Human malaria diagnosis using a single-step direct-PCR based on the Plasmodium cytochrome oxidase III gene

Background: Nested PCRs based on the Plasmodium 18s-rRNA gene have been extensively used for human malaria diagnosis. However, they are not practical when large quantities of samples need to be processed, further there have been challenges in the performance and when interpreting results, especially when submicroscopic infections are analysed. Here the use of "direct PCR" was investigated with the aim of improving diagnosis in the malaria elimination era.\ud \ud Methods: The performance of the Plasmodium cytochrome oxidase III gene (COX-III) based novel malaria detection strategies (direct nested PCR and direct single PCR) were compared using a 18s-rRNA direct nested PCR as a reference tool. Evaluations were based on sensitivity, specificity and the ability to detect mixed infections using control blood spot samples and field collected blood samples with final species diagnosis confirmation by sequencing.\ud \ud Results: The COX-III direct PCR (limit of detection: 0.6–2 parasites/μL) was more sensitive than the 18s-rRNA direct nested PCR (limit of detection: 2–10 parasites/μL). The COX-III direct PCR identified all 21 positive controls (no mixed infections detected) while the 18s-rRNA direct nested PCR identified 18/21 (including four mixed infections). Different concentrations of simulated mixed infections (Plasmodium vivax and Plasmodium falciparum) suggest that the COX-III direct PCR detects only the predominant species. When the 18s-rRNA direct nested PCR was used to detect Plasmodium in field collected bloods spots (n = 3833), there was discrepancy in the results from the genus PCR (16 % positive) and the species-specific PCR (5 % positive). Further, a large portion of a subset of these positive samples (93 % for genus and 60 % for P. vivax), did not align with Plasmodium sequences. In contrast, the COX-III direct PCR clearly identified (single bands confirmed with sequencing) 2 % positive Plasmodium samples including P. vivax, P. falciparum, Plasmodium malariae and Plasmodium ovale wallikeri.\ud \ud Conclusions: The COX-III single direct PCR is an alternative method for accurate detection of Plasmodium microscopic and submicroscopic infections in humans, especially when a large number of samples require screening. This PCR does not require DNA isolation, is sensitive, quick, produces confident/clear results, identifies all the Plasmodium species infecting humans, and is cost-effective.\u

Industrial constructions of publics and public knowledge: a qualitative investigation of practice in the UK chemicals industry

This is a post print version of the article. The official published version can be obtained from the link below - © 2007 by SAGE PublicationsWhile the rhetoric of public engagement is increasingly commonplace within industry, there has been little research that examines how lay knowledge is conceptualized and whether it is really used within companies. Using the chemicals sector as an example, this paper explores how companies conceive of publics and "public knowledge," and how this relates to modes of engagement/communication with them. Drawing on qualitative empirical research in four companies, we demonstrate that the public for industry are primarily conceived as "consumers" and "neighbours," having concerns that should be allayed rather than as groups with knowledge meriting engagement. We conclude by highlighting the dissonance between current advocacy of engagement and the discourses and practices prevalent within industry, and highlight the need for more realistic strategies for industry/public engagement.Funding was received from the ESRC Science in Society Programme

Molecular Characterization Reveals Diverse and Unknown Malaria Vectors in the Western Kenyan Highlands.

The success of mosquito-based malaria control is dependent upon susceptible bionomic traits in local malaria vectors. It is crucial to have accurate and reliable methods to determine mosquito species composition in areas subject to malaria. An unexpectedly diverse set of Anopheles species was collected in the western Kenyan highlands, including unidentified and potentially new species carrying the malaria parasite Plasmodium falciparum. This study identified 2,340 anopheline specimens using both ribosomal DNA internal transcribed spacer region 2 and mitochondrial DNA cytochrome oxidase subunit 1 loci. Seventeen distinct sequence groups were identified. Of these, only eight could be molecularly identified through comparison to published and voucher sequences. Of the unidentified species, four were found to carry P. falciparum by circumsporozoite enzyme-linked immunosorbent assay and polymerase chain reaction, the most abundant of which had infection rates comparable to a primary vector in the area, Anopheles funestus. High-quality adult specimens of these unidentified species could not be matched to museum voucher specimens or conclusively identified using multiple keys, suggesting that they may have not been previously described. These unidentified vectors were captured outdoors. Diverse and unknown species have been incriminated in malaria transmission in the western Kenya highlands using molecular identification of unusual morphological variants of field specimens. This study demonstrates the value of using molecular methods to compliment vector identifications and highlights the need for accurate characterization of mosquito species and their associated behaviors for effective malaria control

Peer-led walking programme to increase physical activity in inactive 60- to 70-year-olds: Walk with Me pilot RCT

Background Levels of physical activity decline with age. Some of the most disadvantaged individuals in society, such as those with a lower rather than a higher socioeconomic position, are also the most inactive. Peer-led physical activity interventions may offer a model to increase physical activity in these older adults and thus help reduce associated health inequalities. This study aims to develop and test the feasibility of a peer-led, multicomponent physical activity intervention in socioeconomically disadvantaged community-dwelling older adults. Objectives The study aimed to develop a peer-led intervention through a rapid review of previous peer-led interventions and interviews with members of the target population. A proposed protocol to evaluate its effectiveness was tested in a pilot randomised controlled trial (RCT). Design A rapid review of the literature and the pilot study informed the intervention design; a pilot RCT included a process evaluation of intervention delivery. Setting Socioeconomically disadvantaged communities in the South Eastern Health and Social Care Trust and the Northern Health and Social Care Trust in Northern Ireland. Participants Fifty adults aged 60–70 years, with low levels of physical activity, living in socioeconomically disadvantaged communities, recruited though community organisations and general practices. Interventions ‘Walk with Me’ is a 12-week peer-led walking intervention based on social cognitive theory. Participants met weekly with peer mentors. During the initial period (weeks 1–4), each intervention group participant wore a pedometer and set weekly step goals with their mentor’s support. During weeks 5–8 participants and mentors met regularly to walk and discuss step goals and barriers to increasing physical activity. In the final phase (weeks 9–12), participants and mentors continued to set step goals and planned activities to maintain their activity levels beyond the intervention period. The control group received only an information booklet on active ageing. Main outcome measures Rates of recruitment, retention of participants and completeness of the primary outcome [moderate- and vigorous-intensity physical activity measured using an ActiGraph GT3X+ accelerometer (ActiGraph, LLC, Pensacola, FL, USA) at baseline, 12 weeks (post intervention) and 6 months]; acceptability assessed through interviews with participants and mentors. Results The study planned to recruit 60 participants. In fact, 50 eligible individuals participated, of whom 66% (33/50) were female and 80% (40/50) were recruited from general practices. At 6 months, 86% (43/50) attended for review, 93% (40/43) of whom returned valid accelerometer data. Intervention fidelity was assessed by using weekly step diaries, which were completed by both mentors and participants for all 12 weeks, and checklists for the level of delivery of intervention components, which was high for the first 3 weeks (range 49–83%). However, the rate of return of checklists by both mentors and participants diminished thereafter. Outcome data indicate that a sample size of 214 is required for a definitive trial. Limitations The sample was predominantly female and somewhat active. Conclusions The ‘Walk with Me’ intervention is acceptable to a socioeconomically disadvantaged community of older adults and a definitive RCT to evaluate its effectiveness is feasible. Some modifications are required to ensure fidelity of intervention delivery is optimised. Future research needs to identify methods to recruit males and less active older adults into physical activity interventions

A Discrete Time Model for the Analysis of Medium-Throughput C. elegans Growth Data

BACKGROUND: As part of a program to predict the toxicity of environmental agents on human health using alternative methods, several in vivo high- and medium-throughput assays are being developed that use C. elegans as a model organism. C. elegans-based toxicological assays utilize the COPAS Biosort flow sorting system that can rapidly measure size, extinction (EXT) and time-of-flight (TOF), of individual nematodes. The use of this technology requires the development of mathematical and statistical tools to properly analyze the large volumes of biological data. METHODOLOGY/PRINCIPAL FINDINGS: Findings A Markov model was developed that predicts the growth of populations of C. elegans. The model was developed using observations from a 60 h growth study in which five cohorts of 300 nematodes each were aspirated and measured every 12 h. Frequency distributions of log(EXT) measurements that were made when loading C. elegans L1 larvae into 96 well plates (t = 0 h) were used by the model to predict the frequency distributions of the same set of nematodes when measured at 12 h intervals. The model prediction coincided well with the biological observations confirming the validity of the model. The model was also applied to log(TOF) measurements following an adaptation. The adaptation accounted for variability in TOF measurements associated with potential curling or shortening of the nematodes as they passed through the flow cell of the Biosort. By providing accurate estimates of frequencies of EXT or TOF measurements following varying growth periods, the model was able to estimate growth rates. Best model fits showed that C. elegans did not grow at a constant exponential rate. Growth was best described with three different rates. Microscopic observations indicated that the points where the growth rates changed corresponded to specific developmental events: the L1/L2 molt and the start of oogenesis in young adult C. elegans. CONCLUSIONS: Quantitative analysis of COPAS Biosort measurements of C. elegans growth has been hampered by the lack of a mathematical model. In addition, extraneous matter and the inability to assign specific measurements to specific nematodes made it difficult to estimate growth rates. The present model addresses these problems through a population-based Markov model

Ex vivo drug response profiling detects recurrent sensitivity patterns in drug-resistant acute lymphoblastic leukemia

Drug sensitivity and resistance testing on diagnostic leukemia samples should provide important functional information to guide actionable target and biomarker discovery. We provide proof of concept data by profiling 60 drugs on 68 acute lymphoblastic leukemia (ALL) samples mostly from resistant disease in cocultures of bone marrow stromal cells. Patient-derived xenografts retained the original pattern of mutations found in the matched patient material. Stromal coculture did not prevent leukemia cell cycle activity, but a specific sensitivity profile to cell cycle-related drugs identified samples with higher cell proliferation both in vitro and in vivo as leukemia xenografts. In patients with refractory relapses, individual patterns of marked drug resistance and exceptional responses to new agents of immediate clinical relevance were detected. The BCL2inhibitor venetoclax was highly active below 10 nM in B-cell precursor ALL (BCP-ALL) subsets, including MLL-AF4 and TCF3-HLF ALL, and in some T-cell ALLs (T-ALLs), predicting in vivo activity as a single agent and in combination with dexamethasone and vincristine. Unexpected sensitivity to dasatinib with half maximal inhibitory concentration values below 20 nM was detected in 2 independent T-ALL cohorts, which correlated with similar cytotoxic activity of the SRC inhibitor KX2-391 and inhibition of SRC phosphorylation. A patient with refractory T-ALL was treated with dasatinib on the basis of drug profiling information and achieved a 5-month remission. Thus, drug profiling captures disease-relevant features and unexpected sensitivity to relevant drugs, which warrants further exploration of this functional assay in the context of clinical trials to develop drug repurposing strategies for patients with urgent medical needs.Peer reviewe

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Application of a Mathematical Model to Describe the Effects of Chlorpyrifos on Caenorhabditis elegans Development

The nematode Caenorhabditis elegans is being assessed as an alternative model organism as part of an interagency effort to develop better means to test potentially toxic substances. As part of this effort, assays that use the COPAS Biosort flow sorting technology to record optical measurements (time of flight (TOF) and extinction (EXT)) of individual nematodes under various chemical exposure conditions are being developed. A mathematical model has been created that uses Biosort data to quantitatively and qualitatively describe C. elegans growth, and link changes in growth rates to biological events. Chlorpyrifos, an organophosphate pesticide known to cause developmental delays and malformations in mammals, was used as a model toxicant to test the applicability of the growth model for in vivo toxicological testing.L1 larval nematodes were exposed to a range of sub-lethal chlorpyrifos concentrations (0-75 microM) and measured every 12 h. In the absence of toxicant, C. elegans matured from L1s to gravid adults by 60 h. A mathematical model was used to estimate nematode size distributions at various times. Mathematical modeling of the distributions allowed the number of measured nematodes and log(EXT) and log(TOF) growth rates to be estimated. The model revealed three distinct growth phases. The points at which estimated growth rates changed (change points) were constant across the ten chlorpyrifos concentrations. Concentration response curves with respect to several model-estimated quantities (numbers of measured nematodes, mean log(TOF) and log(EXT), growth rates, and time to reach change points) showed a significant decrease in C. elegans growth with increasing chlorpyrifos concentration.Effects of chlorpyrifos on C. elegans growth and development were mathematically modeled. Statistical tests confirmed a significant concentration effect on several model endpoints. This confirmed that chlorpyrifos affects C. elegans development in a concentration dependent manner. The most noticeable effect on growth occurred during early larval stages: L2 and L3. This study supports the utility of the C. elegans growth assay and mathematical modeling in determining the effects of potentially toxic substances in an alternative model organism using high-throughput technologies