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Stent grafts (SG) and drug eluting stents (DES) have emerged to combat intimal hyperplasia. It remains unclear which type of stent yields superior outcomes in femoropopliteal (FP) arterial lesions. This study compared the clinical data between the VIABAHN SG and the Eluvia DES two years after endovascular treatment.In this retrospective multicentre study, 504 cases with a lesion length10 cm treated either with SG or DES were analysed. Ankle brachial index (ABI) measurements were conducted before and after the endovascular procedure, and every three months thereafter. When the ABI dropped ≥ 0.15 compared with the baseline value, a duplex ultrasound was conducted to check stent patency. The outcome measures were stent patency rates, freedom from target lesion revascularisation (TLR), stent thrombosis, and acute limb ischaemia (ALI) accompanying loss of patency rates. Propensity score matching (PSM) was performed to adjust for confounding baseline characteristics.PSM extracted 219 limbs in the SG group and 109 limbs in the DES group. Compared with the SG group, the DES group had statistically significantly higher rates of freedom from TLR (86.0 ± 4.2% vs. 73.1 ± 4.8%, p = .040), and ALI accompanying loss of patency (98.9 ± 1.1% vs. 93.5 ± 1.8%, p = .029) at two years. Primary patency (75.9 ± 5.9% vs. 69.5 ± 5.9%, p = .087) and freedom from stent thrombosis (90.4 ± 3.3% vs. 81.2% ± 3.0%, p = .11) were not statistically significantly different. For lesions ≤ 15 cm, primary patency in the DES group was statistically significantly better than the SG group.FP lesions treated with Eluvia DES had a higher primary patency rate in lesions ≤ 15 cm, freedom from clinically driven TLR and ALI accompanying loss of patency than the VIABAHN SG.

Herein, we successfully improved the maximum oscillation frequency and maximum stable gain (MSG) across a wide bias range of surface-oxide-controlled (SOC) InGaAs/InAlAs inverted-type metal-oxide-semiconductor high-electron-mobility transistors (inverted MOS-HEMTs) by reducing the gate leakage current and drain conductance (g d). H2O vapor treatment selectively decreased the narrow band gap indium oxide at the surface of the In-based epitaxial layer via the SOC process before the gate oxide deposition. Furthermore, the calculation of band profiles indicated that the gd reduction was possibly attributed to the suppression of impact ionization in the InGaAs channel by inverted MOS-HEMTs. Consequently, SOC-inverted MOS-HEMTs demonstrated a high MSG of >12 dB at 100 GHz across a wide bias range.

We investigated virus infection in the plant pathogenic oomycete Globisporangium splendens, formerly classified as Pythium splendens, in Japan. From 12 strains investigated, three strains contained virus-like double-stranded (dsRNA). Next-generation sequencing revealed that the G. splendens strain MAFF 425508 and MAFF 305867 contained a virus related to toti-like viruses, that we named Pythium splendens RNA virus 1 (PsRV1). PsRV1 has a ca. 5700 nt-length genome encoding two overlapping open reading frames (ORFs). The ORF2 encodes an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis with deduced RdRp amino acid sequences indicated that PsRV1 was closely related to Pythium polare RNA virus 1 (PpRV1) from G. polare infecting mosses in the Arctic. PsRV1 was vertically transmitted through the hyphal swellings, vegetative organs of G. splendens, in a temperature-dependent manner. Also, we showed that PsRV1 infected in a symptomless manner.

Three peptaibols, trichorzins HA II (1), HA V (2), and HA VI (3), were isolated from okara fermented with Trichoderma harzianum HK-61 as anti-plant viral agents. Their structures were confirmed by spectroscopic and chemical methods. At micro molar concentrations, the trichorzins inhibited infections by Cucumber mosaic virus in the cowpea plant Vigna sesquipedalis.

We investigated virus infection in the oomycete Pythium polare from the Arctic. From 39 isolates investigated, 14 contained virus-like double-stranded RNA (dsRNA). Next generation sequencing revealed that the P. polare isolate OPU1176 contained three different virus-like sequences. We determined the full-length genome sequence of one of them. The 5397 nt-length genome had two overlapped open reading frames (ORFs) consistent with a toti and toti-like viruses, that we named Pythium polare RNA virus 1 (PpRV1). The ORF2 encoded an RNA-dependent RNA polymerase (RdRp). The shifty heptamer motif and RNA pseudoknot were predicted near the stop codon of ORF1, implying that the RdRp could be translated as a fusion protein with the ORF1 protein. Phylogenetic analysis with deduced RdRp amino acid sequences indicated that oomycete virus PpRV1 was closely related to the unclassified arthropod toti-like viruses. The comparison of PpRV1-free and -infected lines suggested that PpRV1 infected in a symptomless manner.

Colletotrichum fructicola is a major causal agent among anthracnose pathogens of strawberry in Nara, Japan. We hypothesized that a wide range of weeds growing in and around strawberry fields are inoculum sources of the disease and investigated their potential as hosts of C. fructicola. We also examined the influence of herbicide treatment on C. fructicola sporulation on weeds. The fungus was detected on 31 of 541 (5.7%) leaves sampled from 13 weed species from 2005 to 2008. The fungus was most frequently isolated from leaves of Amaranthus blitum with an isolation frequency of 17.9%; inoculation of A. blitum with the pathogen caused brown leaf spots. Other weeds such as Digitaria ciliaris, Galinsoga ciliata, Solidago altissima, Erigeron annuus, and Sonchus oleraceus were found to harbor the fungus at lower rates (4.3–8.1%) without symptoms. C. fructicola formed acervuli on leaves of A. blitum, D. ciliaris, and S. oleraceus after plants were killed by a herbicide (glyphosate). These results demonstrated that infected weeds associated with strawberry cultivation are potential inoculum sources of C. fructicola, especially after herbicide treatment.

A rapid diagnostic method for Japanese yam mosaic virus (JYMV) infection in Chinese yam (Dioscorea polystachya) by a print-capture RT-PCR was developed. The cut surface of a rolled leaf of a JYMV-infected Chinese yam was stamped onto a nitrocellulose membrane. The clipped membrane was boiled in eluting solution to elute the RNA for use as the template in RT-nested PCR. Although JYMV RNA was detectable in most infected plants in the first PCR of the print-capture RT-PCR, it was detected from all samples in the nested PCR. The tissue-printed membranes could be preserved for at least 3 months at 4 °C for JYMV detection.

The mycoparasitic oomycete Pythium nunn isolate UZ415 contains two double-stranded RNAs (dsRNAs) of different sizes. The 1707-nt dsRNA1 and the 1475-nt dsRNA2 potentially encode an RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively, with sequence similarity to the RdRp and CP of gammapartitiviruses (< 57% and < 36%). Phylogenetic analysis of the deduced RdRp amino acid sequences indicated that the virus identified from P. nunn is classifiable as a distinct member of the genus Ganmmapartitivirus in the family Partitiviridae. This virus isolate is hereby named Pythium nunn virus 1 (PnV1).

We investigated the synergistic effects of co-infection by zucchini yellow mosaic virus (ZYMV) and cucumber mosaic virus (CMV) on viral distribution in the vascular tissues of cucumber. Immunohistochemical observations indicated that ZYMV was present in both the phloem and xylem tissues. ZYMV-RNA was detected in both the xylem wash and guttation fluid of ZYMV-inoculated cucumber. Steam treatment at a stem internode indicated that ZYMV enters the xylem vessels and moves through them but does not cause systemic infection in the plant. CMV distribution in singly infected cucumbers was restricted to phloem tissue. By contrast, CMV was detected in the xylem tissue of cotyledons in plants co-infected with CMV and ZYMV. Although both ZYMV-RNA and CMV-RNA were detected in the xylem wash and upper internodes of steam-treated, co-infected cucumbers grown at 24 °C, neither virus was detected in the upper leaves using an ELISA assay. Genetically modified CMV harboring the ZYMV HC-Pro gene was distributed in the xylem and phloem tissues of singly inoculated cucumber cotyledons. These results indicate that the ZYMV HC-Pro gene facilitates CMV entry into the xylem vessels of co-infected cucumbers.

Neutral electrolyzed water (NEW: pH 6.5–7.5) applied through an overhead irrigation system was evaluated for control of strawberry anthracnose caused by Colletotrichum fructicola. Conidia of the pathogen were completely killed by a 10-s exposure to 10.0 mg/L of available chlorine in the NEW. Disease suppression was significantly higher using the NEW treatment through overhead irrigation, either alone or combined with fungicides, than using conventional fungicides. Plants had no visible phytotoxicity after the NEW treatment, even when combined with fungicides. Thus, the NEW treatment was effective at controlling anthracnose caused by C. fructicola.

Modified lipoproteins can negatively affect beta cell function and survival. However, the mechanisms behind interactions of modified lipoproteins with beta cells – and in particular, relationships to increased uptake – are only partly clarified. By over-expressing the scavenger receptor CD36 (Tet-on), we increased the uptake of fluorescent low-density modified lipoprotein (oxLDL) into insulin-secreting INS-1 cells. The magnitude of uptake followed the degree of CD36 over-expression. CD36 over-expression increased concomitant efflux of3H-cholesterol in proportion to the cellular contents of3H-cholesterol. Exposure to concentrations of oxLDL from 20 to 100 µg/mL dose-dependently increased toxicity (evaluated by MTT) as well as apoptosis. However, the increased uptake of oxLDL due to CD36 over-expression did not exert additive effects on oxLDL toxicity – neither on viability, nor on glucose-induced insulin release and cellular content. Reciprocally, blocking CD36 receptors by Sulfo-N-Succinimidyl Oleate decreased the uptake of oxLDL but did not diminish the toxicity. Pancreatic islets of CD36−/−mice displayed reduced uptake of3H-cholesterol-labeled oxLDL vs wild type but similar toxicity to oxLDL. OxLDL was found to increase the expression of CD36 in islets and INS-1 cells. In summary, given the experimental conditions, our results indicate that (1) increased uptake of oxLDL is not responsible for toxicity of oxLDL, (2) increased efflux of the cholesterol moiety of oxLDL counterbalances, at least in part, increased uptake and (3) oxLDL participates in the regulation of CD36 in pancreatic islets and in INS-1 cells.

OBJECTIVE: Procedural characteristics, including stent design, may influence the outcome of carotid artery stenting (CAS). A thorough comparison of the effect of stent design on outcome of CAS is thus warranted to allow for optimal evidence-based clinical decision making. This study sought to evaluate the effect of stent design on clinical and radiologic outcomes of CAS. METHODS: A systematic search was conducted in MEDLINE, Embase, and Cochrane databases in May 2018. Included were articles reporting on the occurrence of clinical short- and intermediate-term major adverse events (MAEs; any stroke or death) or radiologic adverse events (new ischemic lesions on postprocedural magnetic resonance diffusion-weighted imaging [MR-DWI], restenosis, or stent fracture) in different stent designs used to treat carotid artery stenosis. Random effects models were used to calculate combined overall effect sizes. Metaregression was performed to identify the effect of specific stents on MAE rates. RESULTS: From 2654 unique identified articles, two randomized, controlled trials and 66 cohort studies were eligible for analysis (including 46,728 procedures). Short-term clinical MAE rates were similar for patients treated with open cell vs closed cell or hybrid stents. Use of an Acculink stent was associated with a higher risk of short-term MAE compared with a Wallstent (risk ratio [RR], 1.51; P = .03), as was true for use of Precise stent vs Xact stent (RR, 1.55; P < .001). Intermediate-term clinical MAE rates were similar for open vs closed cell stents. Use of open cell stents predisposed to a 25% higher chance (RR, 1.25; P = .03) of developing postprocedural new ischemic lesions on MR-DWI. No differences were observed in the incidence of restenosis, stent fracture, or intraprocedural hemodynamic depression with respect to different stent design. CONCLUSIONS: Stent design is not associated with short- or intermediate-term clinical MAE rates in patients undergoing CAS. Furthermore, the division in open and closed cell stent design might conceal true differences in single stent efficacy. Nevertheless, open cell stenting resulted in a significantly higher number of subclinical postprocedural new ischemic lesions detected on MR-DWI compared with closed cell stenting. An individualized patient data meta-analysis, including future studies with prospective homogenous study design, is required to adequately correct for known risk factors and to provide definite conclusions with respect to carotid stent design for specific subgroups.

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.

In tobacco plants, the Cucumber mosaic virus (CMV) pepo strain induces mosaic symptoms, including pale green chlorosis and malformed tissues. Here, we characterized the involvement of 2b protein and coat protein (CP) in the development of mosaic symptoms. A 2b mutant (R46C) that lacks viral suppressor of RNA silencing (VSR) activity showed an asymptomatic phenotype with low levels of virus accumulation. Tomato spotted wilt virus NSs protein did not complement the virulence of the R46C, although it did restore high-level virus accumulation. However, R46C mutants expressing mutated CP in which the amino acid P129 was mutated to A, E, C, Q, or S induced chlorosis that was associated with reduced expression of chloroplast and photosynthesis related genes (CPRGs) and abnormal chloroplasts with fewer thylakoid membranes. These results suggest that the CP of the CMV pepo strain acquires virulence by amino acid mutations, which causes CPRG repression and chloroplast abnormalities.

Cucumber mosaic virus (CMV) causes mosaic disease in inoculated tobacco plants. Coat protein (CP) is one of the major virulence determinants of CMV, and an amino acid substitution at residue 129 in CP alters the severity of chlorosis, such as pale green chlorosis and white chlorosis, in symptomatic tissues of mosaic leaves of infected tobacco. In this study, we compared the transcriptomes of chlorotic tissues infected with the wild-type pepo strain of CMV and two strains carrying CP mutants with diverse chlorosis severity. Differential gene expression analysis showed that CMV inoculation appeared to have similar effects on the transcriptional expression profiles of the symptomatic chlorotic tissues, and only the magnitude of expression differed among the different CMVs. Gene ontology analysis with biological process and cellular component terms revealed that many nuclear genes related to abiotic stress responses, including responses to cadmium, heat, cold and salt, were up-regulated, whereas chloroplast- and photosynthesis-related genes (CPRGs) were down-regulated, in the chlorotic tissues. Interestingly, the level of CPRG down-regulation was correlated with the severity of chlorosis. These results indicate that CP mutation governs the repression level and mRNA accumulation of CPRGs, which are closely associated with the induction of chlorosis.

SUMMARY Taxonomic relationships:Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus in the family Bromoviridae, which also encompasses the Peanut stunt virus (PSV) and the Tomato aspermy virus (TAV). Nucleotide sequence similarity among these three cucumoviruses is 60%–65%. CMV strains are divided into three subgroups, IA, IB and II, based on the sequence of the 5′ untranslated region of the genomic RNA 3. Overall nucleotide sequence similarity among CMV strains is approximately 70%–98%. Geographical distribution, host range and symptomatology: CMV is distributed worldwide, primarily in temperate to tropical climate zones. CMV infects more than 1200 species of 100 plant families, including monocot and dicot plants. Symptoms caused by CMV infection vary with the host species and/or CMV strain, and include mosaic, stunt, chlorosis, dwarfing, leaf malformation and systemic necrosis. CMV disease is spread primarily by aphid transmission in a nonpersistent manner. Physical properties: In tobacco sap, the thermal inactivation point of the viral infectivity is approximately 70 °C (10 min), the dilution end-point is approximately 10−4 and viral infectivity is lost after a few days of exposure to 20 °C. Viral infectivity can be retained in freeze-dried tissues and in the form of virions purified using 5 mm sodium borate, 0.5 mm ethylenediaminetetraacetic acid and 50% glycerol (pH 9.0) at −20 °C. CMV particles are isometric, approximately 28–30 nm in diameter and are composed of 180 capsid subunits arranged in pentamer–hexamer clusters with T= 3 symmetry. The sedimentation coefficient (s20,w) is c. 98 S and the particle weight is (5.8–6.7) × 106 Da. The virions contain 18% RNA. The RNA–protein interactions that stabilize the CMV virions are readily disrupted by sodium dodecylsulphate or neutral chloride salts. Genomic properties: The genomic RNAs are single-stranded messenger sense RNAs with 5′ cap and 3′ tRNA-like structures containing at least five open reading frames. The viral RNA consists of three genomic RNAs, RNA 1 (c. 3.3 kb), RNA 2 (c. 3.0 kb) and RNA 3 (c. 2.2 kb), and two subgenomic RNAs, RNA 4 (c. 1.0 kb) and RNA 4A (c. 0.7 kb). The 3′ untranslated regions are conserved across all viral RNAs. CMV is often accompanied by satellite, noncoding, small, linear RNA that is nonhomologous to the helper CMV. Useful websites:http://www.dpvweb.net/dpv/showdpv.php?dpvno=400 (general information); http://www.rcsb.org/pdb/explore/explore.do?pdbId=1f15 (structure).

Previously, we have reported that purification and characterization of an anti-plant viral serine protease (EF-SP1) from Eisenia foetida . In this study, a new serine protease (EF-SP2) showing strong antiviral activities against cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) was purified from the coelomic fluid of the earthworm E. foetida . The activity of EF-SP2 was suppressed by various known serine protease inhibitors, suggesting that the EF-SP2 is a serine protease. Its molecular weight was estimated to be 26,000 by SDS–PAGE, and its optimal pH and temperature were pH 9.5 and 60 °C, respectively. N-terminal amino acid sequence of EF-SP2 was the same as those of E. foetida serine proteases (EFE-d and EFE-e) with fibrinolytic activity, but different from that of EF-SP1. The enzymatic properties of anti-plant serine proteases (EF-SP1 and EF-SP2) and fibrinolytic enzymes (EFE-d and EFE-e) were similar to each other, e.g., substrate specificity, molecular weight, and effect of inhibitors. Our results suggest that EF-SP2 as well as EF-SP1 can be also applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.

Tobacco plants inoculated with the Cucumber mosaic virus pepo strain (CMV) induced cyclic mosaic symptom expression; mosaic and mottled leaves appeared first among the newly developing leaves (L7-9) above inoculated leaf (L0). Next, symptomless leaves developed (L10-11), and then mosaic leaves appeared again (L12-14). To clarify the formation of the cyclic symptoms by CMV, the relationships between viral amounts and distributions in the leaf primordia (2 to 3 mm) or young developing leaves (1 to 2 cm) and symptom severity in the fully expanded leaves were analyzed. Large amounts of CMV were detected in most cells of the young developing leaves in the early infection periods, which later led to the development of mosaic and mottled leaves. Virtually no signals were detected in the leaf primordia/young developing leaves for L11, which developed into symptomless leaves. However, CMV was already distributed in the young developing leaves at L13 in the later mosaic leaves. The relationship between the viral distributions in the leaf primordia/young developing leaves and the symptoms in the fully developed leaves in CMV-infected tobacco is discussed.

The symptomatic effect of the amino acid type at residue 129 in the coat protein of cucumber mosaic virus was investigated in tobacco using coat protein mutants of the pepo strain in which proline 129 was substituted with 19 other amino acids. These mutants caused six types of symptoms: white mosaic, pale green mosaic, veinal chlorosis, veinal necrosis, systemic necrosis, and necrotic local lesions. Transmission electron microscopy revealed that the chloroplasts of plants showing the three former types of symptoms contained few thylakoid membranes. Cytopathic effects characteristic of cells from plants showing the three latter symptom types were not observed.

It has been reported previously that a 2b protein-defective mutant of the cucumber mosaic virus (CMV) Pepo strain (Delta 2b) induces only mild symptoms in systemically infected tobacco plants. To clarify further the role of the 2b protein as an RNA silencing suppressor in mosaic symptom expression during CMV infection, this study monitored the sequential distribution of Delta 2b in the shoot meristem and leaf primordia (LP) of inoculated tobacco. Time-course histochemical observations revealed that Delta 2b was distributed in the shoot meristem at 7 days post-inoculation (p.i.), but could not invade shoot apical meristem (SAM) and quickly disappeared from the shoot meristem, whereas wild-type (Pepo) transiently appeared in SAM from 4 to 10 days p.i. In LP, Delta 2b signals were detected only at 14 and 21 days p.i., whereas dense Pepo signals were observed in LP from 4 to 18 days p.i. Northern blot analysis showed that small interfering RNA (siRNA) derived from Delta 2b RNA accumulated earlier in the shoot meristem and LP than that of Pepo. However, a similar amount of siRNA was detected in both Pepo- and Delta 2b-infected plants at late time points. Tissue printing analysis of the inoculated leaves indicated that the areas infected by Pepo increased faster than those infected by Delta 2b, whereas accumulation of Delta 2b in protoplasts was similar to that of Pepo. These findings suggest that the 2b protein of the CMV Pepo strain determines virulence by facilitating the distribution of CMV in the shoot meristem and LP via prevention of RNA silencing and/or acceleration of cell-to-cell movement.

We analysed the kinetics of virus distribution in Tobacco ringspot virus (TRSV)-infected tobacco. Infected plants developed asymptomatic leaves above the symptomatic leaves that exhibited ringspots. We detected virus in the shoot apical meristem (SAM). Time-course immunohistochemical microscopy showed that TRSV continuously distributed in the centres of the SAM and in leaf primordia. In contrast, TRSV infection was transient in the root meristem. In western blot and RT-PCR analyses, TRSV infection occurred only within and around the ringspot tissues of the symptomatic leaf; however, TRSV was not or hardly detected in the asymptomatic leaves of inoculated tobacco plants. These results indicate that TRSV persists in the SAM, but that TRSV infection is transient in the root meristem and asymptomatic leaves.

A novel protein showing strong antiviral activities against cucumber mosaic virus (CMV) and tomato mosaic virus (TMV) was purified from the coelomic fluid of the earthworm Eisenia foetida. The protein was characterized as a cold-adapted serine protease. Its molecular weight was estimated to be 27,000 by SDS-PAGE. The enzyme was most active at pH 9.5 and 40-50 degrees C. The protease activity at 4 degrees C was 60% of that obtained at the optimal temperature. The activity was suppressed by various serine protease inhibitors. Partial N-terminal amino acid sequence of the enzyme showed homology with serine proteases of earthworms, E. foetida and Lumbricus rubellus previously studied. Our results suggest that the enzyme can be applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.

A virus that caused a distinct yellow mosaic was isolated in Okayama, Japan from Chinese cabbage (Brassica rapa L., Pekinensis group). The virus, with spherical particles ca. 28 nm in diameter, was mechanically transmissible only to cruciferous species. From the host range, characteristic morphology of virus particles, serology and sequence analysis of coat protein gene, the causal virus was identified as Turnip yellow mosaic virus (TYMV). Seed transmission of TYMV at 0–2.2% in Chinese cabbage was confirmed. This report is the first of TYMV from Chinese cabbage and in Japan.

Apple latent spherical virus (ALSV) expressing green fluorescent protein (GFP-ALSV) was used for analysis of virus-induced gene silencing (VIGS) in tobacco plants expressing GFP (GFP-tobacco). In GFP-tobacco inoculated with GFP-ALSV, small dark spots appeared on inoculated leaves at 5 days post-inoculation (dpi), then expanded, and finally covered the whole area of the leaves after 12 dpi. Most of the fluorescence of upper leaves above the 12th true leaf disappeared at 21 dpi. Thus, GFP-ALSV infection efficiently triggered VIGS of a transgene (GFP gene) in tobacco plants. Analysis of GFP-silenced leaves showed that viral RNAs and proteins accumulated in all leaves where most GFP mRNA had been degraded. The siRNAs derived from ALSV-RNAs were not detected in samples from which siRNA of GFP mRNA could be easily detected. Direct tissue blot analysis showed that the spread of GFP-ALSV always preceded the induction of VIGS in infected leaves of GFP-tobacco. GFP leaf patch tests using Nicotiana benthamiana line 16c showed that Vp20, one of the three capsid proteins, is a silencing suppressor which interferes with systemic silencing.

Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus and its single-stranded, plus-sense RNA genome encodes a 216 kDa protein (P216) involved in replication, a 50 kDa movement protein (P50) and a 21 kDa coat protein (CP). In this study, it was investigated whether these proteins might have RNA silencing-suppressor activities by Agrobacterium-mediated transient assay in the green fluorescent protein-expressing Nicotiana benthamiana line 16c. The results indicated that none of these proteins could suppress local silencing in infiltrated leaves. However, systemic silencing in upper leaves induced by both single- and double-stranded RNA could be suppressed by P50, but not by a frame-shift mutant of P50, P216 or CP. Moreover, when P50 was expressed separately from where silencing signals were generated in a leaf, systemic silencing in upper leaves was inhibited. Collectively, our data indicate that P50 acts as a suppressor of systemic silencing without interfering with local silencing, probably by inhibiting the movement of silencing signals.

Cucumber cotyledons inoculated with Cucumber mosaic virus (CMV, Pepo strain) or Zucchini yellow mosaic virus (ZYMV, Z5-1 isolate) developed either mild chlorotic spots or no symptoms. Cotyledons treated with CMV plus ZYMV also developed mild chlorotic spots. However, plants ZYMV-inoculated cotyledons had veinal yellowing and gradual cell death by 20 days postinoculation (dpi) when co-inoculated with CMV on the other cotyledon. When analyzing this synergism, an enzyme-linked immunosorbent assay showed that CMV gradually increased in CMV-inoculated cotyledons of plants, with the other cotyledon mock- or ZYMV-inoculated. However, CMV significantly increased at 9 to 14 dpi in the ZYMV-inoculated cotyledons of plants co-infected with CMV. ZYMV similarly increased in cotyledon pairs of both co-infected and singly infected plants. Inoculation with PepoΔ2b, a modified Pepo-CMV that lacks translation of the 2b protein, revealed that PepoΔ2b without the 2b protein systemically infected cucumber but induced no symptoms on cotyledons or true leaves. Plants with a ZYMV-inoculated cotyledon and co-infected with PepoΔ2b did not undergo cell death; nevertheless, PepoΔ2b was at high levels comparable to levels of CMV in the ZYMV-inoculated cotyledon. The 2b protein thus seems essential for induction of the novel gradual cell death in ZYMV-inoculated cotyledons of cucumbers co-infected with CMV.

Modeling of neutral transport in the tandem mirror plasmas has been performed based on simulation studies using a Monte-Carlo code. A three-dimensional neutral transport simulation in both the central-cell (an axisymmetric simple mirror) and anchor-cell (a non-axisymmetric minimum-B configuration) was successfully carried out using the DEGAS version 63 code. In order to realize the geometrical structure in the simulation space, a ‘second wall’ is introduced in the code, which enabled us to perform a more detailed simulation. The simulation results agreed well with the Hα measurements of the experiment. The effect of cold gas influx from the beamline of the neutral beam injection (NBI) system on the particle fueling is also investigated and it is found that the azimuthally uniform particle source due to the recycling on the central vacuum chamber wall prevails over the localized fueling from cold gas influx accompanying the NBI.

Results of neutral beam injection (NBI) experiments in the GAMMA 10 tandem mirror plasmas are presented together with the neutral particle behavior observed in the experiments. A hydrogen neural beam was injected into the hot-ion-mode plasmas by using the injector installed in the central-cell for the plasma heating and fueling. High-energy ions produced by NBI were observed and its energy distribution was measured for the first time with a neutral particle analyzer installed in the central-cell. The temporal and spatial behavior of hydrogen was observed with axially aligned H{sub {alpha}} detectors installed from the central midplane to anchor-cell. Enhancement of hydrogen recycling due to the beam injection and the cause of the observed decrease in plasma diamagnetism are discussed. The Monte-Carlo code DEGAS for neutral transport simulation was applied to the GAMMA 10 central-cell and a 3-dimensional simulation was performed in the NBI experiment. Localization of neutral particle during the beam injection is investigated based on the simulation and it was found that the increased recycling due to the beam injection was dominant near the injection port.

Distribution of Cucumber mosaic virus (CMV) in shoot meristem tissue of CMV-inoculated tobacco was successively analyzed with immunohistochemical microscopy and in situ hybridization. CMV signals were detected in the tissue at 7 days postinoculation (dpi), but then they decreased and disappeared after 14 dpi. Detailed observation confirmed CMV invasion of shoot apical meristem at 6–8 dpi. Short interfering RNA corresponding to CMV RNAs was first detected at 7 dpi and was detected up to 24 dpi. These results suggest that the shoot meristem tissue is infected with CMV but subsequently recovers from the infection by RNA silencing.

Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) inoculated with a necrotic strain of Potato virus Y (PVY, T01 isolate) developed necrotic symptoms in some systemically infected leaves, but not in younger leaves. However, PVY expressed distinct symptoms not only in the older leaves, but also in the younger leaves, of plants that had been doubly inoculated with PVY and with Cucumber mosaic virus (CMV, strain Pepo). A tissue blot immunoassay of tissues from various positions of the stem detected PVY weakly in each stem, but not in the shoot apex, of singly infected plants, whereas PVY was detected at high levels in almost all sections of doubly infected plants. CMV was also detected at high levels in sections of singly and doubly infected plants. Immunohistochemistry of stem tissues showed that in singly infected plants, PVY was confined to external phloem cells and was not detected in internal phloem cells. However, in doubly infected plants, PVY was distributed uniformly throughout whole tissues, including the external phloem, xylem parenchyma and internal phloem cells. In plants that were doubly infected with PVY and PepoΔ2b, a modified CMV that cannot translate the 2b protein, the spread of PVY was restricted as in singly infected plants. These results suggested that the plant host has a counterdefence mechanism that restricts systemic spread of PVY T01, and that the 2b protein of CMV strain Pepo negates this restriction.

This work describes two alkaline semicontinuous processes for the conversion of refractory organic materials into biodegradable substances. Newsprint was used as a lignocellulosic waste. Methane conversion efficiencies and cellulose removals were investigated for the two following processes: alkaline subcritical-water treatment (ASWT) coupled with methane fermentation and alkaline heat treatment (newsprint heated with steam in an autoclave; AHT) coupled with methane fermentation with a neutral subcritical-water treatment (NSWT) recycle. Results showed that for ASWT coupled with methane fermentation higher methane conversion efficiencies and higher cellulose removals were achieved as HRT increased. At HRT = 20 days, average CH4 conversion efficiency and average cellulose removal reached 26% and 44%, respectively. After a final HRT of 40 days, average CH4 conversion efficiency and average cellulose removal reached 50% and 60%, respectively. On the other hand, for AHT coupled with methane fermentation, methane conversion efficiencies did not show a greater improvement using this pretreatment process. Average conversion reached 9% with an average cellulose removal of 20%. In order to improve the yield of the reactor, approximately one-third of the effluent was recycled using NSWT (150°C; neutral pH). Methane conversion efficiency of this process increased as more recycles were performed. For the fifth operation, the total average methane conversion efficiency was 44% with a total average cellulose removal of 55%.

Single intrauterine death may occur in twin-twin transfusion syndrome. We investigated why the outcome of the surviving twin is fairly good when the donor twin dies first compared with when the recipient twin dies first. A detailed hemodynamic study was performed using Doppler ultrasound in a twin pregnancy affected by twin-twin transfusion syndrome before and after a single intrauterine death that occurred in the donor twin at 26 weeks' gestation. The recipient twin was expected to die due to severe right cardiac failure with functional stenosis of the pulmonary artery 2 days before the cotwin's death. The donor twin's death caused a prompt resolution of cardiac failure and improvement in other indices, including flow velocity waveform patterns of the umbilical vein, the middle cerebral artery and the ductus venosus. A healthy, premature female neonate weighing 1630 g with a hemoglobin concentration of 17.8 g/dL was delivered by Cesarean section following rupture of the fetal membranes 28 days after the episode. Hemorrhaging from the surviving twin to the dead twin that occurred just before or after the cotwin's death may have contributed to the decrease in volume overload in the recipient twin, leading to a prompt amelioration of the critical hemodynamic indices. The early death of the donor twin may thus have played a significant role in improving the status of the recipient twin in this case of twin-twin transfusion syndrome.

Local symptom expression and systemic movement of Cucumber mosaic virus (CMV) in Tetragonia expansa, Momordica charantia and Physalis floridana were mapped to the amino acid at position 129 of CMV coat protein (CP), using pseudorecombinants, chimeric RNAs, a site-directed mutant of RNA 3 and four strains of CMV : pepo-, SO-, MY17- and Y-CMV. Local and systemic symptoms caused by three strains, pepo-, SO- and MY17-CMV, and those by Y-CMV differed in the three host species. The three strains expressed local chlorotic spots at 24°C and systemic chlorotic spots and ringspots at 36°C, whereas Y-CMV developed local necrotic spots at 24°C but no systemic symptoms at 36°C in T. expansa. In M. charantia the three strains caused systemic chlorotic spots, whereas Y-CMV caused local necrotic spots. The three caused systemic mosaic and Y-CMV systemic necrosis in P. floridana. With pseudorecombinants combined with pepo- and Y-CMV RNAs, CMV RNA 3 was responsible for symptom expression and systemic infection. Inoculation with Y-CMV RNA 1, RNA 2 and chimeric RNA 3s exchanged CP gene fragments between pepo- and Y-CMV showed that NruI-XhoI fragment of CP was essential for symptom expression. Comparative analysis of the NruI-XhoI fragments revealed that only the amino acid at position 129 was common among the three strains but different from that of Y-CMV. Inoculation with a point mutant constructed by substituting one nucleotide resulting in an amino acid change from Ser to Pro at position 129 in Y-CMV CP verified the previous experiments. These results indicate that the amino acid at position 129 of CMV CP is the determinant for local symptom expression and systemic movement in the three host species. CMV CP containing Ser at position 129 may induce resistant responses in these plants.

Most plant viruses do not infect the shoot apical meristem (SAM) of a host plant, and this virus-free region of meristem tissue has been used to obtain virus-free clones by meristem tip culture. Thus, the validation of viral distribution in meristem tissues is important for ensuring the appropriate excision of virus-free meristem tips. Although immunohistochemical microscopy and in situ hybridization are classical techniques, they allow us to determine the presence or absence of plant viruses in the shoot meristem tissues of a host plant. Briefly, meristem tissues are excised from infected plants, fixed, embedded in paraffin medium, and prepared in semithin sections (10-15 μm). By treating these sections with an antibody against viral protein or with a probe complementary to viral RNA, the viral distribution in the meristem tissue can be clearly observed. Importantly, these procedures are broadly applicable to most virus (and viroid) and host plant combinations.

Local lesion formation on cowpea leaves was more than 50% inhibited by treatment with a 23 kDa RNase-like glycoprotein from Cucumis figarei, figaren, from 24 hr before to 1 hr after inoculation with Cucumber mosaic virus (CMV). CMV accumulation detected by ELISA in tobacco leaves treated with figaren 6 or 0 hr before inoculation with CMV was suppressed. When upper leaves of tobacco plants were treated with figaren and inoculated 10 min later with CMV, mosaic symptoms were delayed for 5–7 days on most of the tobacco plants, and some plants remained asymptomatic. From fluorescence in situ hybridization, infection sites were present in figaren-treated cowpea or melon leaves after inoculation with CMV, though the sites were reduced in number and size compared with those in water-treated control leaves. The amount of CMV RNAs and CMV antigen in melon protoplasts inoculated with CMV and subsequently incubated with figaren similarly increased with time as did that in the control. ELISA and local lesion assays indicated that CMV infection on the upper surfaces of the leaves of tobacco, melon, cowpea and C. amaranticolor whose lower surfaces had been treated with figaren 5–10 min before CMV inoculation was almost completely inhibited. Figaren did not inhibit CMV infection on the opposite untreated leaf halves of melon, cowpea and C. amaranticolor, whereas it almost completely inhibited CMV infection on the untreated halves of leaves of tobacco. CMV infection was not inhibited in the untreated upper or lower leaves of the four plants. These data suggest that figaren does not completely prevent CMV invasion but does inhibit the initial infection processes. It may also induce localized acquired resistance in host plants.

An antiviral protein, designated figaren, was purified from leaves of Cucumis figarei and partially characterized. Column chromatography, SDS-polyacrylamide gel electrophoresis and periodic acid-Schiff staining revealed that figaren is a glycoprotein with a molecular weight of 23 kDa. Figaren was stable at pH 2 to 12 and below 90°C. N-terminal amino acid sequencing indicated that figaren contained the conserved region for the S-allele-associated ribonucleases (RNases). In-gel RNase assay showed that figaren digested yeast RNAs. Figaren also digested double-stranded RNAs extracted from Cucumber mosaic virus (CMV)-infected tobacco tissues. Fluorescence in situ hybridization revealed that figaren and RNases (beef pancrease and RNase T1 at 1 μg/ml similarly inhibited CMV infection in cowpea leaves. Figaren and the RNases at 5-500 ng/ml had similar inhibitory effect on local lesion formation by CMV. These data suggest that figaren is a novel RNase-like antiviral protein.