Reverse Transcriptase-Coupled Quantitative Real Time PCR Analysis of Cell-Free Transcription on the Chromatin-Assembled p21 Promoter

Background: Cell-free eukaryotic transcription assays have contributed tremendously to the current understanding of the molecular mechanisms that govern transcription at eukaryotic promoters. Currently, the conventional G-less cassette transcription assay is one of the simplest and fastest methods for measuring transcription in vitro. This method requires several components, including the radioisotope labelling of RNA product during the transcription reaction followed by visualization of transcripts using autoradiography. Methodology/Principal Findings: To further simplify and expedite the conventional G-less cassette transcription assay, we have developed a method to incorporate a reverse transcriptase-coupled quantitative real time PCR (RT-qPCR). By using DNA template depletion steps that include DNA template immobilization, Trizol extraction and DNase I treatment, we have successfully enriched p21 promoter-driven transcripts over DNA templates. The quantification results of RNA transcripts using the RT-qPCR assay were comparable to the results of the conventional G-less cassette transcription assay both in naked DNA and chromatin-assembled templates. Conclusions: We first report a proof-of-concept demonstration that incorporating RT-qPCR in cell-free transcription assays can be a simpler and faster alternative method to the conventional radioisotope-mediated transcription assays. This method will be useful for developing high throughput in vitro transcription assays and provide quantitative data for RNA transcript

The clinical features of the piriformis syndrome: a systematic review

Piriformis syndrome, sciatica caused by compression of the sciatic nerve by the piriformis muscle, has been described for over 70 years; yet, it remains controversial. The literature consists mainly of case series and narrative reviews. The objectives of the study were: first, to make the best use of existing evidence to estimate the frequencies of clinical features in patients reported to have PS; second, to identify future research questions. A systematic review was conducted of any study type that reported extractable data relevant to diagnosis. The search included all studies up to 1 March 2008 in four databases: AMED, CINAHL, Embase and Medline. Screening, data extraction and analysis were all performed independently by two reviewers. A total of 55 studies were included: 51 individual and 3 aggregated data studies, and 1 combined study. The most common features found were: buttock pain, external tenderness over the greater sciatic notch, aggravation of the pain through sitting and augmentation of the pain with manoeuvres that increase piriformis muscle tension. Future research could start with comparing the frequencies of these features in sciatica patients with and without disc herniation or spinal stenosis

Chronic non-specific low back pain - sub-groups or a single mechanism?

Copyright 2008 Wand and O'Connell; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Low back pain is a substantial health problem and has subsequently attracted a considerable amount of research. Clinical trials evaluating the efficacy of a variety of interventions for chronic non-specific low back pain indicate limited effectiveness for most commonly applied interventions and approaches. Discussion: Many clinicians challenge the results of clinical trials as they feel that this lack of effectiveness is at odds with their clinical experience of managing patients with back pain. A common explanation for this discrepancy is the perceived heterogeneity of patients with chronic non-specific low back pain. It is felt that the effects of treatment may be diluted by the application of a single intervention to a complex, heterogeneous group with diverse treatment needs. This argument presupposes that current treatment is effective when applied to the correct patient. An alternative perspective is that the clinical trials are correct and current treatments have limited efficacy. Preoccupation with sub-grouping may stifle engagement with this view and it is important that the sub-grouping paradigm is closely examined. This paper argues that there are numerous problems with the sub-grouping approach and that it may not be an important reason for the disappointing results of clinical trials. We propose instead that current treatment may be ineffective because it has been misdirected. Recent evidence that demonstrates changes within the brain in chronic low back pain sufferers raises the possibility that persistent back pain may be a problem of cortical reorganisation and degeneration. This perspective offers interesting insights into the chronic low back pain experience and suggests alternative models of intervention. Summary: The disappointing results of clinical research are commonly explained by the failure of researchers to adequately attend to sub-grouping of the chronic non-specific low back pain population. Alternatively, current approaches may be ineffective and clinicians and researchers may need to radically rethink the nature of the problem and how it should best be managed

The effect of obesity on spirometry tests among healthy non-smoking adults

<p>Abstract</p> <p>Introduction</p> <p>The effects of obesity on pulmonary functions have not been addressed previously among Saudi population. We aim to study the effects of obesity on spirometry tests among healthy non-smoking adults.</p> <p>Methods</p> <p>A cross sectional study conducted among volunteers healthy non-smoking adults Subjects. We divided the subjects into two groups according to their BMI. The first group consisted of non-obese subjects with BMI of 18 to 24.9 kg/m2 and the second group consisted of obese subjects with BMI of 30 kg/m2 and above. Subjects underwent spirometry tests according to American thoracic society standards with measurement of the following values: the forced vital capacity (FVC), forced expiratory volume in one second (FEV1), peak expiratory flow rate (PEF) and forced mid-expiratory flow (FEF25-75).</p> <p>Results</p> <p>The total subjects were 294 with a mean age of 32 years. There were 178 males and 116 females subjects. We found no significant differences in FEV1 (p value = 0.686), FVC (p value = 0.733), FEV1/FVC Ratio (p value = 0.197) and FEF25-75 (p value = 0.693) between the obese and non-obese subjects. However, there was significantly difference in PEF between the two groups (p value < 0.020).</p> <p>Conclusion</p> <p>Obesity does not have effect on the spirometry tests (except PEF) among health non-smoking adults. We recommend searching for alternative diagnosis in case of findings abnormal spirometry tests results among obese subjects.</p

Clean thermal decomposition of tertiary-alkyl metal thiolates to metal sulfides: Environmentally-benign, non-polar inks for solution-processed chalcopyrite solar cells

We report the preparation of Cu2S, In2S3, CuInS2 and Cu(In,Ga)S2 semiconducting films via the spin coating and annealing of soluble tertiary-alkyl thiolate complexes. The thiolate compounds are readily prepared via the reaction of metal bases and tertiary-alkyl thiols. The thiolate complexes are soluble in common organic solvents and can be solution processed by spin coating to yield thin films. Upon thermal annealing in the range of 200-400 ??C, the tertiary-alkyl thiolates decompose cleanly to yield volatile dialkyl sulfides and metal sulfide films which are free of organic residue. Analysis of the reaction byproducts strongly suggests that the decomposition proceeds via an SN1 mechanism. The composition of the films can be controlled by adjusting the amount of each metal thiolate used in the precursor solution yielding bandgaps in the range of 1.2 to 3.3 eV. The films form functioning p-n junctions when deposited in contact with CdS films prepared by the same method. Functioning solar cells are observed when such p-n junctions are prepared on transparent conducting substrates and finished by depositing electrodes with appropriate work functions. This method enables the fabrication of metal chalcogenide films on a large scale via a simple and chemically clear process.ope

Facial expression training optimises viewing strategy in children and adults

This study investigated whether training-related improvements in facial expression categorization are facilitated by spontaneous changes in gaze behaviour in adults and nine-year old children. Four sessions of a self-paced, free-viewing training task required participants to categorize happy, sad and fear expressions with varying intensities. No instructions about eye movements were given. Eye-movements were recorded in the first and fourth training session. New faces were introduced in session four to establish transfer-effects of learning. Adults focused most on the eyes in all sessions and increased expression categorization accuracy after training coincided with a strengthening of this eye-bias in gaze allocation. In children, training-related behavioural improvements coincided with an overall shift in gaze-focus towards the eyes (resulting in more adult-like gaze-distributions) and towards the mouth for happy faces in the second fixation. Gaze-distributions were not influenced by the expression intensity or by the introduction of new faces. It was proposed that training enhanced the use of a uniform, predominantly eyes-biased, gaze strategy in children in order to optimise extraction of relevant cues for discrimination between subtle facial expressions

The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection

Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract

Physiological models of body composition and human obesity

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Klebsiella pneumoniae related community-acquired acute lower respiratory infections in CAMBODIA: clinical characteristics and treatment

<p>Abstract</p> <p>Background</p> <p>In many Asian countries, <it>Klebsiella pneumoniae </it>(KP) is the second pathogen responsible for community-acquired pneumonia. Yet, very little is known about <it>KP </it>etiology in ALRI in Cambodia, a country that has one of the weakest medical infrastructures in the region. We present here the first clinico-radiological description of <it>KP </it>community-acquired ALRI in hospitalized Cambodian patients.</p> <p>Methods</p> <p>Through ALRI surveillance in two provincial hospitals, <it>KP </it>was isolated from sputum and blood cultures, and identified by API20E gallery from patients ≥ 5 years-old with fever and respiratory symptoms onset ≤14 days. Antibiotics susceptibility testing was provided systematically to clinicians when bacteria were isolated. We collected patients' clinical, radiological and microbiological data and their outcome 3 months after discharge. We also compared <it>KP</it>-related with other bacteria-related ALRI to determine risk factors for <it>KP </it>infection.</p> <p>Results</p> <p>From April 2007 to December 2009, 2315 ALRI patients ≥ 5 years-old were enrolled including 587 whose bacterial etiology could be assigned. Of these, 47 (8.0%) had <it>KP </it>infection; their median age was 55 years and 68.1% were females. Reported prior medication was high (42.5%). Patients' chest radiographs showed pneumonia (61.3% including 39% that were necrotizing), preexisting parenchyma lesions (29.5%) and pleural effusions alone (4.5%) and normal parenchyma (4.5%). Five patients had severe conditions on admission and one patient died during hospitalization. Of the 39 patients that were hospital discharged, 14 died including 12 within 1 month after discharge. Only 13 patients (28%) received an appropriate antibiotherapy. Extended-spectrum beta-lactamases (ESBL) - producing strains were found in 8 (17.0%) patients. Female gender (Odds ratio (OR) 2.1; <it>p </it>= 0.04) and diabetes mellitus (OR 3.1; <it>p </it>= 0.03) were independent risk factors for <it>KP</it>-related ALRI.</p> <p>Conclusions</p> <p><it>KP </it>ALRI in Cambodia has high fatality rate, are more frequently found in women, and should be considered in diabetic patients. The extremely high frequency of ESBL-producing strains in the study is alarming in the context of uncontrolled antibiotic consumption and in absence of microbiology capacity in most public-sector hospitals.</p