults with BCNU plus metronidazole

Summary Twenty-six adult patients with astrocytomas were treated with BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) 180–240 mg/m² 1.V. every 6–9 weeks, with metronidazole 1.5 g/m² p. o. 12 h and 1 h before BCNU and again 6 h and 24 h after BCNU. Of twenty-two evaluable patients, 9 (41%) responded with evidence of reduced tumor size on CT scan, 3 (14%) stabilized and 10 (45%) failed. Patients with no prior chemotherapy or radiotherapy, good performance. status, low grade tumors, and age 50 years had the highest response rates, although differences were not statistically significant. Median survival and duration of response have not been reached with a median follow-up time of ten months. Hematological toxicity was dose-limiting and was probably not augmented by the metronidazole. There was one death from infection that was possibly drug-related. Gastrointestinal toxicity was substantial, and was probably increased by the metronidazole.While the combination of BCNU and metronidazole were tolerable, the response rate seen was no higher than that noted for BCNU alone, and further studies using this dose-schedule are not recommended in astrocytomas.Presented at the 13th International Congress of Chemotherapy, Vienna Austria, August 1983.     

Autonomy and developing physicians: Reimagining supervision using self-determination theory

In this article, we address the question, ‘What is the role of autonomy in physician development?’ Medical education is a developmental process, and autonomy plays a motivational role in physician development. Calls for increased supervision of residents have raised concerns that the resulting decreased autonomy might interfere with resident development, leading the authors to explore the relationship between supervision and autonomy. The medical education literature posits a simple inverse relationship between supervision and autonomy. Within competency frameworks, autonomy is operationalised as independence and viewed as the end goal of training. Alternatively, there is emerging empirical literature describing autonomy and supervision as dynamic and developmental constructs and point towards more complex relationship between supervision and autonomy. Self-determination theory (SDT) presents a framework for understanding this dynamic relationship and the role of autonomy in physician development. Within SDT, autonomy is a fundamental psychological need, associated with motivation for learning, self-regulation and an internal locus of control. Supporting learner autonomy can afford learners the opportunity to internalise the values and norms of the profession, leading to an integrated regulation of their behaviours and actions. Conceptualising autonomy through the lens of SDT provides an avenue for education interventions and future research on supervision and autonomy. Educators can integrate supervision and autonomy support in the clinical setting, seeking to motivate learner development by balancing optimal challenge and support and integrating autonomy support with ‘hands-on’ approaches to supervision. SDT also provides a theoretical framework relevant to current discussions regarding feedback conversations and coaching in medical education. Lastly, conceptualising autonomy using SDT opens new avenues for investigation, exploring the complex relationship between supervision and autonomy and developing efforts to integrate autonomy support with clinical supervision.     

Acceptability and Feasibility of a 13-Week Pilot Randomised Controlled Trial Testing the Effects of Incremental Doses of Beetroot Juice in Overweight and Obese Older Adults

Nitrate-rich food can increase nitric oxide production and improve vascular and brain functions. This study examines the feasibility of a randomised controlled trial (RCT) testing the effects of prolonged consumption of different doses of dietary nitrate (NO₃⁻) in the form of beetroot juice (BJ) in overweight and obese older participants. A single-blind, four-arm parallel pilot RCT was conducted in 62 overweight and obese (30.4 ± 4 kg/m²) older participants (mean ± standard deviation (SD), 66 ± 4 years). Participants were randomized to: (1) high-NO₃⁻ (HN: 2 × 70 mL BJ/day) (2) medium-NO₃⁻ (MN: 70 mL BJ/day), (3) low-NO₃⁻ (LN: 70 mL BJ on alternate days) or (4) Placebo (PL: 70 mL of NO₃⁻-depleted BJ on alternate days), for 13 weeks. Compliance was checked by a daily log of consumed BJ, NO₃⁻ intake, and by measuring NO₃⁻ and NO₂⁻ concentrations in plasma, saliva, and urine samples. Fifty participants completed the study. Self-reported compliance to the interventions was >90%. There were significant positive linear relationships between NO₃⁻ dose and the increase in plasma and urinary NO₃⁻ concentration (R² = 0.71, p < 0.001 and R² = 0.46 p < 0.001, respectively), but relationships between NO₃⁻ dose and changes in salivary NO₃⁻ and NO₂⁻ were non-linear (R² = 0.35, p = 0.002 and R² = 0.23, p = 0.007, respectively). The results confirm the feasibility of prolonged BJ supplementation in older overweight and obese adults.