The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS

In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma

The SITLESS project: Exercise referral schemes enhanced by self-management strategies to battle sedentary behaviour in older adults: Study protocol for a randomised controlled trial

Abstract Background Older adults are the fastest growing segment of the world‘s population. Recent evidence indicates that excessive sitting time is harmful to health, independent of meeting the recommended moderate to vigorous physical activity (PA) guidelines. The SITLESS project aims to determine whether exercise referral schemes (ERS) can be enhanced by self-management strategies (SMSs) to reduce sedentary behaviour (SB), increase PA and improve health, quality of life and function in the long term, as well as psychosocial outcomes in community-dwelling older European citizens from four countries, within a three-armed pragmatic randomised controlled trial, compared with ERS alone and also with general recommendations about PA. Methods A total of 1338 older adults will be included in this study, recruited from four European countries through different existing primary prevention pathways. Participants will be randomly allocated into an ERS of 16 weeks (32 sessions, 45–60 min per session), ERS enhanced by seven sessions of SMSs and four telephone prompts, or a control group. Outcomes will be assessed at baseline, month 4 (end of ERS intervention), month 16 (12 months post intervention) and month 22 (18 months post intervention). Primary outcomes will include measures of SB (time spent sedentary) and PA (counts per minute). Secondary outcomes will include muscle and physical function, health economics’ related outcomes, anthropometry, quality of life, social networks, anxiety and depressive symptoms, disability, fear of falling, executive function and fatigue. A process evaluation will be conducted throughout the trial. The full analysis set will follow an intention-to-treat principle and will include all randomised participants for whom a baseline assessment is conducted. The study hypothesis will be tested with mixed linear models with repeated measures, to assess changes in the main outcomes (SB and PA) over time (baseline to month 22) and between study arms. Discussion The findings of this study may help inform the design and implementation of more effective interventions to reduce SB and increase PA levels, and hence improve long-term health outcomes in the older adult population. SITLESS aims to support policy-makers in deciding how or whether ERS should be further implemented or restructured in order to increase its adherence, impact and cost-effectiveness. Trial registration ClinicalTrials.gov, NCT02629666 . Registered 19 November 2015

Effect of a primary care walking intervention with and without nurse support on physical activity levels in 45- to 75-year-olds: The pedometer and consultation evaluation (PACE-UP) cluster randomised clinical trial

Background Pedometers can increase walking and moderate-to-vigorous physical activity (MVPA) levels, but their effectiveness with or without support has not been rigorously evaluated. We assessed the effectiveness of a pedometer-based walking intervention in predominantly inactive adults, delivered by post or through primary care nurse-supported physical activity (PA) consultations. Methods and Findings A parallel three-arm cluster randomised trial was randomised by household, with 12-mo follow-up, in seven London, United Kingdom, primary care practices. Eleven thousand fifteen randomly selected patients aged 45–75 y without PA contraindications were invited. Five hundred forty-eight self-reporting achieving PA guidelines were excluded. One thousand twenty-three people from 922 households were randomised between 2012–2013 to one of the following groups: usual care (n = 338); postal pedometer intervention (n = 339); and nurse-supported pedometer intervention (n = 346). Of these, 956 participants (93%) provided outcome data (usual care n = 323, postal n = 312, nurse-supported n = 321). Both intervention groups received pedometers, 12-wk walking programmes, and PA diaries. The nurse group was offered three PA consultations. Primary and main secondary outcomes were changes from baseline to 12 mo in average daily step-counts and time in MVPA (in ≥10-min bouts), respectively, measured objectively by accelerometry. Only statisticians were masked to group. Analysis was by intention-to-treat. Average baseline daily step-count was 7,479 (standard deviation [s.d.] 2,671), and average time in MVPA bouts was 94 (s.d. 102) min/wk. At 12 mo, mean steps/d, with s.d. in parentheses, were as follows: control 7,246 (2,671); postal 8,010 (2,922); and nurse support 8,131 (3,228). PA increased in both intervention groups compared with the control group; additional steps/d were 642 for postal (95% CI 329–955) and 677 for nurse support (95% CI 365–989); additional MVPA in bouts (min/wk) were 33 for postal (95% CI 17–49) and 35 for nurse support (95% CI 19–51). There were no significant differences between the two interventions at 12 mo. The 10% (1,023/10,467) recruitment rate was a study limitation. Conclusions A primary care pedometer-based walking intervention in predominantly inactive 45- to 75-y-olds increased step-counts by about one-tenth and time in MVPA in bouts by about one-third. Nurse and postal delivery achieved similar 12-mo PA outcomes. A primary care pedometer intervention delivered by post or with minimal support could help address the public health physical inactivity challenge.The PACE-UP trial was funded by the National Institute for Health Research Health Technology Assessment (NIHR HTA) Programme (project number HTA 10/32/02 ISRCTN42122561) and will be published in full in Health Technology Assessment. The funders had no role in study design (beyond the commissioned call outline), data collection and analysis, decision to publish, or preparation of the manuscript

Promoting the avoidance of high-calorie snacks: priming autonomy moderates message framing effects

The beneficial effects of gain-framed vs. loss-framed messages promoting health protective behaviors have been found to be inconsistent, and consideration of potential moderating variables is essential if framed health promotion messages are to be effective. This research aimed to determine the influence of highlighting autonomy (choice and freedom) and heteronomy (coercion) on the avoidance of high-calorie snacks following reading gain-framed or loss-framed health messages. In Study 1 (N = 152) participants completed an autonomy, neutral, or heteronomy priming task, and read a gain-framed or loss-framed health message. In Study 2 (N = 242) participants read a gain-framed or loss-framed health message with embedded autonomy or heteronomy primes. In both studies, snacking intentions and behavior were recorded after seven days. In both studies, when autonomy was highlighted, the gain-framed message (compared to the loss-framed message) resulted in stronger intentions to avoid high-calorie snacks, and lower self-reported snack consumption after seven days. Study 2 demonstrated this effect occurred only for participants to whom the information was most relevant (BMI>25). The results suggest that messages promoting healthy dietary behavior may be more persuasive if the autonomy-supportive vs. coercive nature of the health information is matched to the message frame. Further research is needed to examine potential mediating processes

The transcriptomic basis of oviposition behaviour in the parasitoid wasp Nasonia vitripennis

Linking behavioural phenotypes to their underlying genotypes is crucial for uncovering the mechanisms that underpin behaviour and for understanding the origins and maintenance of genetic variation in behaviour. Recently, interest has begun to focus on the transcriptome as a route for identifying genes and gene pathways associated with behaviour. For many behavioural traits studied at the phenotypic level, we have little or no idea of where to start searching for "candidate" genes: the transcriptome provides such a starting point. Here we consider transcriptomic changes associated with oviposition in the parasitoid wasp Nasonia vitripennis. Oviposition is a key behaviour for parasitoids, as females are faced with a variety of decisions that will impact offspring fitness. These include choosing between hosts of differing quality, as well as making decisions regarding clutch size and offspring sex ratio. We compared the whole-body transcriptomes of resting or ovipositing female Nasonia using a "DeepSAGE" gene expression approach on the Illumina sequencing platform. We identified 332 tags that were significantly differentially expressed between the two treatments, with 77% of the changes associated with greater expression in resting females. Oviposition therefore appears to focus gene expression away from a number of physiological processes, with gene ontologies suggesting that aspects of metabolism may be down-regulated during egg-laying. Nine of the most abundant differentially expressed tags showed greater expression in ovipositing females though, including the genes purity-of-essence (associated with behavioural phenotypes in Drosophila) and glucose dehydrogenase (GLD). The GLD protein has been implicated in sperm storage and release in Drosophila and so provides a possible candidate for the control of sex allocation by female Nasonia during oviposition. Oviposition in Nasonia therefore clearly modifies the transcriptome, providing a starting point for the genetic dissection of oviposition.Publisher PDFPeer reviewe