An Improved Evans Blue Staining Method for Consistent, Accurate Assessment of Plasmodiophora brassicae Resting Spore Viability.

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly (R2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores (R2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.

Can plant-derived phytochemicals provide symptom relief for hair loss? A critical review.

It is known that hair growth disorders and hair loss can cause personal distress and affect well-being. Whilst clinical conditions remain a target for medical research, current research on hair follicle biology and hair growth control mechanisms also provides opportunities for a range of non-medical and cosmetic interventions that have a modulating effect on the scalp and follicle function. Furthermore, an improvement of the hair fibre characteristics (cuticle structure, cortex size and integrity) could add to the overall positive visual effect of the hair array. Since phytochemicals are a popular choice because of their traditional appeal, this review provides a critical evaluation of the available evidence of their activity for hair benefit, excluding data obtained from animal tests, and offers recommendations on improving study validity and the robustness of data collection in pre-clinical and clinical studies.

La perte des cheveux ou les troubles de la croissance sont connus pour engendrer une grande détresse et affecter le bien-être des individus. Bien que les maladies soient la cible principale de la recherche médicale, de récentes découvertes sur la biologie du follicule pileux et les mécanismes de contrôle de la pousse de cheveux pourraient donner lieu à des interventions non-médicales ou cosmétiques afin d'impacter le cuir chevelu et le follicule. De plus, améliorer les caractéristiques de la fibre capillaire (structure des cuticules, taille et intégrité du cortex) pourrait contribuer à embellir l'apparence de la masse capillaire. Les phytoingrédients sont populaires car traditionnellement utilisés dans les cosmétiques, et cette revue présente une évaluation critique de leurs bénéfices pour les cheveux, en excluant les données issues de tests sur les animaux. Des recommandations afin d'améliorer la collecte de données et la validité des études pré-cliniques et cliniques sont aussi présentées dans cette revue.

Measuring the frequency of consumer hair combing and magnitude of combing forces on individual hairs in a tress and the implications for product evaluation and claims substantiation.

OBJECTIVES: It is commonly assumed that, due to the long growth cycle of hair, multi-cycle combing, and strength and fatigue testing using thousands of cycles is relevant for product evaluation and claim substantiation. The objective was to assess the frequency and magnitude of combing forces on individual hairs against a hypothesis that fibres on a consumer's head rarely experience significant loads during routine combing. METHODS: Single fibres were removed from a tress, attached to a load cell and replaced in the tress. Combing of tresses, guided by in-vivo measurements, measured the frequency of significant loads defined as 'events' ≥1 g over 30 combing sets (set = 10 comb strokes @~25 cm s-1 ) with intermediate tangling. Asian and Caucasian hair was assessed by dry, wet, bleached-wet and bleached-dry combing. A questionnaire of 231 Asian and Caucasian women established daily frequency and number of comb strokes for the whole head. In-vivo combing videos of 10 women (five Asian, five Caucasian) were used to establish in-vivo and tress combing speeds. RESULTS: The questionnaires returned an average combing frequency of 1.7×/day (range 0-5) and average number of strokes 16 ± 2.3 per head/day (95% CI). Video analysis measured combing speeds of 22-35 cm s-1 across hair types. Tress data confirmed individual fibres are unlikely to experience repeated loading or significant loads in all but wet combed persulphate bleached hair. 'Events' of ≥1 g - dry combing gave an event probability of 0.2 and average load of 1.7 g over 30 comb sets. Dry combed bleached samples returned a probability of 0.23 and 0.3 respectively. Wet combed virgin Asian and Caucasian hair gave a probability of 0.1 and 0.47 respectively. Wet combed bleached hair gave a probability of one. The addition of a conditioner to bleached hair reduced the event probability to <0.1. CONCLUSION: During combing, individual fibres may not experience any significant loads and are unlikely to experience repetitive loads >10 g. The low number of comb strokes and low event probability is in keeping with consumers growing their hair long and in good condition. The data indicates the need for a significant rethink of methods used for product evaluation and claim substantiation.

LEADER 2: baseline calcitonin in 9340 people with type 2 diabetes enrolled in the Liraglutide Effect and Action in Diabetes: Evaluation of cardiovascular outcome Results (LEADER) trial: preliminary observations.

AIMS: To report preliminary data on baseline serum calcitonin concentrations and associated clinical characteristics in a global population with type 2 diabetes before liraglutide or placebo randomization. METHODS: The ongoing LEADER trial has enrolled 9340 people with type 2 diabetes and at high risk of cardiovascular disease at 410 centres worldwide. People with baseline serum calcitonin ≤ 50 ng/l were randomized to liraglutide once daily or placebo and will be followed for up to 5 years. Serum calcitonin was measured at baseline and will be measured annually thereafter. An independent committee of thyroid experts will oversee calcitonin monitoring throughout the trial and will review all calcitonin concentrations ≥ 20 ng/l. RESULTS: The mean age of participants was 64.3 ± 7.2 years, 64.3% were men, and mean the body mass index was 32.5 ± 6.3 kg/m(2). The median (interquartile range) baseline serum calcitonin values were 3.9 (1.0 to >7.6) ng/l in men and 1.0 (1.0 to >1) ng/l in women. Serum calcitonin was >10 ng/l in 14.6% of men and in 0.96% of women. In sex-specific multivariable linear analysis of covariance models, a reduced glomerular filtration rate (GFR) was associated with higher serum calcitonin concentrations that were statistically significant. A 20 ml/min/1.73 m(2) decrease in estimated GFR (eGFR) was associated with a 14% increase in serum calcitonin in women and an 11% increase in men. CONCLUSIONS: In the LEADER population, the prevalence of elevated serum calcitonin concentrations at baseline was high, and there was an inverse association between eGFR and serum calcitonin concentrations.

Use of cffDNA to avoid administration of anti-D to pregnant women when the fetus is RhD-negative: implementation in the NHS.

OBJECTIVE: To determine whether a policy of offering cffDNA testing to all RhD-negative women at about 16 weeks' gestation to avoid anti-D administration when the fetus is RhD-negative could be implemented successfully in the NHS without additional funding. DESIGN: Prospectively planned observational service implementation pilot and notes audit. SETTING: Three maternity services in the South West of England. POPULATION: All RhD-negative women in a 6-month period. METHODS: Prospective, intervention, cross-sectional observational study, using pre-intervention data as controls. MAIN OUTCOME MEASURES: Proportion of suitable women who offered and accepted the test. Accuracy of the cffDNA result as assessed by cord blood group result. Fall in anti-D doses administered. RESULTS: 529 samples were received; three were unsuitable. The results were reported as RhD-positive (n = 278), RhD-negative (n = 185) or inconclusive, treat as positive (n = 63). Cord blood results were available in 502 (95%) and the only incorrect result was one case of a false positive (cffDNA reported as positive, cord blood negative - and so given anti-D unnecessarily). The notes audit showed that women who declined this service were correctly managed and that anti-D was not given when the fetus was predicted to be RhD-negative. The total use of anti-D doses fell by about 29% which equated to about 35% of RhD-negative women not receiving anti-D in their pregnancy unnecessarily. CONCLUSIONS: We recommend this service is extended to all UK NHS services.

Three uncommon KEL alleles in one family with unusual Kell phenotypes explain a 35-year old conundrum.

BACKGROUND: Kell is a complex blood group system comprising 35 antigens. Kell antigens are absent from rare red cells of the Ko (null) phenotype and expressed only weakly in the Kmod phenotype. Molecular analysis of three uncommon KEL alleles elucidated the obscure pattern of inheritance of Kell antigens in one family. MATERIALS AND METHODS: Standard serological methods were employed. All exons, flanking intronic sequence and introns 15 and 16 of KEL were sequenced from genomic DNA. cDNA was obtained from erythroid cells cultured from progenitor cells isolated from peripheral blood. RESULTS: The Kmod propositus was heterozygous for two KEL mutations: c.2107G>A, p.Gly703Arg and a synonymous mutation, c.1719C>T, in the codon for p.573Gly. Sequencing of cDNA revealed that c.1719C>T caused skipping of exon 16, resulting in a silent allele. Her KEL:3,-4 brother was heterozygous for KEL*03/04 and c.1719C/T. CONCLUSION: A synonymous mutation caused complete exon skipping, despite being located 16 bases downstream of the 3' splice site, resulting in a null KEL allele. The combined effects of two mod alleles, one responsible for KEL3 expression and the other for p.Gly703Arg, were probably responsible for an unexpected KEL:3,-4 phenotype.

International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology: Cancun report (2012).

The International Society of Blood Transfusion Working Party on red cell immunogenetics and blood group terminology convened during the International congress in Cancun, July 2012. This report details the newly identified antigens in existing blood group systems and presents three new blood group systems.

International reference reagents to standardise blood group genotyping: evaluation of candidate preparations in an international collaborative study.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate, in an international collaboration, four lyophilised genomic DNA preparations, selected from genotyped and phenotyped donors by the study organisers, for their suitability to standardise and control blood group genotyping procedures for common ancestral Caucasian and Black African alleles. MATERIALS AND METHODS: Twenty-nine laboratories performed 'blind' testing of replicated ampoules of the candidate reference reagents, RBC1 (10/232), RBC4 (10/236), RBC5 (10/238) and RBC12 (10/234), using a range of genotyping procedures, most commonly classical PCR using allele or sequence specific primers. RESULTS: The majority of laboratories reported blood group genotypes in accordance with those determined by the study organisers and the serological phenotypes. Despite an overall high level of accuracy in genotyping, the identified errors and inconsistencies, and the limited genotyping capabilities of many laboratories, confirmed the need for validated reference materials to control test procedures. CONCLUSIONS: The establishment of RBC1, RBC4, RBC5 and RBC12 as World Health Organization Reference Reagents will facilitate international standardisation of blood group genotyping and ensure that such tests are sufficiently sensitive and specific.

What can ecological data tell us about reasons for divergence in health status between West Central Scotland and other regions of post-industrial Europe?

BACKGROUND: The link between the effects of de-industrialization (unemployment, poverty) and population health is well understood. Post-industrial decline has, therefore, been cited as an underlying cause of high mortality in Scotland's most de-industrialized region. However, previous research showed other comparably de-industrialized regions in Europe to have better and faster improving health (with, in many cases, a widening gap evident from the early to mid-1980s). OBJECTIVES: To explore whether ecological data can provide insights into reasons behind the poorer, and more slowly improving, health status of West Central Scotland (WCS) compared with other European regions that have experienced similar histories of post-industrial decline. Specifically, this study asked: (1) could WCS's poorer health status be explained purely in terms of socio-economic factors (poverty, deprivation etc.)? and (2) could comparisons with other health determinant information identify important differences between WCS and other regions? These aims were explored alongside other research examining the historical, economic and political context in WCS compared with other de-industrialized regions. STUDY DESIGN AND METHODS: A range of ecological data, derived from surveys and routine administrative sources, were collected and analysed for WCS and 11 other post-industrial regions. Analyses were underpinned by the collection and analysis of more detailed data for four particular regions of interest. In addition, the project drew on accompanying literature-based research, analysing important contextual factors in de-industrialized regions, including histories of economic and welfare policies, and national and regional responses to de-industrialization. RESULTS: The poorer health status of WCS cannot be explained in terms of absolute measures of poverty and deprivation. However, compared with other post-industrial regions in Mainland Europe, the region is distinguished by having wider income inequalities and associated social characteristics (e.g. more single adults, lone parent households, higher rates of teenage pregnancy). Some of these distinguishing features are shared by other UK post-industrial regions which experienced the same economic history as WCS. CONCLUSION: From the collection of data and supporting analyses of important contextual factors, one can argue that poor health in WCS can be attributed to three layers of causation: the effects of de-industrialization (which have impacted on health in all post-industrial regions); the impact of 'neoliberal' UK economic policies, resulting in wider inequalities in WCS and the other UK regions; and an as-yet-unexplained (but under investigation) set of factors that cause WCS to experience worse health outcomes than similar regions within the UK.

Exploring the effects of non-medical versus medical approaches to the management of skin aging in women over sixty.

This study was a pilot project, set up to assess ageing skin using a multi-disciplinary approach. The main aim of this study was to evaluate whether the use of more radical ('medical') treatments in the management of skin ageing would bring superior results and ultimately make people look younger, than the use of cosmetics ('non-medical' treatments). A simple post-hoc study design was used, whereby medical treatments varied within the group, all of them completed at least 2 weeks before the start of the study. In addition, it was of interest to assess the suitability of the proposed combination of methods. A total of 21 female participants were recruited for this study: 11 for the non-medical and 10 for the medical group. The multi-disciplinary approach consisted of instrumental measurements, self-assessment, expert assessment by Merz scales and a public perception survey. The majority of nearly 70 sets of instrumental skin data obtained in this study did not differ significantly between the non-medical and the medical group. However, the medical group gave higher self-assessment scores for their faces. The scores for hands were lower than scores for faces by both groups. This was partly supported by instrumental data (lower skin hydration on hands than on the face). The findings of the public perception survey of nine matched pairs of subjects scored the non-medical group as younger looking. Data analysis has shown that the judgement of youthfulness did not depend on either the gender or the age of observers.

Non-invasive prenatal determination of fetal sex: translating research into clinical practice.

The effectiveness and clinical utility of non-invasive prenatal diagnosis (NIPD) for fetal sex determination using cell-free fetal DNA (cffDNA) was assessed by undertaking a prospective national audit of UK testing. NIPD was performed using real-time polymerase chain reaction analysis of the DYS14 or SRY gene in cffDNA extracted from maternal plasma. All cases referred for fetal sex determination from 1 April 2006 to 31 March 2009 were ascertained from two laboratories offering the test. Fetal gender determined by NIPD was compared with that based on ultrasound, invasive test or phenotype at birth. Indication and rate of invasive testing was ascertained. In the first year, results were issued in 150/161 pregnancies tested. Of the 135 with outcome data, results were concordant in 130/135 [96.3% (95% CI 91.6-98.8%)]. Reporting criteria were changed and in the subsequent 511 pregnancies the concordancy rate increased to 401/403 [99.5% (95% CI 98.2-99.9%)]. Over the 3 years only 32.9% (174/528) underwent invasive testing. NIPD for fetal sex determination using cffDNA is highly accurate when performed in National Health Service laboratories if stringent reporting criteria are applied. Parents should be advised of the small risk of discordant results and possible need for repeat testing to resolve inconclusive results.

Structural modelling of red cell surface proteins.

The red cell surface membranes contain a large variety of proteins, many of which express blood group activity as a result of variation in their oligosaccharide or amino acid sequences. To understand the nature of the blood group epitopes, the functions of the proteins that express them and their relationship to each other, computer modelling has been employed to provide predictions of their structures. Modelling is an excellent method of first resort when experimental structural data for the protein of interest are absent or incomplete. The model can then be used to explain previous experimental data and to direct future experiments. In this review, examples of protein modelling are taken from the Rh, RHAG, Kell, LW, Lutheran, Duffy, Dombrock and RAPH blood group systems.

Report of the fourth International Workshop on molecular blood group genotyping.

The fourth International Society of Blood Transfusion (ISBT) workshop on molecular blood group genotyping was held in 2010, with a feedback meeting at the ISBT Congress in Berlin, Germany. Fifty laboratories participated, 17 more than in 2008. Six samples were distributed. Samples 1-3 were DNA samples for all red cell blood group tests available to the participants. Of the 46 laboratories that tested these samples, 37 obtained completely correct results, although the extent of testing varied considerably. Sample 4, also a DNA sample, was an Rh problem in which RHDΨ and RHCE*ceCF were present, but the participants were only informed that the donor's red cells typed as positive with some monoclonal anti-D. Of the 42 laboratories that participated in this exercise, seven performed the sequencing necessary to obtain the correct result. Samples 5 and 6 were plasma samples from RhD-negative pregnant women, for foetal RhD testing. These were sent to 25 laboratories, and two incorrect results were reported. Overall, the level of accuracy was about equal to that of the previous workshop. The main conclusion for the last two workshops can be reiterated: with greater care and attention to detail, very high standards could be set for molecular blood group genotyping.

A new blood group system, RHAG: three antigens resulting from amino acid substitutions in the Rh-associated glycoprotein.

BACKGROUND AND OBJECTIVES: Rh-associated glycoprotein (RhAG) is closely associated with the Rh proteins in the red cell membrane. Two high frequency antigens (Duclos and DSLK) and one low frequency antigen (Ol(a)) have serological characteristics suggestive of expression on RhAG. MATERIALS AND METHODS: RHAG was sequenced from the DNA of one Duclos-negative, one DSLK-negative, and two Ol(a+) individuals. Recombinant protein was expressed in HEK 293 cells. Protein models with RhAG subunits were constructed. RESULTS: The original Duclos-negative patient was homozygous for RHAG 316C>G, encoding Gln106Glu. HEK 293 cells expressing Gln106Glu mutant RhAG did not react with anti-Duclos. An individual with DSLK-negative red cells was homozygous for 490A>C, encoding Lys164Gln. Two Ol(a+) members of the original Norwegian family were heterozygous for 680C>T, encoding Ser227Leu. A Japanese donor with Rh(mod) phenotype had Ol(a+) red cells and was homozygous for 680C>T. CONCLUSION: The three red cell antigens encoded by RHAG form the RHAG blood group system: Duclos is RHAG1 (030001); Ol(a) is RHAG2 (030002); and DSLK is provisionally RHAG3 (030003).

Controlled propagation of replication-competent Sindbis viral vector using suicide gene strategy.

A major concern of using viral gene therapy is the potential for uncontrolled vector propagation and infection that might result in serious deleterious effects. To enhance the safety, several viral vectors, including vectors based on Sindbis virus, were engineered to lose their capability to replicate and spread after transduction of target cells. Such designs, however, could dramatically reduce the therapeutic potency of the viral vectors, resulting in the need for multiple dosages to achieve treatment goals. Earlier, we showed that a replication-defective (RD) Sindbis vector achieved specific tumor targeting without any adverse effects in vivo. Here, we present a replication-competent Sindbis viral vector that has an hsvtk suicide gene incorporated into ns3, an indispensable non-structural gene for viral survival. The capability of viral propagation significantly increases tumor-specific infection and enhances growth suppression of tumor compared with the conventional RD vectors. Furthermore, in the presence of the prodrug ganciclovir, the hsvtk suicide gene serves as a safety mechanism to prevent uncontrolled vector propagation. In addition to suppressing vector propagation, toxic metabolites, generated by prodrug activation, could spread to neighboring uninfected tumor cells to further enhance tumor killing.

The pure-science ideal and democratic culture.

These early experiences of pure scientists will have an unmistakable ring of familiarity to anyone familiar with the current situation. Charles Sanders Peirce, with characteristic insight, had stated the fundamental dilemma of the pure scientist operating within a democratic framework. How can one ask the public to provide support, much less facilities, for the intellectual gratification of one select group? A part of the answer, of course, is simply that one cannot. As long as a group is dependent upon public support it must seek some means of contact with the values of the enveloping society, and the moment it does this it departs in some measure from the ideal purity. The schizophrenic attitude described by Dubos therefore became a professional necessity as soon as the new ideal was adopted. Since the time of Gould, scientists have been able to tell each other that the man who based science's claim to support on grounds of immediate practical utility was "no loyal follower and true friend of science" and, at the same time, to trust that the popularizers and technicians would convey a different message to the public. On the whole, they have not been disappointed in their expectation, and there has been little need for them to go beyond the standard formula : Utility is not to be a test of scientific work, but all knowledge will ultimately prove useful. Since the continued existence of scientists in this society depends upon the believability of that vague claim, there is little likelihood hood that the schizophrenia will disappear.