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.

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.

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.

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.

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.

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.

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.

Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro.

We have developed a continuous fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA helicase activity in vitro. The assay is tested using the hepatitis C virus (HCV) NS3 helicase as a model. We prepared a double-stranded RNA (dsRNA) substrate with a 5' fluorophore-labeled strand hybridized to a 3' quencher-labeled strand. When the dsRNA is unwound by helicase, the fluorescence of the fluorophore is emitted following the separation of the strands. Unlike in conventional gel-based assays, this new assay eliminates the complex and time-consuming steps, and can be used to simply measure the real-time kinetics in a single helicase reaction. Our results demonstrate that Alexa Fluor 488 and BHQ1 are an effective fluorophore-quencher pair, and this assay is suitable for the quantitative measurement of the RNA helicase activity of HCV NS3. Moreover, we found that several extracts of marine organisms exhibited different inhibitory effects on the RNA and DNA helicase activities of HCV NS3. We propose that this assay will be useful for monitoring the detailed kinetics of RNA unwinding mechanisms and screening RNA helicase inhibitors at high throughput.

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.

High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon.

We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

Pathological and microbiological studies of Japanese Hare (Lepus brachyurus angustidens) naturally infected with Francisella tularensis subsp. holarctica.

An adult male hare (Lepus brachyurus angustidens) was discovered in a moribund condition in the bush in the mountains of Aomori prefecture in Japan. Upon gross inspection, many ticks were found on the neck and the external ear regions, and more than half the ticks contained blood in the intestine. The skin around the tick bite wounds was alopecic and mildly thickened. At necropsy, enlargement of the cervical lymph nodes and spleen were observed. Histologically, acute necrotizing splenitis, lymphadenitis, hepatitis, pneumonia, myelitis, adrenalitis, and encephalitis with bacterial organisms were observed. The cutaneous lesions were chronic and cysts had formed in the areas marked by tick bites. Immunohistochemically, the organisms in the skin, liver, spleen, lymph nodes, lungs, adrenal glands, brain, bone marrow, and ticks were positive for F. tularensis antigen. Microbiological and polymerase chain reaction results were consistent with F. tularensis subsp. holarctica. Because the cutaneous lesions were more chronic than those in the visceral organs and F. tularensis was detected in the ticks, we inferred that F. tularensis was transmitted to the hare via tick bites.

Comparison of whole genome amplification methods for detecting pathogenic bacterial genomic DNA using microarray.

The genetic diagnosis of pathogenic agents using microarrays has the advantage of high-throughput detection, but a relatively large amount of DNA sample is required. To obtain a sufficient amount of DNA for molecular diagnoses, several whole genome amplification (WGA) methods have been proposed. In this study, using Francisella tularensis and Escherichia coli as models, we compared four WGA methods in terms of their efficiency of amplification of whole genomic DNA in order to identify the most suitable method for preparing DNA to be used for microarray analysis. It was possible to obtain more than 1.5 microg of products from 10 ng of F. tularensis and E. coli genomic DNA using four methods, but biases in the amplification of bacterial genes were least prominent in the multiple displacement amplification (MDA) or OmniPlex WGA. When the amplified DNAs were applied to microarray slides consisting of 32 different genes probes, DNAs amplified by Phi29 v2 of MDA and OmniPlex WGA showed high signal intensity as well as a high signal-to-noise ratio for all 32 genes. These results indicate that Phi29 v2 and OmniPlex WGA are useful methods for obtaining sufficient DNA from a limited amount of samples for the detection of microbes using microarrays.

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.

An overview of challenges in modeling heat and mass transfer for living on Mars.

Engineering a life-support system for living on Mars requires the modeling of heat and mass transfer. This report describes the analysis of heat and mass transfer phenomena in a greenhouse dome, which is being designed as a pressurized life-support system for agricultural production on Mars. In this Martian greenhouse, solar energy will be converted into chemical energy in plant biomass. Agricultural products will be harvested for food and plant cultivation, and waste materials will be processed in a composting microbial ecosystem. Transpired water from plants will be condensed and recycled. In our thermal design and analysis for the Martian greenhouse, we addressed the question of whether temperature and pressure would be maintained in the appropriate range for humans as well as plants. Energy flow and material circulation should be controlled to provide an artificial ecological system on Mars. In our analysis, we assumed that the greenhouse would be maintained at a subatmospheric pressure under 1/3-G gravitational force with 1/2 solar light intensity on Earth. Convection of atmospheric gases will be induced inside the greenhouse, primarily by heating from sunlight. Microclimate (thermal and gas species structure) could be generated locally around plant bodies, which would affect gas transport. Potential effects of those environmental factors are discussed on the phenomena including plant growth and plant physiology and focusing on transport processes. Fire safety is a crucial issue and we evaluate its impact on the total gas pressure in the greenhouse dome.

Development of a real-time PCR assay for detection and quantification of Francisella tularensis.

The facultative intracellular bacterium, Francisella tularensis, is an etiological agent of tularemia and is also considered to be a potential biological threat agent due to its extreme infectivity. We established a real-time PCR assay using the LightCycler (LC) system to detect a Francisella-specific sequence of the outer membrane protein (fopA) gene. Twenty-five F. tularensis strains including 16 Japanese isolates were subjected to this LC-PCR assay, and were tested positive, whereas Francisella philomiragia and other bacteria species did not show any specific fluorescent signal. A linear response was observed using F. tularensis genomic DNAs of between 20 fg and 2 ng, corresponding to 1.2 to 1.2 x 10(5) bacteria. The newly established real-time PCR allows the detection of the F. tularensis genome specifically, sensitively, and rapidly. This assay may contribute to the standardization of the laboratory diagnosis of tularemia.

Involvement of STAT3 in bladder smooth muscle hypertrophy following bladder outlet obstruction.

We examined the involvement of the signal transducer and activator of transcription 3 (STAT3) in bladder outlet obstruction (BOO)-induced bladder smooth muscle hypertrophy using a rat in vivo and in vitro study. BOO induced increases in bladder weight and bladder smooth muscle thickness 1 week after the operation. By using antibody microarrays, 64 of 389 proteins blotted on the array met our selection criteria of an INR value between > or = 2.0 and < or = 0.5. This result revealed up-regulation of transcription factors, cell cycle regulatory proteins, apoptosis-associated proteins and so on. On the other hand, down-regulation (INR value < or = 0.5) of proteins was not found. In a profiling study, we found an increase in the expression of STAT3. A significant increase in nuclear phosphorylated STAT3 expression was confirmed in bladder smooth muscle tissue by immunohistochemistry and Western blot analysis. Cyclical stretch-relaxation (1 Hz) at 120% elongation significantly increased the expression of STAT3 and of alpha-smooth muscle actin in primary cultured bladder smooth muscle cells. Furthermore, the blockade of STAT3 expression by the transfection of STAT3 small interfering RNA (siRNA) significantly prevented the stretch-induced increase in alpha-smooth muscle actin expression. These results suggest that STAT3 has an important role in the induction of bladder smooth muscle hypertrophy.

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.