Long-term storage of DNA-free RNA for use in vaccine studies

RNA replicons represent potential vaccine delivery vehicles, but are considered too unstable for such use. This study examined the recovery, integrity and function of in vitro transcribed replicon RNA encoding hepatitis C virus (HCV) proteins. To remove residual template DNA, the RNA was digested with TURBO DNase followed by RNeasy DNase set and purified through an RNeasy column. The RNA was freeze-dried in distilled water or trehalose, stored under nitrogen gas for up to 10 months and analyzed at different time points. The recovery of RNA stored at < or = 4 degrees C that was freeze-dried in distilled water varied between 66% to zero of that recovered from RNA freeze-dried in 10% trehalose, a figure that depended on the duration of storage. In contrast, the recovery of the RNA stored in trehalose was consistently high for all time points. After recovery, both RNAs were translationally competent and expressed high levels of proteins after transfection, although the level of expression from the trehalose-stored RNA was consistently higher. Thus the addition of trehalose permitted stable storage of functional RNA at 4 degrees C for up to 10 months and this permits the development of RNA vaccines, even in developing countries where only minimum storage conditions (e.g., 4 degrees C) can be achieved.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

Determinants of choosing a career in family medicine

Background

Student choice is an important determinant of the distribution of specialties of practising physicians in many countries. Understanding characteristics at entry into medical school that are associated with the choice of residency in family medicine can assist medical schools in admitting an appropriate mix of students to serve the health care needs of their regions.

Methods

From 2002 to 2004, we collected data from students in 15 classes at 8 of 16 Canadian medical schools at entry. Surveys included questions on career choice, attitudes to practice and socio-demographic characteristics. We followed students prospectively with these data linked to their residency choice. We used multiple logistic regression analysis to identify entry characteristics that predicted a student’s ultimate career choice in family medicine.

Results

Of 1941 eligible students in the participating classes, 1542 (79.4%) contributed data to the final analyses. The following 11 entry variables predicted whether a student named family medicine as his or her top residency choice: being older, being engaged or in a long-term relationship, not having parents with postgraduate university education nor having family or close friends practicing medicine, having undertaken voluntary work in a developing nation, not volunteering with elderly people, desire for varied scope of practice, a societal orientation, a lower interest in research, desire for short postgraduate training, and lower preference for medical versus social problems.

Interpretation

Demographic and attitudinal characteristics at entry into medical school predicted whether students chose a career in family medicine.The number of physicians per capita in both Canada and the United States has declined, and this decline is expected to continue. Canada has already experienced a drop of 5.1% in the physician-to-population ratio from a peak in 2000, and in the United States a shortage of up to 200 000 physicians, or 20% of the needed workforce, is predicted to occur by 2025.^1,2 Combined with a growing elderly population and decreasing physician-hours,^3,4 this reduction in the physician-to-population ratio is expected to have implications for the health care systems of both countries. Therefore, health resource planners likely will be looking to expand the role of primary care, and of family medicine in particular.^5–8 To support such an expansion, a commensurate increase in the numbers of domestically trained family physicians will be required. But with as few as 25% (in 2003)⁹ of Canadian medical graduates choosing family medicine as their top career choice in the residency match, it is unlikely the health care system will be able to supply adequate numbers of primary care physicians.In most countries, the number and the distribution of specialties of physicians are determined by numerous factors, including government policies, training opportunities, immigration and emigration of providers, sex and age distribution of providers, and remuneration incentives and disincentives.^10–17 In many medical systems, the career interests of students also have a substantial steering effect on the distribution and number of available physicians.^18,19The purpose of this study was to follow a large cohort of Canadian medical students from school entry through exit to examine how their career aspirations changed over time and to identify, using multiple logistic regression analysis, the variables at entry into medical school that predict a career choice of family medicine at graduation.     

Virus-specific T-cell immunity correlates with control of GB virus B infection in marmosets

GB virus B (GBV-B) is a hepatotropic virus that is closely related to hepatitis C virus (HCV). GBV-B causes acute hepatitis in infected marmosets and tamarins and is therefore a useful small-animal model for the study of HCV. We investigated virus-specific T-cell responses in marmosets infected with GBV-B. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses in the peripheral blood of two marmosets were assessed throughout the course of GBV-B infection. These T-cell responses were directed against the GBV-B nonstructural proteins 3 (NS3), 4A (NS4A), and 5B (NS5B), and their appearance was temporally associated with clearance of viremia. These marmosets were then rechallenged with GBV-B at least 3 months after clearance of the primary infection to determine if the animals were protected from reinfection. There was no detectable viremia following reinfection, although a sharp increase in T-cell responses against GBV-B proteins was observed. Epitope mapping of T-cell responses to GBV-B was performed with liver and blood samples from both marmosets after rechallenge with GBV-B. Three shared, immunodominant T-cell epitopes within NS3 were identified in animals with multiple common major histocompatibility complex class I alleles. IFN-gamma ELISPOT responses were also detected in the livers of two marmosets that had resolved a primary GBV-B infection. These responses were high in frequency and were directed against epitopes within GBV-B NS3, NS4A, and NS5B proteins. These results indicate that virus-specific T-cell responses are detectable in the liver and blood of GBV-B-infected marmosets and that the clearance of GBV-B is associated with the appearance of these responses.     

Inter and intra‐population phenotypic and genotypic structuring in the European whitefish Coregonus lavaretus,a rare freshwater fish in Scotland

This study revealed between‐lake genetic structuring between Coregonus lavaretus collected from the only two native populations of this species in Scotland, U.K. (Lochs Eck and Lomond) evidenced by the existence of private alleles (12 in Lomond and four in Eck) and significant genetic differentiation (F_ST = 0·056) across 10 microsatellite markers. Juvenile C. lavaretus originating from eggs collected from the two lakes and reared in a common‐garden experiment showed clear phenotypic differences in trophic morphology (i.e. head and body shape) between these populations indicating that these characteristics were, at least partly, inherited. Microsatellite analysis of adults collected from different geographic regions within Loch Lomond revealed detectable and statistically significant but relatively weak genetic structuring (F_ST = 0·001–0·024) and evidence of private alleles related to the basin structure of the lake. Within‐lake genetic divergence patterns suggest three possibilities for this observed pattern: (1) differential selection pressures causing divergence into separate gene pools, (2) a collapse of two formerly divergent gene pools and (3) a stable state maintained by balancing selection forces resulting from spatial variation in selection and lake heterogeneity. Small estimates of effective population sizes for the populations in both lakes suggest that the capacity of both populations to adapt to future environmental change may be limited.     

Floristic variation and willow carr development within a southwest England wetland

Abstract. Woodland colonization on wetlands is considered to have a detrimental effect on their ecological value, even though detailed analysis of this process is lacking. This paper provides an evaluation of the ecological changes resulting from succession of poor fen (base‐poor mire) to willow wet woodland on Goss Moor NNR in Cornwall, UK. Different ages of willow carr were associated with eight understorey communities. During willow colonization, in the ground flora, there was a progressive decrease in poor fen species and an associated increase in woodland species, which appeared to be related to an increase in canopy cover and therefore shade. The most diverse community was found to be the most recent willow and was dominated by poor fen species. The oldest willow was the second most diverse and was associated with a reduction in poor fen species and an increase in woodland species. Architectural features were used successfully to assess the general condition and structure of willow. Tree height and DBH were identified as useful parameters to accurately assess willow age in the field. The implications of active intervention to remove willow in order to conserve the full range of communities within the hydrosere are discussed.