Droplet digital PCR as a useful tool for the quantitative detection of Enterovirus 71

Hand, foot and mouth disease (HFMD) is a contagious viral disease that frequently affects infants and children and present with blisters and flu-like symptoms. This disease is caused by a group of enteroviruses such as enterovirus 71 (EV71) and coxsackievirus A16 (CA16). However, unlike other HFMD causing enteroviruses, EV71 have also been shown to be associated with more severe clinical manifestation such as aseptic meningitis, brainstem and cerebellar encephalitis which may lead to cardiopulmonary failure and death. Clinically, HFMD caused by EV71 is indistinguishable from other HFMD causing enteroviruses such as CA16. Molecular diagnosis methods such as the use of real-time PCR has been used commonly for the identification of EV71. In this study, two platforms namely the real-time PCR and the droplet digital PCR were compared for the detection quantitation of known EV71 viral copy number. The results reveal accurate and consistent results between the two platforms. In summary, the droplet digital PCR was demonstrated to be a promising technology for the identification and quantitation of EV71 viral copy number.

A rapid and label-free dual detection of Hg (II) and cysteine with the use of fluorescence switching of graphene quantum dots

A simple and rapid method of analysis for mercury ions (Hg2+) and cysteine (Cys) was developed with the use of graphene quantum dots (GQDs) as a fluorescent probe. In the presence of GQDs, Hg2+ cations are absorbed on their negatively charged surface by means of electrostatic interactions. Thus, the fluorescence (FL) of the GQDs would be significantly quenched as a result of the FL charge transfer, e.g. 92% quenching at 450 nm occurs for a 5 μmol L−1 Hg2+ solution. However, when Cys was added, a significant FL enhancement was observed (510% at 450 nm for a 8.0 μmol L−1 Cys solution), and Hg2+ combined with Cys rather than with the GQDs in an aqueous solution. This occurred because a strong metalsingle bondthiol bond formed, displacing the weak electrostatic interactions, and this resulted in an FL enhancement of the GQDs. The limits of detection (LOD) for Hg2+ and Cys were 0.439 nmol L−1 and 4.5 nmol L−1, respectively. Also, this method was used successfully to analyze Hg2+ and Cys in spiked water samples.

Adjustment of apnea-hypopnea index with severity of obstruction events enhances detection of sleep apnea patients with the highest risk of severe health consequences

Introduction Presently, the severity of obstructive sleep apnea (OSA) is estimated based on the apnea-hypopnea index (AHI). Unfortunately, AHI does not provide information on the severity of individual obstruction events. Previously, the severity of individual obstruction events has been suggested to be related to the outcome of the disease. In this study, we incorporate this information into AHI and test whether this novel approach would aid in discriminating patients with the highest risk. We hypothesize that the introduced adjusted AHI parameter provides a valuable supplement to AHI in the diagnosis of the severity of OSA. Methods This hypothesis was tested by means of retrospective follow-up (mean ± sd follow-up time 198.2 ± 24.7 months) of 1,068 men originally referred to night polygraphy due to suspected OSA. After exclusion of the 264 patients using CPAP, the remaining 804 patients were divided into normal (AHI < 5) and OSA (AHI ≥ 5) categories based on conventional AHI and adjusted AHI. For a more detailed analysis, the patients were divided into normal, mild, moderate, and severe OSA categories based on conventional AHI and adjusted AHI. Subsequently, the mortality and cardiovascular morbidity in these groups were determined. Results Use of the severity of individual obstruction events for adjustment of AHI led to a significant rearrangement of patients between severity categories. Due to this rearrangement, the number of deceased patients diagnosed to have OSA was increased when adjusted AHI was used as the diagnostic index. Importantly, risk ratios of all-cause mortality and cardiovascular morbidity were higher in moderate and severe OSA groups formed based on the adjusted AHI parameter than in those formed based on conventional AHI. Conclusions The adjusted AHI parameter was found to give valuable supplementary information to AHI and to potentially improve the recognition of OSA patients with the highest risk of mortality or cardiovascular morbidity.

Dynamic, invivo, real-time detection of retinal oxidative status in a model of elevated intraocular pressure using a novel, reversibly responsive, profluorescent nitroxide probe

Changes to the redox status of biological systems have been implicated in the pathogenesis of a wide variety of disorders including cancer, Ischemia-reperfusion (I/R) injury and neurodegeneration. In times of metabolic stress e.g. ischaemia/reperfusion, reactive oxygen species (ROS) production overwhelms the intrinsic antioxidant capacity of the cell, damaging vital cellular components. The ability to quantify ROS changes in vivo, is therefore essential to understanding their biological role. Here we evaluate the suitability of a novel reversible profluorescent probe containing a redox-sensitive nitroxide moiety (methyl ester tetraethylrhodamine nitroxide, ME-TRN), as an in vivo, real-time reporter of retinal oxidative status. The reversible nature of the probe's response offers the unique advantage of being able to monitor redox changes in both oxidizing and reducing directions in real time. After intravitreal administration of the ME-TRN probe, we induced ROS production in rat retina using an established model of complete, acute retinal ischaemia followed by reperfusion. After restoration of blood flow, retinas were imaged using a Micron III rodent fundus fluorescence imaging system, to quantify the redox-response of the probe. Fluorescent intensity declined during the first 60 min of reperfusion. The ROS-induced change in probe fluorescence was ameliorated with the retinal antioxidant, lutein. Fluorescence intensity in non-Ischemia eyes did not change significantly. This new probe and imaging technology provide a reversible and real-time response to oxidative changes and may allow the in vivo testing of antioxidant therapies of potential benefit to a range of diseases linked to oxidative stress

Altered formalin-induced pain and Fos induction in the periaqueductal grey of preadolescent rats following neonatal LPS exposure

Animal and human studies have demonstrated that early pain experiences can produce alterations in the nociceptive systems later in life including increased sensitivity to mechanical, thermal, and chemical stimuli. However, less is known about the impact of neonatal immune challenge on future responses to noxious stimuli and the reactivity of neural substrates involved in analgesia. Here we demonstrate that rats exposed to Lipopolysaccharide (LPS; 0.05 mg/kg IP, Salmonella enteritidis) during postnatal day (PND) 3 and 5 displayed enhanced formalin-induced flinching but not licking following formalin injection at PND 22. This LPS-induced hyperalgesia was accompanied by distinct recruitment of supra-spinal regions involved in analgesia as indicated by significantly attenuated Fos-protein induction in the rostral dorsal periaqueductal grey (DPAG) as well as rostral and caudal axes of the ventrolateral PAG (VLPAG). Formalin injections were associated with increased Fos-protein labelling in lateral habenula (LHb) as compared to medial habenula (MHb), however the intensity of this labelling did not differ as a result of neonatal immune challenge. These data highlight the importance of neonatal immune priming in programming inflammatory pain sensitivity later in development and highlight the PAG as a possible mediator of this process

Automated categorisation of patent claims that reference human genome sequences

Debates on gene patents have necessitated the analysis of patents that disclose and reference human sequences. In this study, we built an automated classifier that assigns sequences to one of nine predefined categories according to their functional roles in patent claims by applying natural language processing and supervised learning techniques. To improve its correctness, we experimented with various feature mappings, resulting in the maximal accuracy of 79%.

Electrochemical detection of benzo(a)pyrene and related DNA damage using DNA/hemin/nafion-graphene biosensor

A novel electrochemical biosensor, DNA/hemin/nafion–graphene/GCE, was constructed for the analysis of the benzo(a)pyrene PAH, which can produce DNA damage induced by a benzo(a)pyrene (BaP) enzyme-catalytic product. This biosensor was assembled layer-by-layer, and was characterized with the use of cyclic voltammetry, electrochemical impedance spectroscopy (EIS) and atomic force microscopy. Ultimately, it was demonstrated that the hemin/nafion–graphene/GCE was a viable platform for the immobilization of DNA. This DNA biosensor was treated separately in benzo(a)pyrene, hydrogen peroxide (H2O2) and in their mixture, respectively, and differential pulse voltammetry (DPV) analysis showed that an oxidation peak was apparent after the electrode was immersed in H2O2. Such experiments indicated that in the presence of H2O2, hemin could mimic cytochrome P450 to metabolize benzo(a)pyrene, and a voltammogram of its metabolite was recorded. The DNA damage induced by this metabolite was also detected by electrochemical impedance and ultraviolet spectroscopy. Finally, a novel, indirect DPV analytical method for BaP in aqueous solution was developed based on the linear metabolite versus BaP concentration plot; this method provided a new, indirect, quantitative estimate of DNA damage.

Hot spot detection and spatio-temporal dynamics of dengue in Queensland, Australia

Dengue has been a major public health concern in Australia since it re-emerged in Queensland in 1992-1993. This study explored spatio-temporal distribution and clustering of locally-acquired dengue cases in Queensland State, Australia and identified target areas for effective interventions. A computerised locally-acquired dengue case dataset was collected from Queensland Health for Queensland from 1993 to 2012. Descriptive spatial and temporal analyses were conducted using geographic information system tools and geostatistical techniques. Dengue hot spots were detected using SatScan method. Descriptive spatial analysis showed that a total of 2,398 locally-acquired dengue cases were recorded in central and northern regions of tropical Queensland. A seasonal pattern was observed with most of the cases occurring in autumn. Spatial and temporal variation of dengue cases was observed in the geographic areas affected by dengue over time. Tropical areas are potential high-risk areas for mosquito-borne diseases such as dengue. This study demonstrated that the locally-acquired dengue cases have exhibited a spatial and temporal variation over the past twenty years in tropical Queensland, Australia. There is a clear evidence for the existence of statistically significant clusters of dengue and these clusters varied over time. These findings enabled us to detect and target dengue clusters suggesting that the use of geospatial information can assist the health authority in planning dengue control activities and it would allow for better design and implementation of dengue management programs.

A data mining approach for fault diagnosis: An application of anomaly detection algorithm

Rolling-element bearing failures are the most frequent problems in rotating machinery, which can be catastrophic and cause major downtime. Hence, providing advance failure warning and precise fault detection in such components are pivotal and cost-effective. The vast majority of past research has focused on signal processing and spectral analysis for fault diagnostics in rotating components. In this study, a data mining approach using a machine learning technique called anomaly detection (AD) is presented. This method employs classification techniques to discriminate between defect examples. Two features, kurtosis and Non-Gaussianity Score (NGS), are extracted to develop anomaly detection algorithms. The performance of the developed algorithms was examined through real data from a test to failure bearing. Finally, the application of anomaly detection is compared with one of the popular methods called Support Vector Machine (SVM) to investigate the sensitivity and accuracy of this approach and its ability to detect the anomalies in early stages.

Altered nociceptive, endocrine, and dorsal horn neuron responses in rats following a neonatal immune challenge

Summary The neonatal period is characterized by significant plasticity where the immune, endocrine, and nociceptive systems undergo fine-tuning and maturation. Painful experiences during this period can result in long-term alterations in the neurocircuitry underlying nociception, including increased sensitivity to mechanical or thermal stimuli. Less is known about the impact of neonatal exposure to mild inflammatory stimuli, such as lipopolysaccharide (LPS), on subsequent inflammatory pain responses. Here we examine the impact of neonatal LPS exposure on inflammatory pain sensitivity and HPA axis activity during the first three postnatal weeks. Wistar rats were injected with LPS (0.05 mg/kg IP, Salmonella enteritidis) or saline on postnatal days (PNDs) 3 and 5 and later subjected to the formalin test at PNDs 7, 13, and 22. One hour after formalin injection, blood was collected to assess corticosterone responses. Transverse spinal cord slices were also prepared for whole-cell patch clamp recording from lumbar superficial dorsal horn neurons (SDH). Brains were obtained at PND 22 and the hypothalamus was isolated to measure glucocorticoid (GR) and mineralocorticoid receptor (MR) transcript expression using qRT-PCR. Behavioural analyses indicate that at PND 7, no significant differences were observed between saline- or LPS-challenged rats. At PND 13, LPS-challenged rats exhibited enhanced licking (p < .01), and at PND 22, increased flinching in response to formalin injection (p < .05). LPS-challenged rats also displayed increased plasma corticosterone at PND 7 and PND 22 (p < .001) but not at PND 13 following formalin administration. Furthermore, at PND 22 neonatal LPS exposure induced decreased levels of GR mRNA and increased levels of MR mRNA in the hypothalamus. The intrinsic properties of SDH neurons were similar at PND 7 and PND 13. However, at PND 22, ipsilateral SDH neurons in LPS-challenged rats had a lower input resistance compared to their saline-challenged counterparts (p < .05). These data suggest neonatal LPS exposure produces developmentally regulated changes in formalin-induced behavioural responses, corticosterone levels, and dorsal horn neuron properties following noxious stimulation later in life. These findings highlight the importance of immune activation during the neonatal period in shaping pain sensitivity later in life. This programming involves both spinal cord neurons and the HPA axis.

Multimodal polymer nanoparticles with combined 19F magnetic resonance and optical detection for tunable, targeted, multimodal imagingin vivo

Understanding the complex nature of diseased tissue in vivo requires development of more advanced nanomedicines, where synthesis of multifunctional polymers combines imaging multimodality with a biocompatible, tunable, and functional nanomaterial carrier. Here we describe the development of polymeric nanoparticles for multimodal imaging of disease states in vivo. The nanoparticle design utilizes the abundant functionality and tunable physicochemical properties of synthetically robust polymeric systems to facilitate targeted imaging of tumors in mice. For the first time, high-resolution 19F/1H magnetic resonance imaging is combined with sensitive and versatile fluorescence imaging in a polymeric material for in vivo detection of tumors. We highlight how control over the chemistry during synthesis allows manipulation of nanoparticle size and function and can lead to very high targeting efficiency to B16 melanoma cells, both in vitro and in vivo. Importantly, the combination of imaging modalities within a polymeric nanoparticle provides information on the tumor mass across various size scales in vivo, from millimeters down to tens of micrometers.

Raman spectroscopy techniques for detection of energetic materials at high and low concentration

This thesis presents the development of a rapid, sensitive and reproducible spectroscopic method for the detection of TNT in forensic and environmental applications. Simple nano sensors prepared by cost effective methods were utilized as sensitive platforms for the detection of TNT by surface enhanced Raman spectroscopy. The optimization of the substrate and the careful selection of a suitable recognition molecule contributed to the significant improvements of sensitive and selective targeting over current detection methods. The work presented in this thesis paves the way for effective detection and monitoring of explosives residues in law enforcement and environmental health applications.

Computer-assisted sampling of acoustic data for more efficient determination of bird species richness

Bird species richness survey is one of the most intriguing ecological topics for evaluating environmental health. Here, bird species richness denotes the number of unique bird species in a particular area. Factors affecting the investigation of bird species richness include weather, observation bias, and most importantly, the prohibitive costs of conducting surveys at large spatiotemporal scales. Thanks to advances in recording techniques, these problems have been alleviated by deploying sensors for acoustic data collection. Although automated detection techniques have been introduced to identify various bird species, the innate complexity of bird vocalizations, the background noise present in the recording and the escalating volumes of acoustic data pose a challenging task on determination of bird species richness. In this paper we proposed a two-step computer-assisted sampling approach for determining bird species richness in one-day acoustic data. First, a classification model is built based on acoustic indices for filtering out minutes that contain few bird species. Then the classified bird minutes are ordered by an acoustic index and the redundant temporal minutes are removed from the ranked minute sequence. The experimental results show that our method is more efficient in directing experts for determination of bird species compared with the previous methods.

ELISA for the detection of venoms from four medically important snakes of India

A double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed to detect Echis carinatus venom in various organs (brain, heart, lungs, liver, spleen and kidneys) as well as tissue at the site of injection of mice, at various time intervals (1, 6, 12, 18, 24 h and 12 h intervals up to 72 h) after death. The assay could detect E. carinatus venom levels up to 2.5 ng/ml of tissue homogenate and the venom was detected up to 72 h after death. A highly sensitive and species-specific avidin-biotin microtitre ELISA was also developed to detect venoms of four medically important Indian snakes (Bungarus caeruleus, Naja naja, E. carinatus and Daboia russelli russelli) in autopsy specimens of human victims of snake bite. The assay could detect venom levels as low as 100 pg/ml of tissue homogenate. Venoms were detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area and postmortem blood. In all 12 human victim cadavers tested the culprit species were identified. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart and lungs. Development of a simple, rapid and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

Circulating tumor cell detection in high-risk non-metastatic prostate cancer

Purpose The detection of circulating tumor cells (CTCs) provides important prognostic information in men with metastatic prostate cancer. We aim to determine the rate of detection of CTCs in patients with high-risk non-metastatic prostate cancer using the CellSearch® method. Method Samples of peripheral blood (7.5 mL) were drawn from 36 men with newly diagnosed high-risk non-metastatic prostate cancer, prior to any initiation of therapy and analyzed for CTCs using the CellSearch® method. Results The median age was 70 years, median PSA was 14.1, and the median Gleason score was 9. The median 5-year risk of progression of disease using a validated nomogram was 39 %. Five out of 36 patients (14 %, 95 % CI 5–30 %) had CTCs detected in their circulation. Four patients had only 1 CTC per 7.5 mL of blood detected. One patient had 3 CTCs per 7.5 mL of blood detected, which included a circulating tumor microemboli. Both on univariate analysis and multivariate analysis, there were no correlations found between CTC positivity and the classic prognostic factors including PSA, Gleason score, T-stage and age. Conclusion This study demonstrates that patients with high-risk, non-metastatic prostate cancer present infrequently with small number of CTCs in peripheral blood. This finding is consistent with the limited literature available in this setting. Other CTC isolation and detection technologies with improved sensitivity and specificity may enable detection of CTCs with mesenchymal phenotypes, although none as yet have been validated for clinical use. Newer assays are emerging for detection of new putative biomarkers for prostate cancer. Correlation of disease control outcomes with CTC detection will be important.