The stability during low-temperature storage of an antifreeze protein isolated from the roots of cold-acclimated carrots

Natural antifreeze proteins (AFPs) not only inhibit freezing at high subzero temperatures; they have the additional properties of inhibiting the recrystallization of ice during warming and of preventing devitrification. The natural AFP that occurs in the roots of cold-acclimated carrots can be extracted reasonably simply and is non-toxic: it was selected for study as a possible ingredient of the vitrification mixtures that are being developed for use in tissue cryopreservation. For this application, it would be essential for the AFP to remain active during prolonged storage at very low temperatures. For logistic reasons, it would also be essential to have an effective method of storage of the purified AFP itself. In this study, carrot AFP was isolated and purified, and its ability to inhibit recrystallization was monitored over 40 weeks of storage at -80 or -196 degrees C. The data revealed a progressive decrease in activity during storage, reaching half the original activity in 10-20 weeks and only 2-3% of the original activity at 40 week. These data suggest that carrot AFP will not be effective in tissue cryopreservation.     

Transposable elements reveal the impact of introgression,rather than transposition,in Pisum diversity,evolution, and domestication

The genetic structure and evolutionary history of the genus Pisum were studied exploiting our germplasm collection to compare the contribution of different mechanisms to the generation of diversity. We used sequence-specific amplification polymorphism (SSAP) markers to assess insertion site polymorphism generated by a representative of each of the two major groups of LTR-containing retrotransposons, PDR1 (Ty1/copia-like) and Cyclops (Ty3/gypsy-like), together with Pis1, a member of the En/Spm transposon superfamily. The analysis of extended sets of the four main Pisum species, P. fulvum, P. elatius, P. abyssinicum, and P. sativum, together with the reference set, revealed a distinct pattern of the NJ (Neighbor-Joining) tree for each basic lineage, which reflects the different evolutionary history of each species. The SSAP markers showed that Pisum is exceptionally polymorphic for an inbreeding species. The patterns of phylogenetic relationships deduced from different transposable elements were in general agreement. The retrotransposon-derived markers gave a clearer separation of the main lineages than the Pis1 markers and were able to distinguish the truly wild form of P. elatius from the antecedents of P. sativum. There were more species-specific and unique PDR1 markers than Pis1 markers in P. fulvum and P. elatius, pointing to PDR1 activity during speciation and diversification, but the proportion of these markers is low. The overall genetic diversity of Pisum and the extreme polymorphism in all species, except P. abyssinicum, indicate a high contribution of recombination between multiple ancestral lineages compared to transposition within lineages. The two independently domesticated pea species, P. abyssinicum and P. sativum, arose in contrasting ways from the common processes of hybridization, introgression, and selection without associated transpositional activity.     

Conceptual challenges to the harm threshold

Children are presumptively regarded as incompetent to make their own medical decisions, and the responsibility for making such decisions typically falls to parents. Parental authority is not unlimited, however, and ethical guidelines identifying appropriate bounds on this authority are needed. One proposal currently gaining support is the harm threshold (HT), which asserts that the state may only legitimately intervene in parental decision-making when serious and preventable harm to children is likely. This paper considers two questions: in virtue of what underlying principle or property does the HT gain its purported justification?; and does this underlying principle or property ground the HT as its proponents conceive of it? I identify two separate grounds represented in the literature: (a) J.S. Mill’s Harm Principle; and (b) the liberty interests of parents. I find that the HT is not sufficiently grounded in either of these, revealing a substantial conceptual difficulty for its advocates.     

A multi-centre randomized controlled trial of rehabilitation aimed at improving outdoor mobility for people after stroke: study protocol for a randomized controlled trial

ABSTRACT: BACKGROUND: Up to 42% of all stroke patients do not get out of the house as much as they would like. This can impede a person's quality of life. This study is testing the clinical effectiveness and cost effectiveness of a new outdoor mobility rehabilitation intervention by comparing it to usual care. METHODS: Multi-centre parallel group individually randomised controlled trial. At least 506 participants will be recruited through 15 primary and secondary care settings and will be eligible if they are over 18 years of age, have had a stroke and wish to get out of the house more often. Participants are being randomly allocated to either the intervention group or the control group. Intervention group participants receive up to 12 rehabilitation outdoor mobility sessions over up to 4 months. The main component of the intervention is repeated practice of outdoor mobility with a therapist. Control group participants are receiving the usual intervention for outdoor mobility limitations: verbal advice and provision of leaflets provided over one session. Outcome measures are being collected using postal questionnaires, travel calendars and by independent assessors. The primary outcome measure is the Social Function domain of the SF36v2 quality of life assessment six months after recruitment. The secondary outcome measures include: functional ability, mobility, the number of journeys (monthly travel diaries), satisfaction with outdoor mobility, mood, health-related quality of life, resource use of health and social care. Carer mood is also being collected. The mean Social Function score of the SF-36v2 will be compared between treatment arms using a multiple membership form of mixed effects multiple regression analysis adjusting for centre (as a fixed effect), age and baseline Social Function score as covariates and therapist as a multiple membership random effect. Regression coefficients and 95% confidence intervals will be presented. DISCUSSION: This study protocol describes a pragmatic randomised controlled trial that will hopefully provide robust evidence of the benefit of outdoor mobility interventions after stroke for clinicians working in the community. The results will be available towards the end of 2012.     

The development of a high-content screening binding assay for the smoothened receptor

In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-μM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.     

Post-fledging brood and care division in the roseate tern (Sterna dougallii)

Extended post-fledging parental care is an important aspect of parental care in birds, although little studied due to logistic difficulties. Commonly, the brood is split physically (brood division) and/or preferential care is given to a subset of the brood by one parent or the other (care division). Among gulls and tern (Laridae), males and females generally share parental activities during the pre-fledging period, but the allocation of parental care after fledging is little documented. This study examined the behaviour of male and female roseate terns (Sterna dougallii) during the late chick-rearing and early post-fledging periods, and in particular the amount of feeds and the time spent in attendance given to individual chicks/fledglings. Pre-fledging parental care was biparental in all cases. Post-fledging parental care was dependent on the number of fledglings in the brood. Males and females continued biparental care in clutches with one surviving fledgling, while in two-fledgling clutches, males fed the A-fledgling while females fed the B-fledgling. Overall, there was no difference in attendance, only in feeds. This division of care may be influenced by the male only being certain of the paternity of the A-chick but not by chick sex.     

Impacts of feral horses in the Australian Alps and evidence‐based solutions

New evidence of impacts by feral horses in Australia's alpine parks systems confirms they endanger threatened species and extensively damage critically endangered bog communities that could take millennia to recover. These impacts are not confounded by effects of deer and accumulate over time, even when only a small number of feral horses (~100) are present. With protected areas representing only a small proportion of the area of the Australian states of New South Wales (9.3%) and Victoria (17%), allowing feral horses to degrade reserves is not a reasonable management compromise, is contrary to the purpose of the protected area system and conflicts with international obligations. Modelling and decades of management experience indicate that trapping alone does not control feral horse numbers. Trapping and fertility control can work in small populations, but not when there are several thousand horses in remote areas. Aerial culling is needed to cost‐effectively and humanely control feral horse populations. The relatively small amount of suffering feral horses experience during a cull is outweighed by (i) avoiding suffering and death of horses from starvation and thirst, (ii) avoiding the suffering of native animals displaced by horses and (iii) avoiding the ethical concerns of driving threatened species towards extinction. Objections to aerial culling on welfare and cultural grounds are contradicted by evidence. Improving knowledge in the general community about what is at stake is long overdue because without this knowledge, small groups with vested interests and unfounded claims have been able to dominate debate and dictate management actions. As a result of ineffective management, horse populations are now expanding and causing well‐documented damage to Australia's alpine parks, placing at risk almost $10M spent on restoration after livestock grazing ended. The costs of horse control and restoration escalate the longer large horse populations remain in the alpine parks. It is crucial that feral horse numbers are rapidly reduced to levels where ecosystems begin to recover. Aerial culling is needed as part of the toolbox to achieve that reduction.     

Effects of localised, low-voltage pulsed electric fields on the development and inhibition of Pseudomonas aeruginosa biofilms

This work describes the use of low-voltage (0.5 - 5 V) pulsed electric fields to prevent Pseudomonas aeruginosa biofilm development. Interdigitated electrodes (IDEs) with 29-mum spacing between 22-mum-wide electrodes, were used as a platform where the effect of localised, high-strength electric fields could be tested. Alternating current, square-wave pulses were applied to the IDEs in 1 sec intervals. A two-level, three-variable factorial design experiment was used to detect the effects of applied voltage, frequency, and pulse duty ratio (i.e. percentage of pulsing time over one cycle) on the inhibition of biofilm formation. The observations indicated that a pulse configuration of 1% duty ratio, 5 V, and 200 Hz frequency reduced the area of the electrodes covered by biofilm by 50%. In general, the application of low-duty ratio pulses had a positive effect on preventing biofouling. Comparatively, frequency and applied voltage were observed to have less influence on biofouling.