Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination

Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.     

The Relationship between Fenestrations,Sieve Plates and Rafts in Liver Sinusoidal Endothelial Cells

Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished.     

RNA-Seq vs Dual- and Single-Channel Microarray Data: Sensitivity Analysis for Differential Expression and Clustering

With the fast development of high-throughput sequencing technologies, a new generation of genome-wide gene expression measurements is under way. This is based on mRNA sequencing (RNA-seq), which complements the already mature technology of microarrays, and is expected to overcome some of the latter’s disadvantages. These RNA-seq data pose new challenges, however, as strengths and weaknesses have yet to be fully identified. Ideally, Next (or Second) Generation Sequencing measures can be integrated for more comprehensive gene expression investigation to facilitate analysis of whole regulatory networks. At present, however, the nature of these data is not very well understood. In this paper we study three alternative gene expression time series datasets for the Drosophila melanogaster embryo development, in order to compare three measurement techniques: RNA-seq, single-channel and dual-channel microarrays. The aim is to study the state of the art for the three technologies, with a view of assessing overlapping features, data compatibility and integration potential, in the context of time series measurements. This involves using established tools for each of the three different technologies, and technical and biological replicates (for RNA-seq and microarrays, respectively), due to the limited availability of biological RNA-seq replicates for time series data. The approach consists of a sensitivity analysis for differential expression and clustering. In general, the RNA-seq dataset displayed highest sensitivity to differential expression. The single-channel data performed similarly for the differentially expressed genes common to gene sets considered. Cluster analysis was used to identify different features of the gene space for the three datasets, with higher similarities found for the RNA-seq and single-channel microarray dataset.     

Rabx-5 regulates RAB-5 early endosomal compartments and synaptic vesicles in C. elegans

Early endosomal membrane compartments are required for the formation and recycling of synaptic vesicles, but how these compartments are regulated is incompletely understood. We performed a forward genetic screen in C. elegans for mutations that affect RAB-5 labeled early endosomal compartments in GABAergic motoneurons. Here we report the isolation and characterization of one mutation, rabx-5. The rabx-5 mutation leads to decreased intensity of YFP::RAB-5 in the cell soma but increased intensity in the synaptic and intersynaptic regions of the axon. This effect is due to the bias of the cycling state of RAB-5, and results from a change in the organization of the early endosomal compartment as well as the membrane binding state of RAB-5. Synaptic vesicle accumulation is altered in rabx-5 mutants, and synaptic transmission from cholinergic neurons is decreased. Early endosomal membrane compartments show disorganization with ageing and rabx-5 mutant animals age faster. These results suggest that rabx-5 regulation of RAB-5 compartments is important for maintaining proper synaptic function throughout the lifetime.     

Molecular identification of lyso-glycerophosphocholines as endogenous immunosuppressives in bovine and rat gonadal fluids

The ability of the gametes to escape detection by the immune system is vital to successful human reproduction. Furthermore, the observed capacity of the testis in some species to support tissue grafts without rejection (immunological privilege) indicates that spermatogenic cells are protected by local immunoregulatory mechanisms. One of these mechanisms involves targeting T cells for inactivation and destruction within the testicular environment. Although the fluids of the testis and ovary surrounding the developing gametes contain soluble factors that inhibit T cells, the identity of the molecule(s) responsible for this activity has been unknown. Using a specific T-cell proliferation assay to monitor bioactivity, these molecules were purified from bovine ovarian follicular fluid by methanol extraction and sequential reverse-phase HPLC (RP-HPLC). All purified active fractions coincided with the elution position on RP-HPLC of several small molecules ranging in size from 496 to 522 Da. The same molecules were localized to the immunosuppressive fractions of rat testicular interstitial fluid. The active molecules were identified, using capillary electrophoresis electrospray ionization mass spectroscopy, as lyso-glycerophosphocholines (lyso-GPCs), namely, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-oleoyl-sn-glycero-3-phosphocholine, a 18:2a/lyso-GPC (putatively, 1-linoleoyl-sn-glycero-3-phosphocholine), and a 20:4a/lyso-GPC (putatively, 1-arachidonyl-sn-glycero-3-phosphocholine). Comparison of the bioactivity and mass spectroscopy profiles of two of the purified molecules with their synthetic standards confirmed the identification. These molecules inhibit T-cell proliferation in response to activation and induce apoptosis of these cells in a time- and dose-dependent manner. The emergence of gonadal lyso-GPCs as potential regulators of critical immune events opens up new avenues of inquiry into the origins of autoimmune infertility and more generally into mechanisms of peripheral immunoregulation and the development of novel immunosuppressives.     

Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane

Aquaporin-1 (AQP1) is an integral membrane protein that facilitates osmotic water transport across cell plasma membranes in epithelia and endothelia. AQP1 has no known specific interactions with cytoplasmic or membrane proteins, but its recovery in a detergent-insoluble membrane fraction has suggested possible raft association. We tracked the membrane diffusion of AQP1 molecules labeled with quantum dots at an engineered external epitope at frame rates up to 91 Hz and over times up to 6 min. In transfected COS-7 cells, >75% of AQP1 molecules diffused freely over ∼7 μm in 5 min, with diffusion coefficient, D_1-3 ∼ 9 × 10⁻¹⁰ cm²/s. In MDCK cells, ∼60% of AQP1 diffused freely, with D_1-3 ∼ 3 × 10⁻¹⁰ cm²/s. The determinants of AQP1 diffusion were investigated by measurements of AQP1 diffusion following skeletal disruption (latrunculin B), lipid/raft perturbations (cyclodextrin and sphingomyelinase), and bleb formation. We found that cytoskeletal disruption had no effect on AQP1 diffusion in the plasma membrane, but that diffusion was increased greater than fourfold in protein de-enriched blebs. Cholesterol depletion in MDCK cells greatly restricted AQP1 diffusion, consistent with the formation of a network of solid-like barriers in the membrane. These results establish the nature and determinants of AQP1 diffusion in cell plasma membranes and demonstrate long-range nonanomalous diffusion of AQP1, challenging the prevailing view of universally anomalous diffusion of integral membrane proteins, and providing evidence against the accumulation of AQP1 in lipid rafts.     

Caeoma tsukubaense n. sp., a rhododendron rust fungus of Japan and southern Asia, and its relationship to Chrysomyxa rhododendri

A rust fungus found in Japan on Rhododendron kaempferi, R. kiusianum, and R. dauricum has previously been identified as Chrysomyxa rhododendri. Light and scanning electron microscopy of fresh and herbarium materials of the rust fungus, however, show that the spore surface morphology differs from the urediniospores of C. rhododendri, and the spores are slightly smaller. Furthermore, the DNA sequence of the 5′-end of the large subunit of ribosomal DNA differs from that of C. rhododendri by 3%. Telia have not been found; therefore, it is redescribed as a new anamorphic species, Caeoma tsukubaense. Several specimens from North Korea, Tibet, and Nepal bearing a similar rust fungus are also included in the species.Contribution no.193 from the Laboratory of Plant Parasitic Mycology, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan     

Climate Change Influences on Environmental and Human Health Chemical Standards

Regulatory agencies use many chemical standards to help protect human health and the environment. Some of these standards could potentially be adversely influenced by climate change, so it is important to scope the likelihood of such changes, and develop a plan for addressing them. A review of the literature showed that many complex interactions could occur between climate change factors, chemicals, and receptors. However, the way in which chemical standards are currently set is already likely to take into account predicted climate changes in temperate regions, at least implicitly. In contrast, compliance monitoring to ensure that chemical standards are not breached may be compromised if climate change leads to more variability in measured parameters, with a consequent reduction in the statistical power of monitoring programs.     

A radioimmunoprecipitation method in the serodiagnosis of HIV infection: the development of the optimal conditions for its performance and the evaluation of the possibilities for its use

The optimum conditions for using the method of radioimmunoprecipitation (RIP) for the detection of human immunodeficiency virus (HIV) in serum samples have been established. Out of several available cell lines persistently infected with HIV, specially selected line 17 has been chosen. The characteristic feature of this is the high and stable (under the conditions of prolonged cultivation) accumulation of virus-specific proteins in infected cells. The optimum conditions for making the test and its evaluation have also been established. The data of literature on the advantages of the method of RIP over such traditional methods as the enzyme immunoassay and immunoblotting have been confirmed. Thus, the presence of specific antibodies in several serum samples registered as false negative has been established. The intertypical reactivity of two serotypes of the virus, HIV-1 and HIV-2, has been studied. Cross reactivity of antibodies with respect to the HIV gene gag, but not with respect to viral glycoproteids, has been established. Ideas on the expediency and prospects of using RIP for the serological control of HIV infection are presented.