Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error

The use of thermographic imaging to evaluate therapeutic response in human tumour xenograft models

YesNon-invasive methods to monitor tumour growth are an important goal in cancer drug development. Thermographic imaging systems offer potential in this area, since a change in temperature is known to be induced due to changes within the tumour microenvironment. This study demonstrates that this imaging modality can be applied to a broad range of tumour xenografts and also, for the first time, the methodology’s suitability to assess anti-cancer agent efficacy. Mice bearing subcutaneously implanted H460 lung cancer xenografts were treated with a novel vascular disrupting agent, ICT-2552, and the cytotoxin doxorubicin. The effects on tumour temperature were assessed using thermographic imaging over the first 6 hours post-administration and subsequently a further 7 days. For ICT-2552 a significant initial temperature drop was observed, whilst for both agents a significant temperature drop was seen compared to controls over the longer time period. Thus thermographic imaging can detect functional differences (manifesting as temperature reductions) in the tumour response to these anti-cancer agents compared to controls. Importantly, these effects can be detected in the first few hours following treatment and therefore the tumour is observable non-invasively. As discussed, this technique will have considerable 3Rs benefits in terms of reduction and refinement of animal use.University of Bradfor

Fitness consultations in routine care of patients with type 2 diabetes in general practice: an 18-month non-randomised intervention study

<p>Abstract</p> <p>Background</p> <p>Increasing physical activity is a cornerstone in the treatment of type 2 diabetes and in general practice it is a challenge to achieve long-term adherence to this life style change. The aim of this study was to investigate in a non-randomised design whether the introduction of motivational interviewing combined with fitness tests in the type 2 diabetes care programme was followed by a change in cardio-respiratory fitness expressed by VO_2max, muscle strength of upper and lower extremities, haemoglobin A_1c(HbA_1c) and HDL-cholesterol.</p> <p>Methods</p> <p>Uncontrolled 18-month intervention study with follow-up and effect assessment every 3 months in a primary care unit in Denmark with six general practitioners (GPs). Of 354 eligible patients with type 2 diabetes, 127 (35.9%) were included. Maximum work capacity was tested on a cycle ergometer and converted to VO_2max. Muscle strength was measured with an arm curl test and a chair stand test. The results were used in a subsequent motivational interview conducted by one of the GPs. Patients were encouraged to engage in lifestyle exercise and simple home-based self-managed exercise programmes. Data were analysed with mixed models.</p> <p>Results</p> <p>At end of study, 102 (80.3%) participants remained in the intervention. Over 18 months, VO_2maxincreased 2.5% (p = 0.032) while increases of 33.2% (p < 0.001) and 34.1% (p < 0.001) were registered for the arm curl test and chair stand test, respectively. HDL-cholesterol increased 8.6% (p < 0.001), but HbA_1cremained unchanged (p = 0.57) on a low level (6.8%). Patients without cardiovascular disease or pain from function limitation increased their VO_2maxby 5.2% (p < 0.0001) and 7.9% (p = 0.0008), respectively.</p> <p>Conclusions</p> <p>In this 18-month study, participants who had repeated fitness consultations, including physical testing and motivational interviewing to improve physical activity, improved VO_2max, muscle strength, and lipid profile. Our results indicate that physical testing combined with motivational interviewing is feasible in a primary health care setting. Here, a fitness consultation tailored to the individual patient, his/her comorbidities and conditions in the local area can be incorporated into the diabetes programme to improve patients' muscle strength and cardio-respiratory fitness.</p

An investigation of factors associated with the health and well-being of HIV-infected or HIV-affected older people in rural South Africa

BackgroundDespite the severe impact of HIV in sub-Saharan Africa, the health of older people aged 50+ is often overlooked owing to the dearth of data on the direct and indirect effects of HIV on older people's health status and well-being. The aim of this study was to examine correlates of health and well-being of HIV-infected older people relative to HIV-affected people in rural South Africa, defined as participants with an HIV-infected or death of an adult child due to HIV-related cause. MethodsData were collected within the Africa Centre surveillance area using instruments adapted from the World Health Organization (WHO) Study on global AGEing and adult health (SAGE). A stratified random sample of 422 people aged 50+ participated. We compared the health correlates of HIV-infected to HIV-affected participants using ordered logistic regressions. Health status was measured using three instruments: disability index, quality of life and composite health score. ResultsMedian age of the sample was 60 years (range 50-94). Women HIV-infected (aOR 0.15, 95% confidence interval (CI) 0.08-0.29) and HIV-affected (aOR 0.20, 95% CI 0.08-0.50), were significantly less likely than men to be in good functional ability. Women's adjusted odds of being in good overall health state were similarly lower than men's; while income and household wealth status were stronger correlates of quality of life. HIV-infected participants reported better functional ability, quality of life and overall health state than HIV-affected participants. Discussion and Conclusions The enhanced healthcare received as part of anti-retroviral treatment as well as the considerable resources devoted to HIV care appear to benefit the overall well-being of HIV-infected older people; whereas similar resources have not been devoted to the general health needs of HIV uninfected older people. Given increasing numbers of older people, policy and programme interventions are urgently needed to holistically meet the health and well-being needs of older people beyond the HIV-related care system. <br/

CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases

Beyond Gross-Pitaevskii Mean Field Theory

A large number of effects related to the phenomenon of Bose-Einstein Condensation (BEC) can be understood in terms of lowest order mean field theory, whereby the entire system is assumed to be condensed, with thermal and quantum fluctuations completely ignored. Such a treatment leads to the Gross-Pitaevskii Equation (GPE) used extensively throughout this book. Although this theory works remarkably well for a broad range of experimental parameters, a more complete treatment is required for understanding various experiments, including experiments with solitons and vortices. Such treatments should include the dynamical coupling of the condensate to the thermal cloud, the effect of dimensionality, the role of quantum fluctuations, and should also describe the critical regime, including the process of condensate formation. The aim of this Chapter is to give a brief but insightful overview of various recent theories, which extend beyond the GPE. To keep the discussion brief, only the main notions and conclusions will be presented. This Chapter generalizes the presentation of Chapter 1, by explicitly maintaining fluctuations around the condensate order parameter. While the theoretical arguments outlined here are generic, the emphasis is on approaches suitable for describing single weakly-interacting atomic Bose gases in harmonic traps. Interesting effects arising when condensates are trapped in double-well potentials and optical lattices, as well as the cases of spinor condensates, and atomic-molecular coupling, along with the modified or alternative theories needed to describe them, will not be covered here.Comment: Review Article (19 Pages) - To appear in 'Emergent Nonlinear Phenomena in Bose-Einstein Condensates: Theory and Experiment', Edited by P.G. Kevrekidis, D.J. Frantzeskakis and R. Carretero-Gonzalez (Springer Verlag

Why Do Species Co-Occur? A Test of Alternative Hypotheses Describing Abiotic Differences in Sympatry versus Allopatry Using Spadefoot Toads

Areas of co-occurrence between two species (sympatry) are often thought to arise in regions where abiotic conditions are conducive to both species and are therefore intermediate between regions where either species occurs alone (allopatry). Depending on historical factors or interactions between species, however, sympatry might not differ from allopatry, or, alternatively, sympatry might actually be more extreme in abiotic conditions relative to allopatry. Here, we evaluate these three hypothesized patterns for how sympatry compares to allopatry in abiotic conditions. We use two species of congeneric spadefoot toads, Spea multiplicata and S. bombifrons, as our study system. To test these hypotheses, we created ecological niche models (specifically using Maxent) for both species to create a map of the joint probability of occurrence of both species. Using the results of these models, we identified three types of locations: two where either species was predicted to occur alone (i.e., allopatry for S. multiplicata and allopatry for S. bombifrons) and one where both species were predicted to co-occur (i.e., sympatry). We then compared the abiotic environment between these three location types and found that sympatry was significantly hotter and drier than the allopatric regions. Thus, sympatry was not intermediate between the alternative allopatric sites. Instead, sympatry occurred at one extreme of the conditions occupied by both species. We hypothesize that biotic interactions in these extreme environments facilitate co-occurrence. Specifically, hybridization between S. bombifrons females and S. multiplicata males may facilitate co-occurrence by decreasing development time of tadpoles. Additionally, the presence of alternative food resources in more extreme conditions may preclude competitive exclusion of one species by the other. This work has implications for predicting how interacting species will respond to climate change, because species interactions may facilitate survival in extreme habitats

Microenvironmental Influence on Pre-Clinical Activity of Polo-Like Kinase Inhibition in Multiple Myeloma: Implications for Clinical Translation

Polo-like kinases (PLKs) play an important role in cell cycle progression, checkpoint control and mitosis. The high mitotic index and chromosomal instability of advanced cancers suggest that PLK inhibitors may be an attractive therapeutic option for presently incurable advanced neoplasias with systemic involvement, such as multiple myeloma (MM). We studied the PLK 1, 2, 3 inhibitor BI 2536 and observed potent (IC50<40 nM) and rapid (commitment to cell death <24 hrs) in vitro activity against MM cells in isolation, as well as in vivo activity against a traditional subcutaneous xenograft mouse model. Tumor cells in MM patients, however, don't exist in isolation, but reside in and interact with the bone microenvironment. Therefore conventional in vitro and in vivo preclinical assays don't take into account how interactions between MM cells and the bone microenvironment can potentially confer drug resistance. To probe this question, we performed tumor cell compartment-specific bioluminescence imaging assays to compare the preclinical anti-MM activity of BI 2536 in vitro in the presence vs. absence of stromal cells or osteoclasts. We observed that the presence of these bone marrow non-malignant cells led to decreased anti-MM activity of BI 2536. We further validated these results in an orthotopic in vivo mouse model of diffuse MM bone lesions where tumor cells interact with non-malignant cells of the bone microenvironment. We again observed that BI 2536 had decreased activity in this in vivo model of tumor-bone microenvironment interactions highlighting that, despite BI 2536's promising activity in conventional assays, its lack of activity in microenvironmental models raises concerns for its clinical development for MM. More broadly, preclinical drug testing in the absence of relevant tumor microenvironment interactions may overestimate potential clinical activity, thus explaining at least in part the gap between preclinical vs. clinical efficacy in MM and other cancers

Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer

Background: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis