ICNMM2008-62014 Microchannel Fluorescent Melting Curve Analysis

ABSTRACT I

Effect of substrate thermal resistance on space-domain microchannel

In recent years, Fluorescent Melting Curve Analysis (FMCA) has become an almost ubiquitous feature of commercial quantitative PCR (qPCR) thermal cyclers. Here a micro-fluidic device is presented capable of performing FMCA within a microchannel. The device consists of modular thermally conductive blocks which can sandwich a microfluidic substrate. Opposing ends of the blocks are held at differing temperatures and a linear thermal gradient is generated along the microfluidic channel. Fluorescent measurements taken from a sample as it passes along the micro-fluidic channel permits fluorescent melting curves to be generated. In this study we measure DNA melting temperature from two plasmid fragments. The effects of flow velocity and ramp-rate are investigated, and measured melting curves are compared to those acquired from a commercially available PCR thermocycler

Smoking in film in New Zealand: measuring risk exposure

BACKGROUND: Smoking in film is a risk factor for smoking uptake in adolescence. This study aimed to quantify exposure to smoking in film received by New Zealand audiences, and evaluate potential interventions to reduce the quantity and impact of this exposure. METHODS: The ten highest-grossing films in New Zealand for 2003 were each analysed independently by two viewers for smoking, smoking references and related imagery. Potential interventions were explored by reviewing relevant New Zealand legislation, and scientific literature. RESULTS: Seven of the ten films contained at least one tobacco reference, similar to larger film samples. The majority of the 38 tobacco references involved characters smoking, most of whom were male. Smoking was associated with positive character traits, notably rebellion (which may appeal to adolescents). There appeared to be a low threshold for including smoking in film. Legislative or censorship approaches to smoking in film are currently unlikely to succeed. Anti-smoking advertising before films has promise, but experimental research is required to demonstrate cost effectiveness. CONCLUSION: Smoking in film warrants concern from public health advocates. In New Zealand, pre-film anti-smoking advertising appears to be the most promising immediate policy response

First Isolation of Hepatitis E Virus Genotype 4 in Europe through Swine Surveillance in the Netherlands and Belgium

Hepatitis E virus (HEV) genotypes 3 and 4 are a cause of human hepatitis and swine are considered the main reservoir. To study the HEV prevalence and characterize circulating HEV strains, fecal samples from swine in the Netherlands and Belgium were tested by RT-PCR. HEV prevalence in swine was 7–15%. The Dutch strains were characterized as genotype 3, subgroups 3a, 3c and 3f, closely related to sequences found in humans and swine earlier. The HEV strains found in Belgium belonged to genotypes 3f and 4b. The HEV genotype 4 strain was the first ever reported in swine in Europe and an experimental infection in pigs was performed to isolate the virus. The genotype 4 strain readily infected piglets and caused fever and virus shedding. Since HEV4 infections have been reported to run a more severe clinical course in humans this observation may have public health implications

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

A Real-Time Continuous Flow Polymerase Chain Reactor for DNA Expression Quantification

ABSTRACT Real-time quantitative Polymerase Chain Reaction (PCR) is an extremely sensitive and reliable method for quantifying gene expression, allowing subtle shifts in gene expression to be easily monitored. Currently, stationary real-time PCR is readily achieved using fluorescent labels which increase in fluorescence as the DNA is exponentially amplified. Quantitative PCR is used in a myriad of applications. However currently most commercial real-time PCR devices are batch process stationary well based systems, limiting their throughput. Continuous flow microfluidic PCR devices have allowed for advancement in terms of improved PCR throughput and reduced reagent usage. As part of an overall total analysis system a device integrating all the functional steps of continuous flow realtime quantitative PCR has been designed and fabricated. Initially the PCR reaction mixture is segmented into nanolitre PCR reactors which are then thermally cycled on a two temperature fifty cycle flow-through PCR device, which allows laser induced fluorescent imaging of the nanoreactors. Previous studies into continuous flow PCR have demonstrated endpoint fluorescent measurements, however this research allows PCR nanoreactors to be fluorescently monitored after every PCR thermal cycle. Fluorescent optical monitoring is achieved through laser excitation of the nanoreactors while a Charged Coupled Device (CCD) camera is used to record the fluorescent emissions from the nanoreactors. Intensity analysis of the recorded images is then preformed using MATLAB to accurately determine the fluorescence intensity level, thereby allowing real-time quantitative amplification curves to be generated. This has major advantages over existing continuous flow PCR devices which use endpoint fluorescence and capillary electrophoresis, as the amplification curves allow far more information to be gleaned and allow the initial DNA template concentration to be accurately determined

Rate of Force Development as a Predictor of Mobility in Community-dwelling Older Adults

BACKGROUND AND PURPOSE: Rate of force development (RFD) is influential, and possibly more influential than other muscular performance parameters, for mobility in older adults. However, only a few studies have investigated this matter, and this has not been examined for the plantar flexors (PFs). The purpose of this study was to examine the contribution of PF RFD and other common tests of muscular performance to Up-and-Go (UG) performance and walking speed (WS) in older adults. METHODS: Twenty-six (19 females) healthy, community-dwelling older adults (73.7 ± 4.9 years) were recruited from a senior citizen center for this observational study. Handgrip strength, UG performance, as well as preferred and maximal WS were obtained. Time taken to complete 5-chair rises and the number of chair rises completed in 30 seconds were recorded. Rate of force development of the PFs was obtained during a rapid, bilateral calf raise performed on a force plate. Hierarchical multiple linear regression was used to identify significant predictors, after adjusting for physical activity level and body mass index, of mobility (ie, UG, preferred and maximal WS). RESULTS AND DISCUSSION: No muscular performance variables correlated with preferred WS. Rate of force development (adjusted R2 = 0.356; P = .008) and handgrip strength (adjusted R2 = 0.293; P = .026) were the only predictors of maximal WS and accounted for a 21.7% and 16.1% change in R2, respectively, after accounting for physical activity level and body mass index. Rate of force development was the only predictor of UG performance (adjusted R2 = 0.212; P = .006) and accounted for a 29.2% change in R2 after adjustment variables were applied. CONCLUSIONS: Compared to common assessments of muscular performance, such as handgrip strength and chair rise performance, PF RFD was a greater predictor of mobility in older adults. These findings, in conjunction with recent reports, indicate that the assessment of RFD likely complements strength testing, thereby enabling a more robust assessment of functional decline in older adults

Protein kinase C beta II suppresses colorectal cancer by regulating IGF-1 mediated cell survival.

Despite extensive efforts, cancer therapies directed at the Protein Kinase C (PKC) family of serine/threonine kinases have failed in clinical trials. These therapies have been directed at inhibiting PKC and have, in some cases, worsened disease outcome. Here we examine colon cancer patients and show not only that PKC Beta II is a tumour suppressor, but patients with low levels of this isozyme have significantly decreased disease free survival. Specifically, analysis of gene expression levels of all PKC genes in matched normal and cancer tissue samples from colon cancer patients revealed a striking down-regulation of the gene coding PKC Beta in the cancer tissue (n = 21). Tissue microarray analysis revealed a dramatic down-regulation of PKC Beta II protein levels in both the epithelial and stromal diseased tissue (n = 166). Of clinical significance, low levels of the protein in the normal tissue of patients is associated with a low (10%) 10 year survival compared with a much higher (60%) survival in patients with relatively high levels of the protein. Consistent with PKC Beta II levels protecting against colon cancer, overexpression of PKC Beta II in colon cancer cell lines reveals that PKC Beta II reverses transformation in cell based assays. Further to this, activation of PKC Beta II results in a dramatic downregulation of IGF-I-induced AKT, indicating a role for PKCs in regulating IGF-1 mediated cell survival. Thus, PKC Beta II is a tumour suppressor in colon cancer and low levels serve as a predictor for poor survival outcome

Extracellular matrix gene expression profiling using microfluidics for colorectal carcinoma stratification

In cancer, biomarkers have many potential applications including generation of a differential diagnosis, prediction of response to treatment, and monitoring disease progression. Many molecular biomarkers have been put forward for different diseases but most of them do not possess the required specificity and sensitivity. A biomarker with a high sensitivity has a low specificity and vice versa. The inaccuracy of the biomarkers currently in use has led to a compelling need to identify more accurate markers with diagnostic and prognostic significance. The aim of the present study was to use a novel, droplet-based, microfluidic platform to evaluate the prognostic value of a panel of thirty-four genes that regulate the composition of extracellular matrices in colorectal carcinoma. Our method is a novel approach as it uses using continuous-flowing Polymerase Chain Reaction for the sensitive detection and accurate quantitation of gene expression. We identified a panel of relevant extracellular matrix genes whose expression levels were measured by real-time quantitative polymerase chain reaction using Taqman VR reagents in twenty-four pairs of matched colorectal cancer tumour and associated normal tissue. Differential expression patterns occurred between the normal and malignant tissue and correlated with histopathological parameters and overall surgical staging. The findings demonstrate that a droplet-based microfluidic quantitative PCR system enables biomarker classification. It was further possible to sub-classify colorectal cancer based on extracellular matrix protein expressing groups which in turn correlated with prognosis. (C) 2016 Author(s)