Diagnostic virology and patient care: from vaguely interesting to vitally important

The existence of pathogenic viruses was inferred by experiments at the turn of the twentieth century. Key developments in detection of viruses, including electron microscopy and monolayer cell culture, were made in the middle of that century. However, in terms of patient care, the results from the virology laboratory often arrived the patient was ‘better or dead’. The advent of molecular techniques, particularly polymerase chain reaction and more recently whole genome sequencing made timely and accurate diagnosis of viral infections feasible. A range of approaches have been taken to identify and characterise new viruses. Vaccines against viruses have made it possible to eliminate two pathogenic mammalian viruses altogether, with several others close to eradication. The role of rking in diagnostic virology ismore relevant to patient care than ever

Influence of cell cycle on responses of MCF-7 cells to benzo[a]pyrene

<p>Abstract</p> <p>Background</p> <p>Benzo[a]pyrene (BaP) is a widespread environmental genotoxic carcinogen that damages DNA by forming adducts. This damage along with activation of the aryl hydrocarbon receptor (AHR) induces complex transcriptional responses in cells. To investigate whether human cells are more susceptible to BaP in a particular phase of the cell cycle, synchronised breast carcinoma MCF-7 cells were exposed to BaP. Cell cycle progression was analysed by flow cytometry, DNA adduct formation was assessed by ³²P-postlabeling analysis, microarrays of 44K human genome-wide oligos and RT-PCR were used to detect gene expression (mRNA) changes and Western blotting was performed to determine the expression of some proteins, including cytochrome P450 (CYP) 1A1 and CYP1B1, which are involved in BaP metabolism.</p> <p>Results</p> <p>Following BaP exposure, cells evaded <it>G1 </it>arrest and accumulated in <it>S</it>-phase. Higher levels of DNA damage occurred in <it>S</it>- and <it>G2/M</it>- compared with <it>G0/G1-</it>enriched cultures. Genes that were found to have altered expression included those involved in xenobiotic metabolism, apoptosis, cell cycle regulation and DNA repair. Gene ontology and pathway analysis showed the involvement of various signalling pathways in response to BaP exposure, such as the Catenin/Wnt pathway in <it>G1</it>, the ERK pathway in <it>G1 </it>and <it>S</it>, the Nrf2 pathway in <it>S </it>and <it>G2/M </it>and the Akt pathway in <it>G2/M</it>. An important finding was that higher levels of DNA damage in <it>S- </it>and <it>G2/M</it>-enriched cultures correlated with higher levels of <it>CYP1A1 </it>and <it>CYP1B1 </it>mRNA and proteins. Moreover, exposure of synchronised MCF-7 cells to BaP-7,8-diol-9,10-epoxide (BPDE), the ultimate carcinogenic metabolite of BaP, did not result in significant changes in DNA adduct levels at different phases of the cell cycle.</p> <p>Conclusions</p> <p>This study characterised the complex gene response to BaP in MCF-7 cells and revealed a strong correlation between the varying efficiency of BaP metabolism and DNA damage in different phases of the cell cycle. Our results suggest that growth kinetics within a target-cell population may be important determinants of susceptibility and response to a genotoxic agent.</p

Turbulent mixing of two-layer stratified fluid

Author Posting. © American Institute of Physics, 2007. This is the author's version of the work. It is posted here by permission of American Institute of Physics for personal use, not for redistribution. The definitive version was published in Physics of Fluids 19 (2007): 125104, doi:10.1063/1.2821913.A two-layer salt-stratified tank of water was mixed by turbulence generated by many excursions of a horizontally moving vertical rod. The objective is to observe the timedependent response of the mean density field for ranges of Richardson number Ri>0.9 and Reynolds Number Re>600. As the density profile of the fluid gradually evolves from a single step to a mixed state over a wide range of time, there is almost perfect collapse of all the profiles to one universal profile as a function of a similarity variable. Although the turbulent diffusion is not constant, the value in the limit of small stratification has similar magnitude to values found by others

Stroke survivors’ endorsement of a “stress belief model” of stroke prevention predicts control of risk factors for recurrent stroke

Perceptions that stress causes and stress-reduction controls hypertension have been associated with poorer blood pressure (BP) control in hypertension populations. The current study investigated these “stress-model perceptions” in stroke survivors regarding prevention of recurrent stroke and the influence of these perceptions on patients’ stroke risk factor control. Stroke and transient ischemic attack survivors (N = 600) participated in an in-person interview in which they were asked about their beliefs regarding control of future stroke; BP and cholesterol were measured directly after the interview. Counter to expectations, patients who endorsed a “stress-model” but not a “medication-model” of stroke prevention were in better control of their stroke risk factors (BP and cholesterol) than those who endorsed a medication-model but not a stress-model of stroke prevention (OR for poor control = .54, Wald statistic = 6.07, p = .01). This result was not explained by between group differences in patients’ reported medication adherence. The results have implications for theory and practice, regarding the role of stress belief models and acute cardiac events, compared to chronic hypertension

Time's up. Descriptive epidemiology of multi-morbidity and time spent on health related activity by older Australians: a time use survey

Most Western health systems remain single illness orientated despite the growing prevalence of multi-morbidity. Identifying how much time people with multiple chronic conditions spend managing their health will help policy makers and health service providers make decisions about areas of patient need for support. This article presents findings from an Australian study concerning the time spent on health related activity by older adults (aged 50 years and over), most of whom had multiple chronic conditions. A recall questionnaire was developed, piloted, and adjusted. Sampling was undertaken through three bodies; the Lung Foundation Australia (COPD sub-sample), National Diabetes Services Scheme (Diabetes sub-sample) and National Seniors Australia (Seniors sub-sample). Questionnaires were mailed out during 2011 to 10,600 older adults living in Australia. 2540 survey responses were received and analysed. Descriptive analyses were completed to obtain median values for the hours spent on each activity per month. The mean number of chronic conditions was 3.7 in the COPD sub-sample, 3.4 in the Diabetes sub-sample and 2.0 in the NSA sub-sample. The study identified a clear trend of increased time use associated with increased number of chronic conditions. Median monthly time use was 5-16 hours per month overall for our three sub-samples. For respondents in the top decile with five or more chronic conditions the median time use was equivalent to two to three hours per day, and if exercise is included in the calculations, respondents spent from between five and eight hours per day: an amount similar to full-time work. Multi-morbidity imposes considerable time burdens on patients. Ageing is associated with increasing rates of multi-morbidity. Many older adults are facing high demands on their time to manage their health in the face of decreasing energy and mobility. Their time use must be considered in health service delivery and health system reform.This work was funded by the National Health and Medical Research Council ID (402793, 2006)

Characterization of a 3D matrix bioreactor for scaled production of human mesenchymal stem cells

Human Mesenchymal Stem Cells (hMSCs) are multipotent, immune-privileged, and possess the capacity to proliferate ex-vivo, making them a good candidate for stem cell therapy. However, a reliable scalable production system for hMSCs is needed to fuel the growing field of regenerative medicine. Current growth of hMSCs is achieved through adherent 2D methods using tissue culture flasks or cell factory systems. These processes are labor intensive and can lead to low purity and poor yield of hMSCs due to the limited control of culture conditions inherent in these systems. In this work, we are investigating a novel 3D honeycomb matrix culture system for controlled high density hMSC production. We have assessed compatibility of the hMSCs on the honeycomb matrix and developed a scale down model bioreactor for development and characterization. Computational Fluid Dynamic (CFD) modeling is used in parallel with the described in-vitro experimentation to characterize shear profiles and oxygen transport for optimization of the conditions to support high cell density hMSC cultures. These techniques will potentially allow for higher yield and purity of hMSCs to meet the large quantities of cells needed for emerging whole cell therapies

Identification through microarray gene expression analysis of cellular responses to benzo(a)pyrene and its diol-epoxide that are dependent or independent of p53

Human colon carcinoma cells (HCT116) differing in p53 status were exposed to benzo(a)pyrene (BaP) or anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) and their gene expression responses compared by complementary DNA microarray technology. Exposure of cells to BPDE for up to 24 h resulted in gene expression profiles more distinguishable by duration of exposure than by p53 status, although a subset of genes were identified that had significantly different expression in p53 wild-type (WT) cells relative to p53-null cells. Apoptotic signalling genes were up-regulated in p53-WT cells but not in p53-null cells and, consistent with this, reduced viability and caspase activity were also p53 dependent. BPDE modulated cell cycle and histone genes in both cell lines and, in agreement with this, both cell lines accumulated in S phase. In p53-WT cells, G(2) arrest was also evident, which was associated with accumulation of CDKN1A. Regardless of p53 status, exposure to BaP for up to 48 h had subtle effects on gene transcription and had no influence on cell viability or cell cycle. Interestingly, DNA adduct formation after BaP, but not BPDE, exposure was p53 dependent with 10-fold lower levels detected in p53-null cells. Other cell lines were investigated for BaP-DNA adduct formation and in these the effect of p53 knockdown was also to reduce adduct formation. Taken together, these results give further insight into the role of p53 in the response of human cells to BaP and BPDE and suggest that loss of this tumour suppressor can influence the metabolic activation of BaP

SPEKcheck — fluorescence microscopy spectral visualisation and optimisation: a web application, javascript library, and data resource [version 1; referees: 3 approved]

Advanced fluorescence imaging methods require careful matching of excitation sources, dichroics, emission filters, detectors, and dyes to operate at their best. This complex task is often left to guesswork, preventing optimal dye:filter combinations, particularly for multicolour applications. To overcome this challenge we developed SPEKcheck, a web application to visualise the efficiency of the light path in a fluorescence microscope. The software reports values for the excitation efficiency of a dye, the collection efficiency of the emitted fluorescence, and a "brightness" score, allowing easy comparison between different fluorescent labels. It also displays a spectral plot of various elements in the configuration, enabling users to readily spot potential problems such as low efficiency excitation, emission, or high bleedthrough. It serves as an aid to exploring the performance of different dyes and filter sets