Steady State Analysis of Underground Electromagnetic Field Generated by Dipole Located over Ground

This paper presents the steady state analysis of an underground electromagnetic field induced by a sinusoidally excited dipole located over the ground. Theoretical derivation of the electromagnetic field is carried out in the complex frequency domain. The numerical method to calculate the obtained equation which contains complicated infinite integrals is presented

RNA-seq Transcriptional Profiling of an Arbuscular Mycorrhiza Provides Insights into Regulated and Coordinated Gene Expression in Lotus japonicus and Rhizophagus irregularis

Gene expression during arbuscular mycorrhizal development is highly orchestrated in both plants and arbuscular mycorrhizal fungi. To elucidate the gene expression profiles of the symbiotic association, we performed a digital gene expression analysis of Lotus japonicus and Rhizophagus irregularis using a HiSeq 2000 next-generation sequencer with a Cufflinks assembly and de novo transcriptome assembly. There were 3,641 genes differentially expressed during arbuscular mycorrhizal development in L. japonicus, approximately 80% of which were up-regulated. The up-regulated genes included secreted proteins, transporters, proteins involved in lipid and amino acid metabolism, ribosomes and histones. We also detected many genes that were differentially expressed in small-secreted peptides and transcription factors, which may be involved in signal transduction or transcription regulation during symbiosis. Coregulated genes between arbuscular mycorrhizal and root nodule symbiosis were not particularly abundant, but transcripts encoding for membrane traffic-related proteins, transporters and iron transport-related proteins were found to be highly co-up-regulated. In transcripts of arbuscular mycorrhizal fungi, expansion of cytochrome P450 was observed, which may contribute to various metabolic pathways required to accommodate roots and soil. The comprehensive gene expression data of both plants and arbuscular mycorrhizal fungi provide a powerful platform for investigating the functional and molecular mechanisms underlying arbuscular mycorrhizal symbiosis.ArticlePLANT AND CELL PHYSIOLOGY. 56(8):1490-1511 (2015)journal articl

RNA-seq Transcriptional Profiling of an Arbuscular Mycorrhiza Provides Insights into Regulated and Coordinated Gene Expression in Lotus japonicus

Gene expression during arbuscular mycorrhizal development is highly orchestrated in both plants and arbuscular mycorrhizal fungi. To elucidate the gene expression profiles of the symbiotic association, we performed a digital gene expression analysis of Lotus japonicus and Rhizophagus irregularis using a HiSeq 2000 next-generation sequencer with a Cufflinks assembly and de novo transcriptome assembly. There were 3,641 genes differentially expressed during arbuscular mycorrhizal development in L. japonicus, approximately 80% of which were up-regulated. The up-regulated genes included secreted proteins, transporters, proteins involved in lipid and amino acid metabolism, ribosomes and histones. We also detected many genes that were differentially expressed in small-secreted peptides and transcription factors, which may be involved in signal transduction or transcription regulation during symbiosis. Coregulated genes between arbuscular mycorrhizal and root nodule symbiosis were not particularly abundant, but transcripts encoding for membrane traffic-related proteins, transporters and iron transport-related proteins were found to be highly co-up-regulated. In transcripts of arbuscular mycorrhizal fungi, expansion of cytochrome P450 was observed, which may contribute to various metabolic pathways required to accommodate roots and soil. The comprehensive gene expression data of both plants and arbuscular mycorrhizal fungi provide a powerful platform for investigating the functional and molecular mechanisms underlying arbuscular mycorrhizal symbiosis.ArticlePLANT AND CELL PHYSIOLOGY. 56(8):1490-1511 (2015)journal articl

Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles

<div><p>Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites <i>via</i> GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.</p></div

Role of Xkr8 in PS exposure on the surface of virus particles.

<p>Xkr8 is transported to the budding sites together with GP <i>via</i> intracellular vesicles (A). VP40 is independently transported to the PM (B). Transported GP and Xkr8 are incorporated into virus particles (C). Incorporated Xkr8 is activated by a caspase (D), which leads to externalization of PS in the envelope of the EBOV particles (1–3).</p

Role of GP in the incorporation of Xkr8 in Ebola VLPs.

<p>(A) Characterization of incorporated Xkr8 in Ebola VLPs by means of flow cytometry. FBS- (left), Ebola VLP- (middle), or VLPΔGP- (right) conjugated beads incubated with the rabbit anti-VP40, GP, or Xkr8 polyclonal antibodies. For the binding of antibodies against VP40 and Xkr8, the beads were pre-treated with Triton X-100. 2^nd Ab indicates samples that were not treated with primary antibody. As a control, the rabbit anti-LASV GPC polyclonal antibody was used. The percentages of the positive populations are indicated. X-axis: fluorescence intensity, Y-axis: forward scatter corner signals. The results are representative of three individual experiments. (B) Characterization of incorporated Xkr8 in Ebola VLPs by western blotting. 293T cells were transfected with the expression plasmids of VP40 and NP, with or without GP. At 48 h.p.t., the cells and culture medium were harvested. Viral particles were purified from the culture medium. Total cell lysates and VLPs were analyzed by western blotting with the rabbit polyclonal antibodies against VP40, GP, Xkr8, or β-actin. (C) Binding of AF-ANX V to the beads. FBS- (left), Ebola VLP- (middle), or VLPΔGP- (right) conjugated beads were incubated with AF-ANX V. The percentages of the positive populations are indicated. X-axis: fluorescence intensity, Y-axis: forward scatter corner signals. The results are representative of three individual experiments. (D) Summary of the binding of the anti-Xkr8 antibody (left) and AF ANX V (right) to the Ebola VLP- or VLPΔGP-conjugated beads. Each experiment was performed in triplicate and the percentages of the positive populations are presented as the mean ± SD. **, <i>P</i> < 0.01 versus respective control (Student’s <i>t</i> test).</p

Effect of a pan-caspase inhibitor on the externalization of PS on the surface of Ebola VLPs and their internalization.

<p>(A) The effect of a pan-caspase inhibitor on Xkr8 in cell lysate and VLPs. 293T cells were transfected with the expression plasmids of EBOV VP40, GP, and NP and incubated for 48 h in the absence or presence of 20 μM Z-VAD-FMK. At 48 h.p.t., the cells and culture medium were harvested. Viral particles were purified from culture medium. Total cell lysates and VLPs were analyzed by western blotting with rabbit polyclonal antibodies against VP40, Xkr8, or β-actin. % of cleaved Xkr8 was analyzed as the ratio of the intensities of the cleaved bands to total Xkr8 bands. (B) The effect of Z-VAD-FMK treatment on the morphology of VLPs. 293T cells were transfected with the expression plasmids of EBOV VP40, NP, and GP and incubated for 48 h in the absence or presence of 20 μM Z-VAD-FMK. At 48 h.p.t., the culture medium was harvested. Viral particles were purified from the culture medium by ultracentrifugation followed by negative staining. Scale bars; 500 nm. (C–E) The effect of Z-VAD-FMK treatment on the incorporation of viral proteins and Xkr8 into VLPs and on the externalization of PS on the surface of Ebola VLPs. (C) Ebola VLPs obtained from untreated- (left) or Z-VAD-FMK-treated cells (right) were conjugated with latex beads. The beads were then incubated with rabbit polyclonal antibodies against EBOV GP, VP40, or Xkr8 followed by flow cytometric analysis. 2^nd Ab indicates samples that were not treated with primary antibody. As a control, the rabbit anti-LASV GPC polyclonal antibody was used. (D) For detection of externalized PS on the VLPs, the beads were incubated with AF-ANX V and subsequently subjected to flow cytometric analysis. The percentages of the positive populations are indicated. X-axis: fluorescence intensity, Y-axis: forward scatter corner signals. The results are representative of three individual experiments. (E) Summary of the binding of the anti-Xkr8 antibody (left) and AF ANX V (right) to VLPs released from untreated- or Z-VAD-FMK-treated cells. Each experiment was performed in triplicate and the percentages of the positive populations are presented as the mean ± SD. **, <i>P</i> < 0.01 versus respective control (Student’s <i>t</i> test). (F, G) The effect of Z-VAD-FMK treatment on VLP internalization. (F) Purified VLPs were labeled with DiI and adsorbed to Vero-E6 cells for 30 min at room temperature. After incubation for 2 h at 37°C, surface-bound virions were removed by trypsinization for 5 min at 37°C and the internalization of the Ebola VLPs was analyzed by using a confocal laser scanning microscope. (G) The number of internalized DiI-VLPs in 10 individual cells was measured. Each experiment was performed in triplicate and the relative uptake efficiencies are presented as the mean ± SD. **, <i>P</i> < 0.01 versus respective control (Student’s <i>t</i> test).</p