Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR.

BACKGROUND: Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification. FINDINGS: We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. CONCLUSIONS: These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Community perspectives on food insecurity and obesity: Focus groups with caregivers of metis and Off-reserve first nations children

Introduction: Aboriginal children in Canada are at a higher risk for overweight and obesity than other Canadian children. In Northern and remote areas, this has been linked to a lack of affordable nutritious food. However, the majority of Aboriginal children live in urban areas where food choices are more plentiful. This study aimed to explore the experiences of food insecurity among Métis and First Nations parents living in urban areas, including the predictors and perceived connections between food insecurity and obesity among Aboriginal children. Methods: Factors influencing children\u27s diets, families\u27 experiences with food insecurity, and coping strategies were explored using focus group discussions with 32 parents and caregivers of Métis and off-reserve First Nations children from Midland-Penetanguishene and London, Ontario. Four focus groups were conducted and transcribed verbatim between July 2011 and March 2013. A thematic analysis was conducted using NVivo software, and second coders ensured reliability of the results. Results: Caregivers identified low income as an underlying cause of food insecurity within their communities and as contributing to poor nutrition among their children. Families reported a reliance on energy-dense, nutrient-poor foods, as these tended to be more affordable and lasted longer than more nutritious, fresh food options. A lack of transportation also compromised families\u27 ability to purchase healthful food. Aboriginal caregivers also mentioned a lack of access to traditional foods. Coping strategies such as food banks and community programming were not always seen as effective. In fact, some were reported as potentially exacerbating the problem of overweight and obesity among First Nations and Métis children. Conclusion: Food insecurity manifested itself in different ways, and coping strategies were often insufficient for addressing the lack of fruit and vegetable consumption in Aboriginal children\u27s diets. Results suggest that obesity prevention strategies should take a family-targeted approach that considers the unique barriers facing urban Aboriginal populations. This study also reinforces the importance of low income as an important risk factor for obesity among Aboriginal peoples

Community perspectives on food insecurity and obesity: Focus groups with caregivers of metis and Off-reserve first nations children

Introduction: Aboriginal children in Canada are at a higher risk for overweight and obesity than other Canadian children. In Northern and remote areas, this has been linked to a lack of affordable nutritious food. However, the majority of Aboriginal children live in urban areas where food choices are more plentiful. This study aimed to explore the experiences of food insecurity among Métis and First Nations parents living in urban areas, including the predictors and perceived connections between food insecurity and obesity among Aboriginal children. Methods: Factors influencing children\u27s diets, families\u27 experiences with food insecurity, and coping strategies were explored using focus group discussions with 32 parents and caregivers of Métis and off-reserve First Nations children from Midland-Penetanguishene and London, Ontario. Four focus groups were conducted and transcribed verbatim between July 2011 and March 2013. A thematic analysis was conducted using NVivo software, and second coders ensured reliability of the results. Results: Caregivers identified low income as an underlying cause of food insecurity within their communities and as contributing to poor nutrition among their children. Families reported a reliance on energy-dense, nutrient-poor foods, as these tended to be more affordable and lasted longer than more nutritious, fresh food options. A lack of transportation also compromised families\u27 ability to purchase healthful food. Aboriginal caregivers also mentioned a lack of access to traditional foods. Coping strategies such as food banks and community programming were not always seen as effective. In fact, some were reported as potentially exacerbating the problem of overweight and obesity among First Nations and Métis children. Conclusion: Food insecurity manifested itself in different ways, and coping strategies were often insufficient for addressing the lack of fruit and vegetable consumption in Aboriginal children\u27s diets. Results suggest that obesity prevention strategies should take a family-targeted approach that considers the unique barriers facing urban Aboriginal populations. This study also reinforces the importance of low income as an important risk factor for obesity among Aboriginal peoples

Characterisation of an in Vitro Aged Cell Culture Model

The present study aims to provide a detailed protocol to obtain an aged cell culture model, which is conveniently characterised in terms of morphology, metabolism, and key biochemical markers of senescence. Commercial Human Dermal Fibroblasts (HDF) (67-year-old female Asian donor) were cultured over a span of 35 passages to determine at what passages they express the phenotypes found in vivo in the geriatric population. The growth rate and density (counting, Trypan Blue) at confluence declined 4- and 3-fold, respectively. During 15 passages in vitro ageing the fibroblast metabolic capacity (XTT) was heavily reduced (by 75%), while conversely the content of cellular proteins (SRB) slightly increased (2 fold). Senescent cells (SA-β-gal positive) were continuously emerging from 0 to 20%. Freezing/thawing at this stage restored metabolic performance to original values which declined at a similar rate as before. Passages 5 - 10 of our selected aged HDFs were found to mimic in vivo tissue of older adults to the greatest extent

Quality and clinical supply considerations of Paediatric Investigation Plans for IV preparations-A case study with the FP7 CloSed project

A Paediatric Investigation Plan (PIP) is a development plan that aims to ensure that sufficient data are obtained through studies in paediatrics to support the generation of marketing authorisation of medicines for children. This paper highlights some practical considerations and challenges with respect to PIP submissions and paediatric clinical trials during the pharmaceutical development phase, using the FP7-funded Clonidine for Sedation of Paediatric Patients in the Intensive Care Unit (CloSed) project as a case study. Examples discussed include challenges and considerations regarding formulation development, blinding and randomisation, product labelling and shipment and clinical trial requirements versus requirements for marketing authorisation. A significant quantity of information is required for PIP submissions and it is hoped that future applicants may benefit from an insight into some critical considerations and challenges faced in the CloSed project

Investigation into the dosage form attributes of currently UK licensed cardiovascular and Parkinson's disease drug products

Globally, there is a continuous rise in the older population (over 65 years), particularly in developed countries. As many diseases are age-related, older adults represent a highly heterogeneous cohort. This presents a major challenge for both the pharmaceutical industry and healthcare professionals. The purpose of this research was to attract attention towards the appropriateness of geriatric formulations by investigating the dosage form attributes of currently UK licensed cardiovascular and Parkinson's disease drug products. Medication available in the UK for cardiovascular disorders and Parkinson's disease were screened and the available formulations, packaging and patient information leaflets of these medicines were analysed, with the goal of raising awareness of the need to cater for elderly patients with increasing difficulty in managing their medication. It emerged that although cardiovascular disorders and Parkinson's disease are more prevalent in older people, many treatment options have not been optimised for this cohort. In particular, older patient centred dosage forms, specific dosing requirements, excipients, patient-friendly packaging and easy-to-follow patient information were highlighted as areas to be considered in order to optimise health outcomes in the ageing population

Investigation of photoplethysmographic signals and blood oxygen saturation values on healthy volunteers during cuff-induced hypoperfusion using a multimode PPG/SpO2 sensor

Photoplethysmography (PPG) is a technique widely used to monitor volumetric blood changes induced by cardiac pulsations. Pulse oximetry uses the technique of PPG to estimate arterial oxygen saturation values (SpO2). In poorly perfused tissues, SpO2 readings may be compromised due to the poor quality of the PPG signals. A multimode finger PPG probe that operates simultaneously in reflectance, transmittance and a combined mode called “transreflectance”’ was developed, in an effort to improve the quality of the PPG signals in states of hypoperfusion. Experiments on 20 volunteers were conducted to evaluate the performance of the multimode PPG sensor and compare the results with a commercial transmittance pulse oximeter. A brachial blood pressure cuff was used to induce artificial hypoperfusion. Results showed that the amplitude of the transreflectance AC PPG signals were significantly different (p\0.05) than the AC PPG signals obtained from the other two conventional PPG sensors (reflectance and transmittance). At induced brachial pressures between 90 and 135 mmHg, the reflectance finger pulse oximeter failed 25 times (failure rate 42.2 %) to estimate SpO2 values, whereas the transmittance pulse oximeter failed 8 times (failure rate 15.5 %). The transreflectance pulse oximeter failed only 3 times (failure rate 6.8 %) and the commercial pulse oximeter failed 17 times (failure rate 29.4 %)