Cellular target genes of Epstein-Barr virus nuclear antigen 2

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines.     

A mutation in aminopeptidase N (CD13) isolated from a patient suffering from leukemia leads to an arrest in the endoplasmic reticulum

Human aminopeptidase N (APN) is used as a routine marker for myelomonocytic cells in hematopoietic malignant disorders. Its gene and surface expressions are increased in cases of malignant transformation, inflammation, or T cell activation, whereas normal B and resting T cells lack detectable APN protein expression. In this study we elucidated the intracellular distribution, expression pattern, and enzymatic activity of a naturally occurring mutation in the coding region of the APN gene. At physiological temperatures the mutant protein is enzymatically inactive, persists as a mannose-rich polypeptide in the endoplasmic reticulum, and is ultimately degraded by an endoplasmic reticulum-associated degradation pathway. It shows in part the distinct behavior of a temperature-sensitive mutant with a permissive temperature of 32 degrees C, leading to correct sorting of the Golgi compartment accompanied by the acquisition of proper glycosylation but without reaching the cell-surface membrane and without regaining its enzymatic activity. Because the patient bearing this mutation suffered from leukemia, possible links to the pathogenesis of leukemia are discussed.     

Signal peptide peptidase-catalyzed cleavage of hepatitis C virus core protein is dispensable for virus budding but destabilizes the viral capsid

The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell.     

How to Create and Use Binocular Rivalry

Each of our eyes normally sees a slightly different image of the world around us. The brain can combine these two images into a single coherent representation. However, when the eyes are presented with images that are sufficiently different from each other, an interesting thing happens: Rather than fusing the two images into a combined conscious percept, what transpires is a pattern of perceptual alternations where one image dominates awareness while the other is suppressed; dominance alternates between the two images, typically every few seconds. This perceptual phenomenon is known as binocular rivalry. Binocular rivalry is considered useful for studying perceptual selection and awareness in both human and animal models, because unchanging visual input to each eye leads to alternations in visual awareness and perception. To create a binocular rivalry stimulus, all that is necessary is to present each eye with a different image at the same perceived location. There are several ways of doing this, but newcomers to the field are often unsure which method would best suit their specific needs. The purpose of this article is to describe a number of inexpensive and straightforward ways to create and use binocular rivalry. We detail methods that do not require expensive specialized equipment and describe each method''s advantages and disadvantages. The methods described include the use of red-blue goggles, mirror stereoscopes and prism goggles.     

Virus–host cell interactions in vaccine production cell lines infected with different human influenza A virus variants: A proteomic approach

A proteomic approach was used to investigate the dynamic cellular host cell response induced by influenza virus infection in two different vaccine production cell lines, MDCK and Vero. For identification of proteins possibly involved in global host cell response mechanisms and virus–host cell interactions in these producer cell lines, quantitative 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were performed. In particular, host cell proteome alterations caused by infection with influenza virus variants showing differences in replication characteristics in MDCK cells were compared. Moreover, the host cell response to virus infection in Vero cells with respect to their deficiency in interferon (IFN) production and the need for virus adaptation to optimize productivity of this cell line were analyzed. Several proteins with differential abundance profiles were identified and Western blot analysis was performed for further confirmation of selected proteins. IFN-induced signal transduction, cytoskeleton remodeling, vesicle transport and proteolysis were the principal pathways that changed in infected MDCK cells. In Vero cells alterations of cell interactions, heat shock and oxidative stress response were detected. The findings will improve understanding of host cell response mechanisms during influenza vaccine production and viral strategies to evade these responses and to replicate efficiently in different cell lines.     

Immigrational Background Affects the Effectiveness of a School‐based Overweight Prevention Program Promoting Water Consumption

We tested whether a simple overweight prevention program promoting water consumption in elementary schools is equally effective in children with an immigrational background (MIG) and in those without (non‐MIG). Thus, a secondary analysis of a controlled cluster trial, lasting one school year, was conducted. Thirty‐two elementary schools located in low socioeconomic districts in two German cities were included. Of the 2,950 school children analyzed, 1,306 were MIG children. Water fountains were installed in the schools of the intervention group (IG) and teachers held lessons to promote water consumption. Control schools (control group (CG)) did not receive any intervention. Before and after intervention, body weight and height was measured. Overweight was defined by age‐ and sex‐specific BMI cutoffs that are linked to an adult BMI of 25 kg/m². Beverage consumption was assessed in questionnaires. Modification of intervention effects by immigrational background was tested by interaction terms. The immigrational background modified the intervention effect on prevalence and remission of overweight (interaction term: P = 0.03 and P = 0.02), but not on the incidence of overweight (P = 0.06). After intervention, the risk of being overweight was reduced in the IG compared to the CG among non‐MIG (odds ratio = 0.51, 95% confidence interval (CI): 0.31–0.83), but not among MIG children (odds ratio = 1.02, 95% CI: 0.63–1.65). After intervention, water consumption significantly increased in the IG equally among both, non‐MIG and MIG, by ～1 glass/day. A simple school‐based intervention promoting water consumption prevented overweight in non‐MIG children, but failed in MIG children. Different beverage consumption, among other lifestyle factors, may account for this effect but scientific discussion remains open.     

Pdro,a Protein Associated with Late Endosomes and Lysosomes and Implicated in Cellular Cholesterol Homeostasis

Background

Cellular cholesterol is a vital component of the cell membrane. Its concentration is tightly controlled by mechanisms that remain only partially characterized. In this study, we describe a late endosome/lysosomes–associated protein whose expression level affects cellular free cholesterol content.

Methodology/Principal Findings

Using a restricted proteomic analysis of detergent-resistant membranes (DRMs), we have identified a protein encoded by gene C11orf59. It is mainly localized to late endosome/lysosome (LE/LY) compartment through N-terminal myristoylation and palmitoylation. We named it Pdro for protein associated with DRMs and endosomes. Very recently, three studies have reported on the same protein under two other names: the human p27RF-Rho that regulates RhoA activation and actin dynamics, and its rodent orthologue p18 that controls both LE/LY dynamics through the MERK-ERK pathway and the lysosomal activation of mammalian target of rapamycin complex 1 by amino acids. We found that, consistent with the presence of sterol-responsive element consensus sequences in the promoter region of C11orf59, Pdro mRNA and protein expression levels are regulated positively by cellular cholesterol depletion and negatively by cellular cholesterol loading. Conversely, Pdro is involved in the regulation of cholesterol homeostasis, since its depletion by siRNA increases cellular free cholesterol content that is accompanied by an increased cholesterol efflux from cells. On the other hand, cells stably overexpressing Pdro display reduced cellular free cholesterol content. Pdro depletion-mediated excess cholesterol results, at least in part, from a stimulated low-density lipoprotein (LDL) uptake and an increased cholesterol egress from LE/LY.

Conclusions/Significance

LDL-derived cholesterol release involves LE/LY motility that is linked to actin dynamics. Because Pdro regulates these two processes, we propose that modulation of Pdro expression in response to sterol levels regulates LDL-derived cholesterol through both LDL uptake and LE/LY dynamics, to ultimately control free cholesterol homeostasis.     

Adolescence as risk factor for adverse pregnancy outcome in Central Africa--a cross-sectional study

Background

Sub-Saharan Africa has the highest rates of maternal and neonatal mortality worldwide. Young maternal age at delivery has been proposed as risk factor for adverse pregnancy outcome, yet there is insufficient data from Sub-Saharan Africa. The present study aimed to investigate the influence of maternal adolescence on pregnancy outcomes in the Central African country Gabon.

Methodology and Principal Findings

Data on maternal age, parity, birth weight, gestational age, maternal Plasmodium falciparum infection, use of bednets, and intake of intermittent preventive treatment of malaria in pregnancy were collected in a cross-sectional survey in 775 women giving birth in three mother-child health centers in Gabon. Adolescent women (≤16 years of age) had a significantly increased risk to deliver a baby with low birth weight in univariable analysis (22.8%, 13/57, vs. 9.3%, 67/718, OR: 2.9, 95% CI: 1.5–5.6) and young maternal age showed a statistically significant association with the risk for low birth weight in multivariable regression analysis after correction for established risk factors (OR: 2.7; 95% CI: 1.1–6.5). In further analysis adolescent women were shown to attend significantly less antenatal care visits than adult mothers (3.3±1.9 versus 4.4±1.9 mean visits, p<0.01, n = 356) and this difference accounted at least for part of the excess risk for low birth weight in adolescents.

Conclusion

Our data demonstrate the importance of adolescent age as risk factor for adverse pregnancy outcome. Antenatal care programs specifically tailored for the needs of adolescents may be necessary to improve the frequency of antenatal care visits and pregnancy outcomes in this risk group in Central Africa.     

Bottlenecks for High Coverage of Intermittent Preventive Treatment in Pregnancy: The Case of Adolescent Pregnancies in Rural Burkina Faso

Background

While IPTp-SP is currently being scaled up in sub-Saharan Africa (SSA), the coverage with the required ≥2 doses of SP remains considerably short of the Roll Back Malaria (RBM) goal of 80%, not to mention of the recently advocated universal coverage.

Methods

The study triangulates quantitative data from a health center randomized community-based trial on IPTp-SP effectiveness and the additional benefit of a promotional campaign with qualitative data from focused ethnography.

Findings

In rural Burkina Faso, despite the significantly higher risk of malaria infection among adolescent primigravidae (PG) (OR 2.44 95%CI 1.81–3.28, p<0.001), making them primary target beneficiaries of IPTp-SP, adolescents adhered to the required three or more ANC visits significantly less (PG: 46.6%; SG 43.7%) than adults (PG: 61.9%; SG 54.9%) and had lower SP uptake during the malaria transmission season, further showing the difficulty of reaching this age group. Adolescents'' structural constraints (such as their social position and household labor requirements) and needs (such as anonymity in the health encounter) leave them highly vulnerable during their pregnancies and, especially, during the high malaria transmission season.

Conclusion

Our study shows that adolescents need to be targeted specifically, prior to their first pregnancy and with measures adapted to their social context, addressing their structural constraints and needs and going beyond standard health promotion campaigns. Unless such specific measures are taken, adolescents'' social vulnerability will present a serious bottleneck for the effectiveness of IPTi-SP.