HIV-1 Packaging Visualised by In-Gel SHAPE.

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA-protein complex. Here, we apply the in-gel SHAPE (selective 2'OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag-dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.This work was funded by an Eric Reid fund for Methodology grant from the Biochemical Society (to JCK), UK Medical Research Council, Grant/Award Number: MR/N022939/1 (to AML and JCK). AMLL is supported by the Clinical Academic Reserve and his laboratory by the NIHR Cambridge BRC (Grant RCAG/18). Research in the Kappei laboratory was supported by the National Research Foundation Singapore and the Singapore Ministry of Education under its Re-search Centres of Excellence initiative

HIV-1 Packaging Visualised by In-Gel SHAPE.

HIV-1 packages two copies of its gRNA into virions via an interaction with the viral structural protein Gag. Both copies and their native RNA structure are essential for virion infectivity. The precise stepwise nature of the packaging process has not been resolved. This is largely due to a prior lack of structural techniques that follow RNA structural changes within an RNA-protein complex. Here, we apply the in-gel SHAPE (selective 2'OH acylation analysed by primer extension) technique to study the initiation of HIV-1 packaging, examining the interaction between the packaging signal RNA and the Gag polyprotein, and compare it with that of the NC domain of Gag alone. Our results imply interactions between Gag and monomeric packaging signal RNA in switching the RNA conformation into a dimerisation-competent structure, and show that the Gag-dimer complex then continues to stabilise. These data provide a novel insight into how HIV-1 regulates the translation and packaging of its genome.This work was funded by an Eric Reid fund for Methodology grant from the Biochemical Society (to JCK), UK Medical Research Council, Grant/Award Number: MR/N022939/1 (to AML and JCK). AMLL is supported by the Clinical Academic Reserve and his laboratory by the NIHR Cambridge BRC (Grant RCAG/18). Research in the Kappei laboratory was supported by the National Research Foundation Singapore and the Singapore Ministry of Education under its Re-search Centres of Excellence initiative

Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells

Undifferentiated nasopharyngeal carcinomas (NPCs) are commonly present with latent EBV infection. However, events regulating EBV infection at early stages of the disease and the role of EBV in disease pathogenesis are largely undefined. Genetic alterations leading to activation of cyclin D1 signaling in premalignant nasopharyngeal epithelial (NPE) cells have been postulated to predispose cells to EBV infection. We previously reported that loss of p16, a negative regulator of cyclin D1 signaling, is a frequent feature of NPC tumors. Here, we report that early premalignant lesions of nasopharyngeal epithelium overexpress cyclin D1. Furthermore, overexpression of cyclin D1 is closely associated with EBV infection. Therefore we investigated the potential role of cyclin D1 overexpression in dysplastic NPE cells in vitro. In human telomerase reverse transcriptase-immortalized NPE cells, overexpression of cyclin D1 or a p16-resistant form of CDK4 (CDK4(R24C)) suppressed differentiation. This suppression may have implications for the close association of EBV infection with undifferentiated NPC. In these in vitro models, we found that cellular growth arrest and senescence occurred in EBV-infected cell populations immediately after infection. Nevertheless, overexpression of cyclin D1 or a p16-resistant form of CDK4 or knockdown of p16 in the human telomerase reverse transcriptase-immortalized NPE cell lines could counteract the EBV-induced growth arrest and senescence. We conclude that dysregulated expression of cyclin D1 in NPE cells may contribute to NPC pathogenesis by enabling persistent infection of EBV.published_or_final_versio

Enhanced IL-6/IL-6R Signaling Promotes Growth and Malignant Properties in EBV-Infected Premalignant and Cancerous Nasopharyngeal Epithelial Cells

Nasopharyngeal carcinoma (NPC) is etiologically associated with Epstein-Barr virus (EBV) infection. However, the exact role of EBV in NPC pathogenesis remains elusive. Activation of signal transducer and activator of transcription 3 (STAT3) is common in human cancers including NPC and plays an important role in the pathogenesis and progression of human cancers. Interleukin-6 (IL-6), a major inflammatory cytokine, is a potent activator of STAT3. In this study, we report that EBV-infected immortalized nasopharyngeal epithelial (NPE) cells often acquire an enhanced response to IL-6-induced STAT3 activation to promote their growth and invasive properties. Interestingly, this enhanced IL-6/STAT3 response was mediated by overexpression of IL-6 receptor (IL-6R). Furthermore, IL-6R overexpression enhanced IL-6-induced STAT3 activation in uninfected immortalized NPE cells in vitro, and promoted growth and tumorigenicity of EBV-positive NPC cell line (C666-1) in vivo. Moreover, it is shown for the first time that IL-6R was overexpressed in clinical specimens of NPC. IL-6 expression could also be strongly detected in the stromal cells of NPC and a higher circulating level of IL-6 was found in the sera of advance-staged NPC patients compared to the control subjects. Therefore, IL-6R overexpression, coupled with enhanced IL-6/STAT3 signaling may facilitate the malignant transformation of EBV-infected premalignant NPE cells into cancer cells, and enhance malignant properties of NPC cells. © 2013 Zhang et al.published_or_final_versio

Potential of a cyclone prototype spacer to improve in vitro dry powder delivery

Copyright The Author(s) 2013. This article is published with open access at Springerlink.com. This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are creditedPurpose: Low inspiratory force in patients with lung disease is associated with poor deagglomeration and high throat deposition when using dry powder inhalers (DPIs). The potential of two reverse flow cyclone prototypes as spacers for commercial carrierbased DPIs was investigated. Methods: Cyclohaler®, Accuhaler® and Easyhaler® were tested with and without the spacers between 30-60 Lmin-1. Deposition of particles in the next generation impactor and within the devices was determined by high performance liquid chromatography. Results: Reduced induction port deposition of the emitted particles from the cyclones was observed due to the high retention of the drug within the spacers (e.g. salbutamol sulphate (SS): 67.89 ± 6.51 % at 30 Lmin-1 in Cheng 1). Fine particle fractions of aerosol as emitted from the cyclones were substantially higher than the DPIs alone. Moreover, the aerodynamic diameters of particles emitted from the cyclones were halved compared to the DPIs alone (e.g. SS from the Cyclohaler® at 4 kPa: 1.08 ± 0.05 μm vs. 3.00 ± 0.12 μm, with and without Cheng 2, respectively) and unaltered with increased flow rates. Conclusion: This work has shown the potential of employing a cyclone spacer for commercial carrier-based DPIs to improve inhaled drug delivery.Peer reviewe

Diet and Cell Size Both Affect Queen-Worker Differentiation through DNA Methylation in Honey Bees (Apis mellifera, Apidae)

Young larvae of the honey bee (Apis mellifera) are totipotent; they can become either queens (reproductives) or workers (largely sterile helpers). DNA methylation has been shown to play an important role in this differentiation. In this study, we examine the contributions of diet and cell size to caste differentiation.We measured the activity and gene expression of one key enzyme involved in methylation, Dnmt3; the rates of methylation in the gene dynactin p62; as well as morphological characteristics of adult bees developed either from larvae fed with worker jelly or royal jelly; and larvae raised in either queen or worker cells. We show that both diet type and cell size contributed to the queen-worker differentiation, and that the two factors affected different methylation sites inside the same gene dynactin p62.We confirm previous findings that Dnmt3 plays a critical role in honey bee caste differentiation. Further, we show for the first time that cell size also plays a role in influencing larval development when diet is kept the same

Identification of plasma lipid biomarkers for prostate cancer by lipidomics and bioinformatics

Background: Lipids have critical functions in cellular energy storage, structure and signaling. Many individual lipid molecules have been associated with the evolution of prostate cancer; however, none of them has been approved to be used as a biomarker. The aim of this study is to identify lipid molecules from hundreds plasma apparent lipid species as biomarkers for diagnosis of prostate cancer. Methodology/Principal Findings: Using lipidomics, lipid profiling of 390 individual apparent lipid species was performed on 141 plasma samples from 105 patients with prostate cancer and 36 male controls. High throughput data generated from lipidomics were analyzed using bioinformatic and statistical methods. From 390 apparent lipid species, 35 species were demonstrated to have potential in differentiation of prostate cancer. Within the 35 species, 12 were identified as individual plasma lipid biomarkers for diagnosis of prostate cancer with a sensitivity above 80%, specificity above 50% and accuracy above 80%. Using top 15 of 35 potential biomarkers together increased predictive power dramatically in diagnosis of prostate cancer with a sensitivity of 93.6%, specificity of 90.1% and accuracy of 97.3%. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) demonstrated that patient and control populations were visually separated by identified lipid biomarkers. RandomForest and 10-fold cross validation analyses demonstrated that the identified lipid biomarkers were able to predict unknown populations accurately, and this was not influenced by patient's age and race. Three out of 13 lipid classes, phosphatidylethanolamine (PE), ether-linked phosphatidylethanolamine (ePE) and ether-linked phosphatidylcholine (ePC) could be considered as biomarkers in diagnosis of prostate cancer. Conclusions/Significance: Using lipidomics and bioinformatic and statistical methods, we have identified a few out of hundreds plasma apparent lipid molecular species as biomarkers for diagnosis of prostate cancer with a high sensitivity, specificity and accuracy

Genome-wide transcriptional analysis of salinity stressed japonica and indica rice genotypes during panicle initiation stage

Rice yield is most sensitive to salinity stress imposed during the panicle initiation (PI) stage. In this study, we have focused on physiological and transcriptional responses of four rice genotypes exposed to salinity stress during PI. The genotypes selected included a pair of indicas (IR63731 and IR29) and a pair of japonica (Agami and M103) rice subspecies with contrasting salt tolerance. Physiological characterization showed that tolerant genotypes maintained a much lower shoot Na(+) concentration relative to sensitive genotypes under salinity stress. Global gene expression analysis revealed a strikingly large number of genes which are induced by salinity stress in sensitive genotypes, IR29 and M103 relative to tolerant lines. We found 19 probe sets to be commonly induced in all four genotypes. We found several salinity modulated, ion homeostasis related genes from our analysis. We also studied the expression of SKC1, a cation transporter reported by others as a major source of variation in salt tolerance in rice. The transcript abundance of SKC1 did not change in response to salinity stress at PI stage in the shoot tissue of all four genotypes. However, we found the transcript abundance of SKC1 to be significantly higher in tolerant japonica Agami relative to sensitive japonica M103 under control and stressed conditions during PI stage. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s11103-006-9112-0 and is accessible for authorized users

The what and where of adding channel noise to the Hodgkin-Huxley equations

One of the most celebrated successes in computational biology is the Hodgkin-Huxley framework for modeling electrically active cells. This framework, expressed through a set of differential equations, synthesizes the impact of ionic currents on a cell's voltage -- and the highly nonlinear impact of that voltage back on the currents themselves -- into the rapid push and pull of the action potential. Latter studies confirmed that these cellular dynamics are orchestrated by individual ion channels, whose conformational changes regulate the conductance of each ionic current. Thus, kinetic equations familiar from physical chemistry are the natural setting for describing conductances; for small-to-moderate numbers of channels, these will predict fluctuations in conductances and stochasticity in the resulting action potentials. At first glance, the kinetic equations provide a far more complex (and higher-dimensional) description than the original Hodgkin-Huxley equations. This has prompted more than a decade of efforts to capture channel fluctuations with noise terms added to the Hodgkin-Huxley equations. Many of these approaches, while intuitively appealing, produce quantitative errors when compared to kinetic equations; others, as only very recently demonstrated, are both accurate and relatively simple. We review what works, what doesn't, and why, seeking to build a bridge to well-established results for the deterministic Hodgkin-Huxley equations. As such, we hope that this review will speed emerging studies of how channel noise modulates electrophysiological dynamics and function. We supply user-friendly Matlab simulation code of these stochastic versions of the Hodgkin-Huxley equations on the ModelDB website (accession number 138950) and http://www.amath.washington.edu/~etsb/tutorials.html.Comment: 14 pages, 3 figures, review articl

Functionally Orthologous Viral and Cellular MicroRNAs Studied by a Novel Dual-Fluorescent Reporter System

Recent research raised the possibility that some viral microRNAs (miRNAs) may function as orthologs of cellular miRNAs. In the present work, to study the functional orthologous relationships of viral and cellular miRNAs, we first constructed a dual-fluorescent protein reporter vector system for the easy determination of miRNA function. By expressing the miRNAs and the indicator and internal control fluorescent proteins individually from a single vector, this simple reporter system can be used for miRNA functional assays that include visualizing miRNA activity in live cells. Sequence alignments indicated that the simian virus 40 (SV40) encoded miRNA sv40-mir-S1-5p contains a seed region identical to that of the human miRNA hsa-miR423-5p. Using the new reporter system, it was found that sv40-mir-S1-5p and hsa-miR423-5p downregulate the expression of common artificial target mRNAs and some predicted biological targets of hsa-miR423-5p, demonstrating that they are functional orthologs. The human immunodeficiency virus 1 (HIV-1) encoded hiv1-miR-N367 also contains a seed sequence identical to that of the human miRNA hsa-miR192. Functional assays showed that hiv1-miR-N367 and hsa-miR192 could downregulate common artificial and predicted biological targets, suggesting that these miRNAs may also act as functional orthologs. Thus, this study presents a simple and universal system for testing miRNA function and identifies two new pairs of functional orthologs, sv40-mir-S1-5p and hsa-miR423-5p as well as hiv-1-miR-N367 and hsa-miR192. These findings also expand upon our current knowledge of functional homology and imply that a more general phenomenon of orthologous relationships exists between viral and cellular miRNAs