Expression of GM3 microdomains on the surfaces of murine fibroblasts correlates with inhibition of cell proliferation

The expression and surface distribution of monosialoganglioside GM3 on the plasma membranes of NIH3T3 fibroblasts cultured at semiconfluence were analyzed by immunofluorescence as well as by immunogold electron microscopy on thin sections and surface replicas. The GM3 expression was highly variable from cell to cell and the distribution of the ganglioside on the positive cells appeared punctate. Quantitative immunogold electron microscopy showed the existence of well-defined GM3 clusters of different sizes scattered all over the cell surfaces. Double immunofluorescence analysis of 5-bromo-2’-deoxyuridine incorporation to identify proliferating cells and of GM3 expression indicated that most of the GM3-positive cells appear unable to synthesize DNA and demonstrated a growth-dependent expression of GM3. Accepted: 16 November 1999     

Cutting edge: functional role for proline-rich tyrosine kinase 2 in NK cell-mediated natural cytotoxicity

Protein tyrosine kinase activation is one of the first biochemical events in the signaling pathway leading to activation of NK cell cytolytic machinery. Here we investigated whether proline-rich tyrosine kinase 2 (Pyk2), the nonreceptor protein tyrosine kinase belonging to the focal adhesion kinase family, could play a role in NK cell-mediated cytotoxicity. Our results demonstrate that binding of NK cells to sensitive target cells or ligation of beta2 integrins results in a rapid induction of Pyk2 phosphorylation and activation. By contrast, no detectable Pyk2 tyrosine phosphorylation is found upon CD16 stimulation mediated by either mAb or interaction with Ab-coated P815 cells. A functional role for Pyk2 in natural but not Ab-mediated cytotoxicity was demonstrated by the use of recombinant vaccinia viruses encoding the kinase dead mutant of Pyk2. Finally, we provide evidence that Pyk2 is involved in the beta2 integrin-triggered extracellular signal-regulated kinase activation, supporting the hypothesis that Pyk2 plays a role in the natural cytotoxicity by controlling extracellular signal-regulated kinase activation.     

Aberrant spermatogenesis and the peculiar mechanism of sex determination in Symphypleonan Collembola (Insecta)

Light and electron microscopy evidence have been obtained to describe the peculiar spermatogenesis in the collembolan species Sminthurus viridis and Allacma fusca (Sminthuridae). In these two species, the two sexes differ for the lack of two chromosomes (the sex chromosomes) in males (males, 2n = 10; females, 2n = 12). While oogenesis seems to proceed normally, spermatogenesis is peculiar because the two daughter cells of the first meiotic division have different chromosome numbers (six and four). The cell receiving four chromosomes degenerates, while the cell receiving six chromosomes completes meiosis and produces identical spermatozoa (n = 6). At fertilization, pronuclei with six chromosomes fuse together to form zygotes with 2n = 12. Male embryos must lose two sex chromosomes during the first zygotic mitosis, as all male cells have 2n = 10 chromosomes. The sex chromosome system of these species can be identified as X1X1X2X2:X1X20. Electron microscopy observations show that the same peculiar spermatogenesis occurs also in two others species of the same family, Caprainea marginata and Lipothrix lubbocki. The peculiar sex determination system described is similar but not identical to what is observed in other insect orders, and it may represent an evolutionary step toward parthenogenesis. It is suggested that this peculiar spermatogenesis is common to all Symphypleona.     

Characterization of caveolae from rat heart: localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse alpha- and beta-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states.     

Lipido‐sterolic extract of Serenoa repens (LSESr,Permixon®) treatment affects human prostate cancer cell membrane organization

The molecular mechanism by which the lipido‐sterolic extract of Serenoa repens (LSESr, Permixon®) affects prostate cells remains to be fully elucidated. In androgen‐independent PC3 prostate cancer cells, the LSESr‐induced effects on proliferation and apoptosis were evaluated by counting cells and using a FACScan cytofluorimeter. PC3 cells were stained with JC‐1 dye to detect mitochondrial membrane potential. Cell membrane lipid composition was evaluated by thin layer chromatography and gas chromatographic analysis. Akt phosphorylation was analyzed by Western blotting and cellular ultrastructure through electron microscopy. LSESr (12.5 and 25 µg/ml) administration exerted a biphasic action by both inhibiting proliferation and stimulating apoptosis. After 1 h, it caused a marked reduction in the mitochondrial potential, decreased cholesterol content and modified phospholipid composition. A decrease in phosphatidylinositol‐4,5‐bisphosphate (PIP2) level was coupled with reduced Akt phosphorylation. After 24 h, all of these effects were restored to pre‐treatment conditions; however, the saturated (SFA)/unsaturated fatty acid (UFA) ratio increased, mainly due to a significant decrease in ω6 content. The reduction in cholesterol content could be responsible for both membrane raft disruption and redistribution of signaling complexes, allowing for a decrease of PIP2 levels, reduction of Akt phosphorylation and apoptosis induction. The decrease in ω6 content appears to be responsible for the prolonged and more consistent increase in the apoptosis rate and inhibition of proliferation observed after 2–3 days of LSESr treatment. In conclusion, LSESr administration results in complex changes in cell membrane organization and fluidity of prostate cancer cells that have progressed to hormone‐independent status. J. Cell. Physiol. 219: 69–76, 2009. © 2008 Wiley‐Liss, Inc.     

Are Movements Necessary for the Sense of Body Ownership? Evidence from the Rubber Hand Illusion in Pure Hemiplegic Patients

A question still debated within cognitive neuroscience is whether signals present during actions significantly contribute to the emergence of human’s body ownership. In the present study, we aimed at answer this question by means of a neuropsychological approach. We administered the classical rubber hand illusion paradigm to a group of healthy participants and to a group of neurological patients affected by a complete left upper limb hemiplegia, but without any propriceptive/tactile deficits. The illusion strength was measured both subjectively (i.e., by a self-report questionnaire) and behaviorally (i.e., the location of one’s own hand is shifted towards the rubber hand). We aimed at examining whether, and to which extent, an enduring absence of movements related signals affects body ownership. Our results showed that patients displayed, respect to healthy participants, stronger illusory effects when the left (affected) hand was stimulated and no effects when the right (unaffected) hand was stimulated. In other words, hemiplegics had a weaker/more flexible sense of body ownership for the affected hand, but an enhanced/more rigid one for the healthy hand. Possible interpretations of such asymmetrical distribution of body ownership, as well as limits of our results, are discussed. Broadly speaking, our findings suggest that the alteration of the normal flow of signals present during movements impacts on human’s body ownership. This in turn, means that movements have a role per se in developing and maintaining a coherent body ownership.     

Health and Economic Outcomes of Introducing the New MenB Vaccine (Bexsero) into the Italian Routine Infant Immunisation Programme

Introduction

In January 2013 a novel type of multicomponent protein-based vaccine against group B meningococcal disease was licensed by the European Medicines Agency. With the widespread use of the meningococcal serogroup C conjugate vaccines, serogroup B remains now the major cause of bacterial meningitis and septicaemia in young children in Europe. The aim of this study is to investigate the health and the economic outcomes of MenB vaccine introduction into the Italian routine mass vaccination programme.

Methods

The present work is structured in two main parts. Firstly, we assess the epidemiological burden of group B meningococcal disease using official hospitalisation and notification data from two of the most populated Italian regions (Lombardia and Piemonte) during a 6-year study period (2007-2012). Secondly, we evaluate the cost-effectiveness of the immunisation programme in Italy from the public health payer perspective under base case parameters assumptions and performing a comprehensive sensitivity analysis to assess the robustness and the uncertainty of our model results.

Results

MenB serotype is responsible for 59% of the 341 cases of Invasive Meningococcal Disease in Lombardia and Piemonte. Incidence rate for MenB infection is estimated to be 0.21/100,000/y resulting at the highest level in children ≤4 years of age. Although the new MenB vaccine can potentially prevent about one third of the disease cases in the Italian population, model results show this strategy is unlikely to be cost-effective (ICER value over €350,000/QALY) with a vaccine that prevents disease only. These results are robust under most of the sensitivity scenarios except when allowing for lower discount rates.

Discussion

The introduction of the novel vaccine into the routine immunisation schedule needs to be carefully evaluated. The new MenB vaccine has the potential to reduce the disease burden at the population level. However, from the Italian Health Service perspective, the immunisation programme is unlikely to be cost-effective at the current incidence levels and vaccine price.     

Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.     

Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis

During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.     

Genetic Origins of Lactase Persistence and the Spread of Pastoralism in Africa

In humans, the ability to digest lactose, the sugar in milk, declines after weaning because of decreasing levels of the enzyme lactase-phlorizin hydrolase, encoded by LCT. However, some individuals maintain high enzyme amounts and are able to digest lactose into adulthood (i.e., they have the lactase-persistence [LP] trait). It is thought that selection has played a major role in maintaining this genetically determined phenotypic trait in different human populations that practice pastoralism. To identify variants associated with the LP trait and to study its evolutionary history in Africa, we sequenced MCM6 introns 9 and 13 and ∼2 kb of the LCT promoter region in 819 individuals from 63 African populations and in 154 non-Africans from nine populations. We also genotyped four microsatellites in an ∼198 kb region in a subset of 252 individuals to reconstruct the origin and spread of LP-associated variants in Africa. Additionally, we examined the association between LP and genetic variability at candidate regulatory regions in 513 individuals from eastern Africa. Our analyses confirmed the association between the LP trait and three common variants in intron 13 (C-14010, G-13907, and G-13915). Furthermore, we identified two additional LP-associated SNPs in intron 13 and the promoter region (G-12962 and T-956, respectively). Using neutrality tests based on the allele frequency spectrum and long-range linkage disequilibrium, we detected strong signatures of recent positive selection in eastern African populations and the Fulani from central Africa. In addition, haplotype analysis supported an eastern African origin of the C-14010 LP-associated mutation in southern Africa.