An automated multiwell plate reading film microscope for live cell autofluorescence lifetime assays

Fluorescence lifetime imaging (FLIM) is increasingly used to read out cellular autofluorescence originating from the coenzyme NADH in the context of investigating cell metabolic state. We present here an automated multiwell plate reading FLIM microscope optimized for UV illumination with the goal of extending high content fluorescence lifetime assays to readouts of metabolism. We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation. In particular, we illustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators, thereby illustrating the potential of the instrument for cytotoxicity studies, assays for drug discovery and stratified medicine

Estimation of Influenza Vaccine Effectiveness from Routine Surveillance Data

BACKGROUND: Influenza vaccines are reviewed each year, and often changed, in an effort to maintain their effectiveness against drifted influenza viruses. There is however no regular review of influenza vaccine effectiveness during, or at the end of, Australian influenza seasons. It is possible to use a case control method to estimate vaccine effectiveness from surveillance data when all patients in a surveillance system are tested for influenza and their vaccination status is known. METHODOLOGY/PRINCIPAL FINDINGS: Influenza-like illness (ILI) surveillance is conducted during the influenza season in sentinel general practices scattered throughout Victoria, Australia. Over five seasons 2003-7, data on age, sex and vaccination status were collected and nose and throat swabs were offered to patients presenting within three days of the onset of their symptoms. Swabs were tested using a reverse transcriptase polymerase chain reaction (RT-PCR) test. Those positive for influenza were sent to the World Health Organization (WHO) Collaborating Centre for Reference and Research on Influenza where influenza virus culture and strain identification was attempted. We used a retrospective case control design in five consecutive influenza seasons, and estimated influenza vaccine effectiveness (VE) for patients of all ages to be 53% (95% CI 38-64), but 41% (95% CI 19-57) adjusted for age group and year. The adjusted VE for all adults aged at least 20 years, the age groups for whom a benefit of vaccination could be shown, was 51% (95% CI 34-63). Comparison of VE estimates with vaccine and circulating strain matches across the years did not reveal any significant differences. CONCLUSIONS/SIGNIFICANCE: These estimates support other field studies of influenza vaccine effectiveness, given that theoretical considerations suggest that these values may underestimate true effectiveness, depending on test specificity and the ratio of the influenza ILI attack rate to the non-influenza ILI attack rate. Incomplete recording of vaccination status and under-representation of children in patients from whom a swab was collected limit the data. Improvements have been implemented for prospective studies

Listeners feel the beat: Entrainment to English and French speech rhythms

Can listeners entrain to speech rhythms? Monolingual speakers of English and French and balanced English–French bilinguals tapped along with the beat they perceived in sentences spoken in a stress-timed language, English, and a syllable-timed language, French. All groups of participants tapped more regularly to English than to French utterances. Tapping performance was also influenced by the participants’ native language: English-speaking participants and bilinguals tapped more regularly and at higher metrical levels than did French-speaking participants, suggesting that long-term linguistic experience with a stress-timed language can differentiate speakers’ entrainment to speech rhythm

Identification and functional characterisation of CRK12:CYC9, a novel cyclin-dependent kinase (CDK)-cyclin complex in Trypanosoma brucei

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively

Piperidinols that show anti-tubercular activity as inhibitors of arylamine N-acetyltransferase: an essential enzyme for mycobacterial survival inside macrophages

Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3-16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs

Time-calibrated phylogenetic trees establish a lag between polyploidisation and diversification in Nicotiana (Solanaceae)

We investigate the timing of diversification in allopolyploids of Nicotiana (Solanaceae) utilising sequence data of maternal and paternal origin to look for evidence of a lag phase during which diploidisation took place. Bayesian relaxed clock phylogenetic methods show recent allopolyploids are a result of several unique polyploidisation events, and older allopolyploid sections have undergone subsequent speciation at the polyploid level (i.e. a number of these polyploid species share a singular origin). The independently formed recent polyploid species in the genus all have mean age estimates below 1 million years ago (Ma). Nicotiana  section Polydicliae (two species) evolved 1.5 Ma, N. section Repandae (four species) formed 4 Ma, and N. section Suaveolentes (*35 species) is about 6 million years old. A general trend of higher speciation rates in older polyploids is evident, but diversification dramatically increases at approximately 6 Ma (in section Suaveolentes). Nicotiana sect. Suaveolentes has spectacularly radiated to form 35 species in Australia and some Pacific islands following a lag phase of almost 6 million years. Species have filled new ecological niches and undergone extensive diploidisation (e.g. chromosome fusions bringing the ancestral allotetraploid number, n = 24, down to n = 15 and ribosomal loci numbers back to diploid condition). Considering the progenitors of Suaveolentes inhabit South America, this represents the colonisation of Australia by polyploids that have subsequently undergone a recent radiation into new environments. To our knowledge, this study is the first report of a substantial lag phase being investigated below the family level

“F*ck it! Let’s get to drinking – poison our livers!”: a thematic analysis of alcohol content in contemporary YouTube music videos

Purpose: To describe the portrayal of alcohol content in popular YouTube music videos. Methods: We used inductive thematic analysis to explore the lyrics and visual imagery in 49 UK Top 40 songs and music videos previously found to contain alcohol content, and watched by many British adolescents aged between 11-18 years, and to examine if branded content contravened alcohol industry advertising codes of practice. Results: The analysis generated three themes. First, alcohol content was associated with sexualised imagery or lyrics and the objectification of women. Second, alcohol was associated with image, lifestyle and sociability. Finally, some videos showed alcohol overtly encouraging excessive drinking and drunkenness, including those containing branding, with no negative consequences to the drinker. Conclusion: Our results suggest that YouTube music videos promote positive associations with alcohol use. Further, several alcohol companies adopt marketing strategies in the video medium that are entirely inconsistent with their own or others agreed advertising codes of practice. We conclude that, as a harm reduction measure, policies should change to prevent adolescent exposure to the positive promotion of alcohol and alcohol branding in music videos

CONFIRM: a double-blind, placebo controlled phase III clinical trial investigating the effect of nivolumab in patients with relapsed mesothelioma: study protocol for a randomised controlled trial

Background: Mesothelioma is an incurable, apoptosis-resistant cancer caused in most cases by previous exposure to asbestos and is increasing in incidence. It represents a growing health burden but remains under-researched, with limited treatment options. Early promising signals of activity relating to both PD-L1- and PD-1-targeted treatment in mesothelioma implicate a dependency of mesothelioma on this immune checkpoint. There is a need to evaluate checkpoint inhibitors in patients with relapsed mesothelioma where treatment options are limited. Methods: The addition of 12 months of nivolumab (anti-PD1 antibody) to standard practice will be conducted in the UK using a randomised, placebo-controlled phase III trial (the Cancer Research UK CONFIRM trial). A total of 336 patients with pleural or peritoneal mesothelioma who have received at least two prior lines of therapy will be recruited from UK secondary care sites. Patients will be randomised 2:1 (nivolumab:placebo), stratified according to epithelioid/non-epithelioid, to receive either 240 mg nivolumab monotherapy or saline placebo as a 30-min intravenous infusion. Treatment will be for up to 12 months. We will determine whether the use of nivolumab increases overall survival (the primary efficacy endpoint). Secondary endpoints will include progression-free survival, objective response rate, toxicity, quality of life and cost-effectiveness. Analysis will be performed according to the intention-to-treat principle using a Cox regression analysis for the primary endpoint (and for other time-to-event endpoints). Discussion: The outcome of this trial will provide evidence of the potential benefit of the use of nivolumab in the treatment of relapsed mesothelioma. If found to be clinically effective, safe and cost-effective it is likely to become the new standard of care in the UK

Additive and multiplicative hazards modeling for recurrent event data analysis

<p>Abstract</p> <p>Background</p> <p>Sequentially ordered multivariate failure time or recurrent event duration data are commonly observed in biomedical longitudinal studies. In general, standard hazard regression methods cannot be applied because of correlation between recurrent failure times within a subject and induced dependent censoring. Multiplicative and additive hazards models provide the two principal frameworks for studying the association between risk factors and recurrent event durations for the analysis of multivariate failure time data.</p> <p>Methods</p> <p>Using emergency department visits data, we illustrated and compared the additive and multiplicative hazards models for analysis of recurrent event durations under (i) a varying baseline with a common coefficient effect and (ii) a varying baseline with an order-specific coefficient effect.</p> <p>Results</p> <p>The analysis showed that both additive and multiplicative hazards models, with varying baseline and common coefficient effects, gave similar results with regard to covariates selected to remain in the model of our real dataset. The confidence intervals of the multiplicative hazards model were wider than the additive hazards model for each of the recurrent events. In addition, in both models, the confidence interval gets wider as the revisit order increased because the risk set decreased as the order of visit increased.</p> <p>Conclusions</p> <p>Due to the frequency of multiple failure times or recurrent event duration data in clinical and epidemiologic studies, the multiplicative and additive hazards models are widely applicable and present different information. Hence, it seems desirable to use them, not as alternatives to each other, but together as complementary methods, to provide a more comprehensive understanding of data.</p

Detecting and staging podoconiosis cases in North West Cameroon: positive predictive value of clinical screening of patients by community health workers and researchers

Background The suitability of using clinical assessment to identify patients with podoconiosis in endemic communities has previously been demonstrated. In this study, we explored the feasibility and accuracy of using Community Health Implementers (CHIs) for the large scale clinical screening of the population for podoconiosis in North-west Cameroon. Methods Before a regional podoconiosis mapping, 193 CHIs and 50 health personnel selected from 6 health districts were trained in the clinical diagnosis of the disease. After training, CHIs undertook community screening for podoconiosis patients under health personnel supervision. Identified cases were later re-examined by a research team with experience in the clinical identification of podoconiosis. Results Cases were identified by CHIs with an overall positive predictive value (PPV) of 48.5% [34.1–70%]. They were more accurate in detecting advanced stages of the disease compared to early stages; OR 2.07, 95% CI = 1.15–3.73, p = 0.015 for all advanced stages). Accuracy of detecting cases showed statistically significant differences among health districts (χ2 = 25.30, p = 0.0001). Conclusion Podoconiosis being a stigmatized disease, the use of CHIs who are familiar to the community appears appropriate for identifying cases through clinical diagnosis. However, to improve their effectiveness and accuracy, more training, supervision and support are required. More emphasis must be given in identifying early clinical stages and in health districts with relatively lower PPVs