Investigation of Combustion of the Gas Turbine Engine from Kerosene and Biokerosene and Their Soot Characteristics in Diffusion Flames.

Performances, emissions from the gas turbine engine, and soot formations in diffusion flames of kerosene (Jet A1) and its mixture with 5% by volume bioparaffins (known as BK-5) are reported in the present study. A Rover 1S/60 gas turbine engine was used for recording performance parameters and emissions. Soot characteristics were investigated in smoke-free coannular wick-fed diffusion flames. This study is the next step that must be performed in the certification process of a new aviation biofuel before it is tested in the aircraft. The results show that BK-5 produced a similar performance against Jet A1. Throughout the whole power range under investigation, BK-5 emitted 3.4% NOx higher than Jet A1, while Jet A1 released CO and HC at the rates that are, respectively, 1.8 and 4.5% greater than its counterpart. The soot emissions from the BK-5 and Jet A1 were comparable across the measured flame height range. The results encouraged future studies to carry out the modern engine and flight tests. The production process for bioparaffins employed in this work has been demonstrated to be viable and appropriate for tropical developing nations. The current process should also continue to be improved by eliminating high-distillation temperature components in bioparaffins.

Lethal Disease in Dogs Naturally Infected with Severe Fever with Thrombocytopenia Syndrome Virus.

Severe fever with the thrombocytopenia syndrome virus (SFTSV) causes fatal disease in humans, cats, and cheetahs. In this study, the information on seven dogs with SFTS was summarized. All dogs showed anorexia, high fever, leukopenia, and thrombocytopenia, two dogs showed vomiting and loose stool, and five dogs had tick parasites. All dogs also had a history of outdoor activity. The SFTSV gene was detected in all dogs. Remarkably, three dogs (43%) died. SFTSV was isolated from six dogs and the complete genomes were determined. A significant increase in anti-SFTSV-IgG antibodies was observed in two dogs after recovery, and anti-SFTSV-IgM antibodies were detected in four dogs in the acute phase. Using an ELISA cut-off value of 0.410 to discriminate between SFTSV-negative and positive dogs, the detection of anti-SFTSV-IgM antibodies was useful for the diagnosis of dogs with acute-phase SFTS. Four out of the ninety-eight SFTSV-negative dogs possessed high anti-SFTSV IgG antibody titers, indicating that some dogs can recover from SFTSV infection. In conclusion, SFTSV is lethal in some dogs, but many dogs recover from SFTSV infection.

Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble those of SFTS patients, whereas SFTS-contracted cats have high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals. In this study, a reverse transcription polymerase chain reaction (RT-PCR) was newly developed to diagnose SFTS-suspected animals without non-specific reactions. METHODOLOGY/PRINCIPLE FINDINGS: Four primer sets were newly designed from consensus sequences constructed from 108 strains of SFTSV. A RT-PCR with these four primer sets successfully and specifically detected four clades of SFTSV. Their limits of detection are 1-10 copies/reaction. Using this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients. CONCLUSION/SIGNIFICANCE: This newly developed RT-PCR could detect SFTSV RNA of several clades and from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study.

Virulence of Francisella tularensis Subspecies holarctica Biovar japonica and Phenotypic Change during Serial Passages on Artificial Media.

Francisella tularensis (F. tularensis) is the etiological agent of the zoonotic disease tularemia. F. tularensis subspecies holarctica biovar japonica has rarely been isolated in Japan and is considered to have moderate virulence, although the biological properties of fresh isolates have not been analyzed in detail. Here, we analyzed the virulence of two strains of F. tularensis subspecies holarctica biovar japonica (NVF1 and KU-1) and their phenotypic stability during serial passages in Eugon chocolate agar (ECA) and Chamberlain's chemically defined medium (CDM) based agar (CDMA). C57BL/6 mice intradermally inoculated with 101 colony-forming units of NVF1 or KU-1 died within 9 days, with a median time to death of 7.5 and 7 days, respectively. Both NVF1 and KU-1 strains passaged on ECA 10 times had comparable virulence prior to passaging, whereas strains passaged on ECA 20 times and on CDMA 50 times were attenuated. Attenuated strains had decreased viability in 0.01% H2O2 and lower intracellular growth rates, suggesting both properties are important for F. tularensis virulence. Additionally, passage on ECA of the KU-1 strains altered lipopolysaccharide antigenicity and bacterial susceptibility to ß-lactam antibiotics. Our data demonstrate F. tularensis strain virulence in Japan and contribute to understanding phenotypic differences between natural and laboratory environments.

Complete Genome Sequences of Two Strains of Francisella tularensis subsp. holarctica bv. japonica.

Francisella tularensis, a highly infectious bacterium, is the etiological agent of the zoonotic disease tularemia. It is widely distributed in the Northern Hemisphere, including Japan. Here, we have determined the complete genome sequences of two strains of F. tularensis subsp. holarctica bv. japonica isolated from hares in 2008 and 2009.

Risk factors for the onset of dependence and chronic psychosis due to cannabis use: Survey of patients with cannabis-related psychiatric disorders.

AIM: The objective of the current study was to identify risk factors that affect the onset of dependence and chronic psychosis due to cannabis use. METHODS: We examined clinical genetic factors, psychiatric disorders prior to cannabis use, starting age of cannabis use, duration and frequency of cannabis use, types of cannabis products used, combined use of other psychoactive substances, and the psychiatric diagnosis of 71 patients with cannabis-related psychiatric disorders who underwent treatment at nine mental health hospitals in Japan. Information was collected from cross-sectional interview surveys conducted by each patient's attending psychiatrist. RESULTS: For the diagnosis of dependence syndrome due to the use of cannabis, we found associations with the number of years of cannabis use and the use of cannabis products with a high Δ9-tetrahydrocannabinol (THC) content. However, we found no association between diagnosis of residual and late-onset psychotic disorders and clinical genetic factors, presence of preceding psychiatric disorders, duration and frequency of cannabis use, starting age of cannabis use, or combined use of other psychoactive substances; an association was found only for the absence of use of cannabis products other than dried cannabis. CONCLUSION: The onset of cannabis dependence was related to long-term cannabis use and the use of cannabis products with a high THC content. However, chronic psychosis was not associated with total THC intake or psychiatric vulnerability. Thus, unknown factors appear to be involved in the onset of chronic psychosis.

Evaluation of Francisella tularensis ΔpdpC as a candidate live attenuated vaccine against respiratory challenge by a virulent SCHU P9 strain of Francisella tularensis in a C57BL/6J mouse model.

Francisella tularensis, which causes tularemia, is an intracellular gram-negative bacterium. F. tularensis has received significant attention in recent decades because of its history as a biological weapon. Thus, development of novel vaccines against tularemia has been an important goal. The attenuated F. tularensis strain ΔpdpC, in which the pathogenicity determinant protein C gene (pdpC) has been disrupted by TargeTron mutagenesis, was investigated as a potential vaccine candidate for tularemia in the present study. C57BL/6J mice immunized s.c. with 1 × 106 CFUs of ΔpdpC were challenged intranasally with 100× the median lethal dose (LD50 ) of a virulent SCHU P9 strain 21 days post immunization. Protection against this challenge was achieved in 38% of immunized C57BL/6J mice administered 100 LD50 of this strain. Conversely, all unimmunized mice succumbed to death 6 days post challenge. Survival rates were significantly higher in vaccinated than in unimmunized mice. In addition, ΔpdpC was passaged serially in mice to confirm its stable attenuation. Low bacterial loads persisted in mouse spleens during the first to tenth passages. No statistically significant changes in the number of CFUs were observed during in vivo passage of ΔpdpC. The inserted intron sequences for disrupting pdpC were completely maintained even after the tenth passage in mice. Considering the stable attenuation and intron sequences, it is suggested that ΔpdpC is a promising tularemia vaccine candidate.

Genetic subtyping of Listeria monocytogenes via multiple-locus sequence typing using iap, sigB and actA.

Pulse field gel electrophoresis (PFGE) is widely used for listeriosis surveillance. Although this technique is effective for epidemiology, the data among laboratories are inconsistent. We previously reported a method for Listeria monocytogenes subtyping combined with sequence analysis of partial iap and whole genome restriction fragment length polymorphism (RFLP) using XbaI, ClaI (BanIII) and PstI. However, distinguishing subtypes was challenging, because the output comprised complicated fragment patterns. In this study, we aimed to establish a simple genotyping method that does not depend on visual observation, rather it focuses on multi-locus sequence typing (MLST) using three genes, iap, sigB and actA. Sixty-eight strains of L. monocytogenes including EGD-e as a reference strain were investigated to ensure consistency with previous data on the genetic characterization. All strains were grouped into 29 types by both analyses. Although there are some differences in classification, major clades included the same strains. Simpson's indices of diversity (SID) by MLST and iap-RFLP-based typing were 0.967 (95% confidence interval [CI]: 0.955/0.978) and 0.967 (95% CI: 0.955/0.979), respectively. The discriminatory power of both methods can be considered almost identical. Compared with the results of 38 selected strains, the strains within the MLST clusters in this study coincided with those obtained using PFGE. Thus, the MLST strategy could help differentiate among L. monocytogenes isolates during epidemiological studies.

Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis.

Pullulanase, an enzyme that catalyzes the hydrolysis of polysaccharides, has been identified in a broad range of organisms, including bacteria, yeasts, fungi, and animals. The pullulanase (pulB; FTT_0412c) of F. tularensis subspecies tularensis Schu S4 is considered to be a homologue of the type I pullulanase (pulA) of the other Francisella subspecies. The significance of Francisella pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of F. tularensis SCHU P9, which was expressed as a his-tagged protein in Escherichia coli. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity in vitro. A pulB gene knockout mutant of F. tularensis SCHU P9 (ΔpulB) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of F. tularensis in macrophages. The intracellular growth of the ΔpulB mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in ΔpulB, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of F. tularensis in macrophages. To assess the role of PulB in virulence, the knockout and parent bacterial strains were used to infect C57BL/6J mice. Histopathological analyses showed that tissues from ΔpulB-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and ΔpulB showed the similar levels of bacterial loads in their tissues. The results suggest that PulB plays a significant role in bacterial growth within murine macrophage but does not contribute to bacterial virulence in vivo.

Virulence of representative Japanese Francisella tularensis and immunologic consequences of infection in mice.

Francisella tularensis, which causes tularemia, is widely distributed in the Northern hemisphere. F. tularensis strains isolated in Japan are genetically unique from non-Japanese strains; however, their phenotypic properties have not been well studied. Thus, mice were infected with representative Japanese strains of F. tularensis and their virulence and mouse immune responses to them assessed. Of four representative Japanese strains, the Ebina, Jap and Tsuchiya strains were susceptible to H2 O2 and did not grow well intracellularly. Only Yama strain grew intracellularly and was lethal to mice. Infection with Yama strain resulted in drastic increases in IFN-γ, CD4 and CD8 double-positive T cells and Th1 cells (CD3, CD4 and Tim3-positive cells), and a decrease in the ratio of CD8-positive CD4-negative T cells in mice. C57BL/6J mice that survived infection produced IgM antibodies to LPS and IgG2c antibodies to 43, 19 and 17 kDa proteinase K-sensitive components. These data are valuable for understanding the phenotypic properties of F. tularensis in Japan.

Survey of Francisella tularensis in Wild Animals in Japan in Areas Where Tularemia is Endemic.

Samples taken from 428 wild animals and 126 ticks, collected from a tularemia-endemic area in Japan between 2005 and 2013, were analyzed for the presence of Francisella tularensis. F. tularensis was isolated from a Japanese hare carcass whereas the samples from live animals and ticks were negative for F. tularensis by real-time PCR. Our results suggest that F. tularensis is still present in Japan although its prevalence is considerably low even in areas where tularemia is endemic.

Serosurveillance for Francisella tularensis among wild animals in Japan using a newly developed competitive enzyme-linked immunosorbent assay.

Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild.

Role of pathogenicity determinant protein C (PdpC) in determining the virulence of the Francisella tularensis subspecies tularensis SCHU.

Francisella tularensis subspecies tularensis, the etiological agent of tularemia, is highly pathogenic to humans and animals. However, the SCHU strain of F. tularensis SCHU P0 maintained by passaging in artificial media has been found to be attenuated. To better understand the molecular mechanisms behind the pathogenicity of F. tularensis SCHU, we attempted to isolate virulent bacteria by serial passages in mice. SCHU P5 obtained after 5th passages in mice remained avirulent, while SCHU P9 obtained after 9th passages was completely virulent in mice. Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5. Comparison of the nucleotide sequences of the whole genomes of SCHU P0, P5, and P9 revealed only 1 nucleotide difference among P0, P5 and P9 in 1 of the 2 copies of pathogenicity determinant protein C (pdpC) gene. An adenine residue deletion was observed in the pdpC1 gene of SCHU P0, P5, and P9 and in the pdpC2 gene of SCHU P0, and P5, while P9 was characterized by the wild type pdpC2 gene. Thus, SCHU P0 and P5 expressed only truncated forms of PdpC protein, while SCHU P9 expressed both wild type and truncated versions. To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis. SCHU P9 mutants with inactivated pdpC gene showed low intracellular growth in J774.1 cells and did not induce severe disease in experimentally infected mice, while virulence of the mutants was restored by complementation with expression of the intact PdpC. These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.

In vitro antibiotic susceptibility of Francisella tularensis isolates from Japan.

The antibiotic susceptibilities of 36 isolates of Japanese Francisella tularensis, an etiological agent of the zoonotic disease tularemia, were analyzed using the E test. All the isolates were susceptible to ciprofloxacin, doxycycline, erythromycin, and gentamicin but resistant to benzylpenicillin and cephalothin. The susceptibility to seven other ß-lactams (aztreonam, cefotaxime, cefoxitin, ceftriaxone, cefuroxime, imipenem, and meropenem) varied among the isolates. These findings suggest that the guidelines for the antibiotic treatment of tularemia issued by the World Health Organization are appropriate for Japanese tularemia patients.

Identification of the source of Francisella tularensis infection by multiple-locus variable-number tandem repeat analysis.

Tularemia is a zoonotic disease caused by Francisella tularensis. Most patients in Japan have reportedly acquired such infections through direct contact with infected Japanese hares. We recently encountered a patient who contracted tularemia after skinning and butchering a dead hare. Because the remains of the hare were available, we attempted to determine whether the patient actually contracted infection by handling the carcass. F. tularensis-specific sequences were successfully amplified by PCR from the patient specimens as well as from the remnants of discarded hare carcass. PCR amplification of the ISFtu2 and RD1 regions indicated infection by F. tularensis subsp. holarctica, which was considered as a prevalent strain in Japan. Furthermore, high-resolution multiple-locus variable-number tandem repeat analysis (MLVA) showed that the combination of repeat numbers in sequences from the patient and hare were indistinguishable, thus indicating that the patient had been infected with F. tularensis strain that had also infected the hare. These findings demonstrated that MLVA is a useful epidemiological investigational tool to identify possible sources of certain zoonotic diseases such as tularemia.

The effect of acute exercise in hypoxia on flow-mediated vasodilation.

The purpose of this study was to clarify the effect of acute exercise in hypoxia on flow-mediated vasodilation (FMD). Eight males participated in this study. Two maximal exercise tests were performed using arm cycle ergometry to estimate peak oxygen uptake [Formula: see text] while breathing normoxic [inspired O(2) fraction (FIO(2)) = 0.21] or hypoxic (FIO(2) = 0.12) gas mixtures. Next, subjects performed submaximal exercise at the same relative exercise intensity [Formula: see text] in normoxia or hypoxia for 30 min. Before (Pre) and after exercise (Post 5, 30, and 60 min), brachial artery FMD was measured during reactive hyperemia by ultrasound under normoxic conditions. FMD was estimated as the percent (%) rise in the peak diameter from the baseline value at prior occlusion at each FMD measurement (%FMD). The area under the curve for the shear rate stimulus (SR(AUC)) was calculated in each measurement, and each %FMD value was normalized to SR(AUC) (normalized FMD). %FMD and normalized FMD decreased significantly (P < 0.05) immediately after exercise in both condition (mean ± SE, FMD, normoxic trial, Pre: 8.85 ± 0.58 %, Post 5: -0.01 ± 1.30 %, hypoxic trial, Pre: 8.84 ± 0.63 %, Post 5: 2.56 ± 0.83 %). At Post 30 and 60, %FMD and normalized FMD returned gradually to pre-exercise levels in both trials (FMD, normoxic trial, Post 30: 1.51 ± 0.68 %, Post 60: 2.99 ± 0.79 %; hypoxic trial, Post 30: 4.57 ± 0.78 %, Post 60: 6.15 ± 1.20 %). %FMD and normalized FMD following hypoxic exercise (at Post 5, 30, and 60) were significantly (P < 0.05) higher than after normoxic exercise. These results suggest that aerobic exercise in hypoxia has a significant impact on endothelial-mediated vasodilation.

Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

Inhibition of both protease and helicase activities of hepatitis C virus NS3 by an ethyl acetate extract of marine sponge Amphimedon sp.

Combination therapy with ribavirin, interferon, and viral protease inhibitors could be expected to elicit a high level of sustained virologic response in patients infected with hepatitis C virus (HCV). However, several severe side effects of this combination therapy have been encountered in clinical trials. In order to develop more effective and safer anti-HCV compounds, we employed the replicon systems derived from several strains of HCV to screen 84 extracts from 54 organisms that were gathered from the sea surrounding Okinawa Prefecture, Japan. The ethyl acetate-soluble extract that was prepared from marine sponge Amphimedon sp. showed the highest inhibitory effect on viral replication, with EC50 values of 1.5 and 24.9 µg/ml in sub-genomic replicon cell lines derived from genotypes 1b and 2a, respectively. But the extract had no effect on interferon-inducing signaling or cytotoxicity. Treatment with the extract inhibited virus production by 30% relative to the control in the JFH1-Huh7 cell culture system. The in vitro enzymological assays revealed that treatment with the extract suppressed both helicase and protease activities of NS3 with IC50 values of 18.9 and 10.9 µg/ml, respectively. Treatment with the extract of Amphimedon sp. inhibited RNA-binding ability but not ATPase activity. These results suggest that the novel compound(s) included in Amphimedon sp. can target the protease and helicase activities of HCV NS3.

Inhibition of hepatitis C virus replication and viral helicase by ethyl acetate extract of the marine feather star Alloeocomatella polycladia.

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC(50) of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC(50) value of 23 to 44 µg/mL, which is similar to the IC(50) value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.

Renal cell carcinoma with a tumor thrombus in the ureter: a case report.

BACKGROUND: Renal cell carcinoma (RCCs) is the most common malignancy of the kidney. When RCC progresses, it is known to form tumor thrombus in the renal vein and/or inferior vena cava. However, RCC does not normally form tumor thrombus in the ureter or renal pelvis. CASE PRESENTATION: A 43-year-old man presented to our department for the treatment of a renal tumor with asymptomatic gross hematuria. In a dynamic CT study, contrast enhancement revealed a tumor suspected to be RCC, but atypical finding as a tumor thrombus that filled the renal pelvis and the whole ureter was also observed. Nephroureterectomy was performed, and the tumor was diagnosed histopathologically as RCC. CONCLUSION: We report here a very rare case of RCC with a tumor thrombus in the whole ureter.