Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

<p>Abstract</p> <p>Background</p> <p>Chandipura virus (CHPV), a member of family <it>Rhabdoviridae </it>was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR) using TaqMan technology was developed for rapid diagnosis.</p> <p>Methods</p> <p>Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [<it>in vivo </it>(mice) <it>in ovo </it>(eggs), <it>in vitro </it>(Vero E6, PS, RD and Sand fly cell line)] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard.</p> <p>Results</p> <p>Real-time one step RT-PCR was optimized using <it>in vitro </it>transcribed (IVT) RNA. Standard curve showed linear relationship for wide range of 10²-10¹⁰(r²= 0.99) with maximum Coefficient of variation (CV = 5.91%) for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice) used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 10⁰PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 10²PFU/ml). Vero and PS cell-lines (1.2 × 10³PFU/ml) were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy individual was used.</p> <p>Conclusion</p> <p>On account of the high sensitivity, reproducibility and specificity, the assay can be used for the rapid detection and quantitation of CHPV RNA from clinical samples during epidemics and from endemic areas. The assay may also find application in screening of antiviral compounds, understanding of pathogenesis as well as evaluation of vaccine.</p

Towards a high precision calculation for the pion-nucleus scattering lengths

We calculate the leading isospin conserving few-nucleon contributions to pion scattering on $^2$H, $^3$He, and $^4$He. We demonstrate that the strong contributions to the pion-nucleus scattering lengths can be controlled theoretically to an accuracy of a few percent for isoscalar nuclei and of 10% for isovector nuclei. In particular, we find the $\pi$-$^3$He scattering length to be $(62 \pm 4\pm 7)\times 10^{-3} m_{\pi}^{-1}$ where the uncertainties are due to ambiguities in the $\pi$-N scattering lengths and few-nucleon effects, respectively. To establish this accuracy we need to identify a suitable power counting for pion-nucleus scattering. For this purpose we study the dependence of the two-nucleon contributions to the scattering length on the binding energy of $^2$H. Furthermore, we investigate the relative size of the leading two-, three-, and four-nucleon contributions. For the numerical evaluation of the pertinent integrals, aMonte Carlo method suitable for momentum space is devised. Our results show that in general the power counting suggested by Weinberg is capable to properly predict the relative importance of $N$-nucleon operators, however, it fails to capture the relative strength of $N$- and $(N+1)$-nucleon operators, where we find a suppression by a factor of 5 compared to the predicted factor of 50. The relevance for the extraction of the isoscalar $\pi$-N scattering length from pionic $^2$H and $^4$He is discussed. As a side result, we show that beyond the calculation of the $\pi$-$^2$H scattering length is already beyond the range of applicability of heavy pion effective field theory.Comment: 24 pages, 14 figures, 10 table

Study of pallial neurogenesis in shark embryos and the evolutionary origin of the subventricular zone

The dorsal part of the developing telencephalon is one of the brain areas that has suffered most drastic changes throughout vertebrate evolution. Its evolutionary increase in complexity was thought to be partly achieved by the appearance of a new neurogenic niche in the embryonic subventricular zone (SVZ). Here, a new kind of amplifying progenitors (basal progenitors) expressing Tbr2, undergo a second round of divisions, which is believed to have contributed to the expansion of the neocortex. Accordingly, the existence of a pallial SVZ has been classically considered exclusive of mammals. However, the lack of studies in ancient vertebrates precludes any clear conclusion about the evolutionary origin of the SVZ and the neurogenic mechanisms that rule pallial development. In this work, we explore pallial neurogenesis in a basal vertebrate, the shark Scyliorhinus canicula, through the study of the expression patterns of several neurogenic markers. We found that apical progenitors and radial migration are present in sharks, and therefore, their presence must be highly conserved throughout evolution. Surprisingly, we detected a subventricular band of ScTbr2-expressing cells, some of which also expressed mitotic markers, indicating that the existence of basal progenitors should be considered an ancestral condition rather than a novelty of mammals or amniotes. Finally, we report that the transcriptional program for the specification of glutamatergic pallial cells (Pax6, Tbr2, NeuroD, Tbr1) is also present in sharks. However, the segregation of these markers into different cell types is not clear yet, which may be linked to the lack of layering in anamniotesThis work was supported by the Spanish Ministerio de Economía y Competitividad-FEDER (BFU2014-5863-1P)S

Consensus recommendations on the use of 18F-FDG PET/CT in lung disease

Positron emission tomography (PET) with 18F-fluorodeoxyglucose (18F-FDG) has been increasingly applied, predominantly in the research setting, to study drug effects and pulmonary biology and monitor disease progression and treatment outcomes in lung diseases, disorders that interfere with gas exchange through alterations of the pulmonary parenchyma, airways and/or vasculature. To date, however, there are no widely accepted standard acquisition protocols and imaging data analysis methods for pulmonary 18F-FDG PET/CT in these diseases, resulting in disparate approaches. Hence, comparison of data across the literature is challenging. To help harmonize the acquisition and analysis and promote reproducibility, acquisition protocol and analysis method details were collated from seven PET centers. Based on this information and discussions among the authors, the consensus recommendations reported here on patient preparation, choice of dynamic versus static imaging, image reconstruction, and image analysis reporting were reached.                   </p

An exploratory study to assess the knowledge, attitudes and practices of Lebanese residents towards acrylamide

Introduction For years, heat treatment has been an essential method for ensuring mature food that meet the desired quality and safety characteristics. However, this process could lead to the formation of harmful compounds such as acrylamide. In this study we aimed to investigate the knowledge, attitudes and practices (KAPs) of the Lebanese population toward the potential risk associated with acrylamide. Materials & methods An online survey (n = 598) was conducted among residents in Lebanon aged 18 years and above. The survey was divided into five sections including participants’ sociodemographic characteristics, knowledge, attitude and practice sections, and some questions related to consumer’s preferences. Results & discussion The results showed that the majority of the participants had low food safety knowledge regarding acrylamide. Specifically, 82.9% of the consumers had no idea about the chemical, its formation, the foods with a high risk of acrylamide formation and the health risks associated with its exposure. Despite lack of knowledge, good domestic food practices (storage, pre-treatment) were noticed among participants. Moreover, the majority of consumers (> 80%) showed positive attitude towards proper acrylamide labeling. Participants with a bachelor’s degree appeared to have a more positive attitude toward food safety compared to those with no qualifications (p<0.001). Conclusion Despite the high consumption of acrylamide by the consumers in Lebanon through fried potatoes, bread, and coffee, the majority have no idea about acrylamide’s presence in food, its sources and its adverse health effects. Raising awareness among the public, involving policy makers in addressing the issue of clear labeling and encouraging the adoption of alternative practices to reduce acrylamide are all crucial to protect consumers’ health in Lebanon and promote healthier food consumption habits

Few-nucleon contribution of pi-scattering on light nuclei

We present our new results of the few-nucleon contributions to π-2H, π-3He and π-4He scattering lengths. To calculate these within the framework of Chiral Perturbation Theory we used a Monte-Carlo algorithm, which is briefly introduced. The size of the two-, three- and four-nucleon corrections in view of the expectations from two different power counting schemes is discussed. Based on our results, we estimate short range contributions, which limit the accuracy of the πN scattering lengths extracted from few-nucleon data. Additionally, we study how the π-4He system constrains the determination of the isoscalar s-wave scattering length a(+) and its isovector counter part a(−)

Genome-wide gene expression analysis of a murine model of prostate cancer progression: Deciphering the roles of IL-6 and p38 MAPK as potential therapeutic targets.

BACKGROUND:Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths among adult males globally. The poor prognosis of PCa is largely due to late diagnosis of the disease when it has already progressed to an advanced stage marked by androgen-independence, thus necessitating new strategies for early detection and treatment. We construe that these direly needed advances are limited by our poor understanding of early events in the progression of PCa and that would thus represent ideal targets for early intervention. To begin to fill this void, we interrogated molecular "oncophenotypes" that embody the transition of PCa from an androgen-dependent (AD) to-independent (AI) state. METHODS:To accomplish this aim, we used our previously established AD and AI murine PCa cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PCa morphologically and molecularly. We statistically surveyed global gene expressions in these cell lines by microarray analysis. Differential profiles were functionally interrogated by pathways, gene set enrichment and topological gene network analyses. RESULTS:Gene expression analysis of PLum-AD and PLum-AI transcriptomes (n = 3 each), revealed 723 differentially expressed genes (392 upregulated and 331 downregulated) in PLum-AI compared to PLum-AD cells. Gene set analysis demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to tumor aggressiveness including cell migration and invasion facilitated by epithelial-to-mesenchymal transition (EMT). Further analysis demonstrated that the p38 mitogen-activated protein kinase (MAPK) was predicted to be significantly activated in the PLum-AI cells, whereas gene sets previously associated with favorable response to the p38 inhibitor SB203580 were attenuated (i.e., inversely enriched) in the PLum-AI cells, suggesting that these aggressive cells may be therapeutically vulnerable to p38 inhibition. Gene set and gene-network analysis also alluded to activation of other signaling networks particularly those associated with enhanced EMT, inflammation and immune function/response including, but not limited to Tnf, IL-6, Mmp 2, Ctgf, and Ptges. Accordingly, we chose SB203580 and IL-6 to validate their effect on PLum-AD and PLum-AI. Some of the common genes identified in the gene-network analysis were validated at the molecular and functional level. Additionally, the vulnerability to SB203580 and the effect of IL-6 were also validated on the stem/progenitor cell population using the sphere formation assay. CONCLUSIONS:In summary, our study highlights pathways associated with an augmented malignant phenotype in AI cells and presents new high-potential targets to constrain the aggressive malignancy seen in the castration-resistant PCa