Induction of humoral and CD8+ T cell responses are required for protection against lethal Ebola virus infection

Ebola virus (EBOV)-like particles (eVLP), composed of the EBOV glycoprotein and matrix viral protein (VP)40 with a lipid membrane, are a highly efficacious method of immunization against EBOV infection. The exact requirements for immunity against EBOV infection are poorly defined at this time. The goal of this work was to determine the requirements for EBOV immunity following eVLP vaccination. Vaccination of BALB/c or C57BL/6 mice with eVLPs in conjunction with QS-21 adjuvant resulted in mixed IgG subclass responses, a Th1-like memory cytokine response, and protection from lethal EBOV challenge. Further, this vaccination schedule led to the generation of both CD4(+) and CD8(+) IFN-gamma(+) T cells recognizing specific peptides within glycoprotein and VP40. The transfer of both serum and splenocytes, but not serum or splenocytes alone, from eVLP-vaccinated mice conferred protection against lethal EBOV infection in these studies. B cells were required for eVLP-mediated immunity to EBOV because B cell-deficient mice vaccinated with eVLPs were not protected from lethal EBOV challenge. We also found that CD8(+), but not CD4(+), T cells are absolutely required for eVLP-mediated protection against EBOV infection. Further, eVLP-induced protective mechanisms were perforin-independent, but IFN-gamma-dependent. Taken together, both EBOV-specific humoral and cytotoxic CD8(+) T cell responses are critical to mediate protection against filoviruses following eVLP vaccination.     

Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning

The amoeba Dictyostelium discoideum is a well-established model organism for studying numerous aspects of cellular and developmental functions. Its rather small (~34Mb) chromosomal genome and the high efficiency of gene disruption by homologous recombination have enabled researchers to dissect various specific gene functions. We describe here the use of one-step cloning for the fast and efficient generation of deletion vectors that are produced in a one-step reaction by inserting two PCR products into an organism-specific, generic acceptor system. This worked efficiently for all 16 tested constructs directed against genes in the amoeba Dictyostelium discoideum. Saving cost and time, the used protocol represents a significant advancement in the generation of such plasmids compared to the conventionally applied restriction enzyme/ligation approach. Using appropriate selection markers, similar systems could also be useful in other organisms, where genes can be knocked out by homologous recombination.     

Association of ebola virus matrix protein VP40 with microtubules

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.     

Cell cycle mutation in Paramecium tetraurelia discriminates between sexual and vegetative functions

The temperature-sensitive mutation cc1 blocks a number of cell cycle processes in Paramecium including macronuclear DNA synthesis, oral morphogenesis, and the later stages of micronuclear mitosis. Oral morphogenesis and micronuclear mitosis also occur in the sexual pathway. This study shows that cc1 cells can proceed through conjugation or autogamy under restrictive conditions; neither stomatogenesis nor micronuclear mitosis is blocked. Fertilization and macronuclear determination occur normally, but DNA synthesis in macronuclear anlagen is blocked. Therefore, this mutation discriminates between oral replacement during meiosis and vegetative prefission stomatogenesis, and between mitotic spindle elongation during the pregamic and postzygotic divisions and spindle elongation during the vegetative cell cycle. These results point to a fundamental regulatory difference between morphogenesis in the vegetative and sexual pathways. © 1994 Wiley-Liss, Inc.     

Using Stable Isotopes to Differentiate Trophic Feeding Channels within Soil Food Webs

The soil is probably the most diverse habitat there is, with organisms ranging in sizes from less than 1 μm to several metres in length. However, it is increasingly evident that we know little about the interactions occurring between these organisms, the functions that they perform as individual species, or together within their different feeding guilds. These interactions between groups of organisms and physical and chemical processes shape the soil as a habitat and influence the nature of the soil food web with consequences for the above‐ground vegetation and food web. Protists are known as one of the most abundant groups of bacterivores within the soil; however, they are also consumers of a number of other food sources. Even though they are responsible for a large proportion of the mineralisation of bacterial biomass and have a large impact on the C and N cycles within the soil they are regularly overlooked when investigating the complete soil food web. Recently, stable isotopes have been used to determine trophic interactions and here we describe how this technique has been used to highlight linkages between protists and the soil food web.     

Nonhuman Transferrin Receptor 1 Is an Efficient Cell Entry Receptor for Ocozocoautla de Espinosa Virus

Ocozocoautla de Espinosa virus (OCEV) is a novel, uncultured arenavirus. We found that the OCEV glycoprotein mediates entry into grivet and bat cells through transferrin receptor 1 (TfR1) binding but that OCEV glycoprotein precursor (GPC)-pseudotyped retroviruses poorly entered 53 human cancer cell lines. Interestingly, OCEV and Tacaribe virus could use bat, but not human, TfR1. Replacing three human TfR1 amino acids with their bat ortholog counterparts transformed human TfR1 into an efficient OCEV and Tacaribe virus receptor.     

Functional Dissection of the Drosophila melanogaster Condensin Subunit Cap-G Reveals Its Exclusive Association with Condensin I

The heteropentameric condensin complexes have been shown to participate in mitotic chromosome condensation and to be required for unperturbed chromatid segregation in nuclear divisions. Vertebrates have two condensin complexes, condensin I and condensin II, which contain the same structural maintenance of chromosomes (SMC) subunits SMC2 and SMC4, but differ in their composition of non–SMC subunits. While a clear biochemical and functional distinction between condensin I and condensin II has been established in vertebrates, the situation in Drosophila melanogaster is less defined. Since Drosophila lacks a clear homolog for the condensin II–specific subunit Cap-G2, the condensin I subunit Cap-G has been hypothesized to be part of both complexes. In vivo microscopy revealed that a functional Cap-G-EGFP variant shows a distinct nuclear enrichment during interphase, which is reminiscent of condensin II localization in vertebrates and contrasts with the cytoplasmic enrichment observed for the other EGFP-fused condensin I subunits. However, we show that this nuclear localization is dispensable for Cap-G chromatin association, for its assembly into the condensin I complex and, importantly, for development into a viable and fertile adult animal. Immunoprecipitation analyses and complex formation studies provide evidence that Cap-G does not associate with condensin II–specific subunits, while it can be readily detected in complexes with condensin I–specific proteins in vitro and in vivo. Mass-spectrometric analyses of proteins associated with the condensin II–specific subunit Cap-H2 not only fail to identify Cap-G but also the other known condensin II–specific homolog Cap-D3. As condensin II–specific subunits are also not found associated with SMC2, our results question the existence of a soluble condensin II complex in Drosophila.     

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.     

A record of N2O and CH4 emissions and underlying soil processes of Korean rice paddies as affected by different water management practices

Rice is staple food of half of mankind and paddy soils account for the largest anthropogenic wetlands on earth. Ample of research is being done to find cultivation methods under which the integrative greenhouse effect caused by emitted CH₄ and N₂O would be mitigated. Whereas most of the research focuses on quantifying such emissions, there is a lack of studies on the biogeochemistry of paddy soils. In order to deepen our mechanistic understanding of N₂O and CH₄ fluxes in rice paddies, we also determined NO₃ ^? and N₂O concentrations as well as N₂O isotope abundances and presence of O₂ along soil profiles of paddies which underwent three different water managements during the rice growing season(s) in (2010 and) 2011 in Korea. Largest amounts of N₂O (2 mmol m^?2) and CH₄ (14.5 mol m^?2) degassed from the continuously flooded paddy, while paddies with less flooding showed 30–60 % less CH₄ emissions and very low to negative N₂O balances. In accordance, the global warming potential (GWP) was lowest for the Intermittent Irrigation paddy and highest for the Traditional Irrigation paddy. The N₂O emissions could the best be explained (*P < 0.05) with the δ¹⁵N values and N₂O concentrations in 40–50 cm soil depth, implying that major N₂O production/consumption occurs there. No significant effect of NO₃ ^? on N₂O production has been found. Our study gives insight into the soil of a rice paddy and reveals areas along the soil profile where N₂O is being produced. Thereby it contributes to our understanding of subsoil processes of paddy soils.