Biological characteristics of staphylococcal enterotoxin Q and its potential risk for food poisoning.

AIMS: To elucidate the biological characteristics and stability of a newly identified staphylococcal enterotoxin Q (SEQ) against heating and digestive enzymes and to evaluate the risk of seq-harbouring Staphylococcus aureus in food poisoning. METHODS AND RESULTS: Purified SEQ was treated with heating, pepsin and trypsin which are related to food cooking, stomach and intestine conditions, respectively. Superantigenic activity of SEQ was assessed by determining the ability of IL-2 induction in mouse spleen cells. The emetic activity of SEQ was assessed using house musk shrew, a small emetic animal model. The results revealed that SEQ exhibits a remarkable resistance to heat treatment and pepsin digestion and has significant superantigenic and emetic activities. Furthermore, a sandwich ELISA for detection of SEQ production was developed, and the results showed that seq-harboring S. aureus isolates produce a large amount of SEQ. CONCLUSIONS: The newly identified SEQ had remarkable stability to heat treatment and digestive enzyme degradation and exhibited significant superantigenic and emetic activities. In addition, seq-harbouring S. aureus isolated from food poisoning outbreaks produced a large amount of SEQ, suggesting that seq-harbouring S. aureus could potentially be a hazard for food safety. SIGNIFICANCE AND IMPACT OF THE STUDY: This study found, for the first time, that SEQ, a nonclassical SE, had remarkable stability to heat treatment and enzyme degradation and exhibited significant emetic activity, indicating that SEQ is a high-risk toxin in food poisoning.

Immunization with outer membrane protein A from Salmonella enterica serovar Enteritidis induces humoral immune response but no protection against homologous challenge in chickens.

Vaccination of poultry is one promising strategy to mitigate Salmonella infection in poultry and, in turn, humans as well. We evaluated the efficacy of outer membrane protein A (OmpA) as a novel vaccine candidate against Salmonella in poultry. Native OmpA purified from Salmonella enterica serovar Enteritidis was mixed with adjuvant and administered intramuscularly to 41-d-old chicks. The vaccinated birds showed no decrease in cecal excretion and tissue colonization compared with the unvaccinated birds after oral challenge with 10(9) cfu of the homologous strain at 28 d postimmunization. However, this vaccination induced an increased level of serum anti-OmpA IgG. Similar results were obtained in the replication experiments using a recombinant OmpA with single and double doses. For the development of more effective component vaccines for avian salmonellosis, the vaccine efficacy of outer membrane proteins other than OmpA and route of immunization other than parenteral administration should be evaluated with regard to protection and immune responses, including mucosal IgA.

Factors associated with silent cerebral microbleeds in hemodialysis patients.

BACKGROUND: The recent development of gradient-echo T2*-weighted magnetic resonance imaging (MRI) has enabled the highly accurate detection of prior cerebral microbleeds (CMBs), which might indicate a higher risk of future intracerebral hemorrhage (ICH) and be a marker of cerebral small-vessel disease in the general population. The present study investigated the clinical factors associated with the presence of CMBs in hemodialysis (HD) patients. METHODS: Cranial MRI, including T2*-weighted MRI, was performed on 179 HD patients without symptomatic cerebrovascular disease and 58 healthy control subjects, and we investigated the prevalence of CMBs and clinical factors associated with the presence of CMBs. We also investigated the relationship between CMBs and other cerebral small-vessel diseases. RESULTS: The prevalence of CMBs was significantly higher in the HD patients than in the healthy subjects (45 patients (25.1%) vs. none in the healthy controls (0%), p < 0.0001). Multiple logistic regression analysis showed that independent and significant factors associated with the presence of CMBs were age, systolic blood pressure, diastolic blood pressure and pulse pressure. Moreover, the presence of CMBs correlated significantly with the presence of lacunar infarcts, periventricular hyperintensity and deep and subcortical white matter hyperintensity. CONCLUSIONS: These findings indicated a high prevalence of CMBs among HD patients, and that older age and high blood pressure were strong factors associated with the presence of CMBs. Moreover, CMBs were closely associated with other cerebral small-vessel diseases.

Potential egg contamination by Salmonella enterica serovar Typhimurium definitive type 104 following experimental infection of pullets at the onset of lay.

The present study investigated the potential of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) definitive type 104 (DT104) to contaminate eggs in vivo. Of 10 strains of Salmonella Typhimurium DT104, none caused egg contamination when hens were inoculated orally. Three passages of the strains through recovery from the reproductive organs of intravenously infected laying hens resulted in no egg contamination after oral infection of the hens. Feed and water withdrawal for 24 h at 5 and 10 d after oral infection with Salmonella Typhimurium DT104 slightly decreased egg production but did not lead to egg contamination. Finally, oral infection of pullets at the onset of lay (approximately 50% of egg production) resulted in egg contamination (1.7%) in 2 wk. In conclusion, the Salmonella Typhimurium DT104 strains used in the present study have a low possibility of causing egg contamination; however, because infection at the onset of lay can cause egg contamination, the introduction of Salmonella Typhimurium DT104 into the layer houses should be prevented, especially when hens start laying eggs.

EPR and ENDOR Investigation of Rhodosemiquinone in Bacterial Reaction Centers Formed by B-Branch Electron Transfer.

In photosynthetic bacteria, light-induced electron transfer takes place in a protein called the reaction center (RC) leading to the reduction of a bound ubiquinone molecule, Q(B), coupled with proton binding from solution. We used electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) to study the magnetic properties of the protonated semiquinone, an intermediate proposed to play a role in proton coupled electron transfer to Q(B). To stabilize the protonated semiquinone state, we used a ubiquinone derivative, rhodoquinone, which as a semiquinone is more easily protonated than ubisemiquinone. To reduce this low-potential quinone we used mutant RCs modified to directly reduce the quinone in the Q(B) site via B-branch electron transfer (Paddock et al. in Biochemistry 44:6920-6928, 2005). EPR and ENDOR signals were observed upon illumination of mutant RCs in the presence of rhodoquinone. The EPR signals had g values characteristic of rhodosemiquinone (g(x) = 2.0057, g(y) = 2.0048, g(z) ∼ 2.0018) at pH 9.5 and were changed at pH 4.5. The ENDOR spectrum showed couplings due to solvent exchangeable protons typical of hydrogen bonds similar to, but different from, those found for ubisemiquinone. This approach should be useful in future magnetic resonance studies of the protonated semiquinone.

Design of target irradiation and diagnostic chamber to study ps-laser generated plasma as a source of singly charged ions for external injection into an electron beam ion source.

High repetition-rate (∼10 kHz) ps-lasers are becoming available on the market with reasonable cost and may offer several advantages compared to ns-lasers by generating nearly continuous beams of singly charged ions appropriate for the "slow" injection mode into the Electron Beam Ion Source (EBIS). To evaluate these advantages, we will perform studies of a ps-laser generated plasma using a laser with a pulse duration of 8 ps and energy up to 5 mJ per pulse. A vacuum chamber equipped with a 3D target positioner, a focusing lens, and a Faraday Cup has been designed and built for this study. Lens-to-target distance variations have been measured using a laser tracker over the whole range of horizontal and vertical translation for all five targets we will use. The variations were found to be within ±150 µm. This degree of "target flatness" should be acceptable for our experimental conditions. Ion currents and ion pulse durations of various elements (from Al to Ta) will be measured for different target irradiation conditions (focal spot size and laser pulse energy). The results obtained will allow us to specify all parameters and geometry of a laser ion source based on a ps-laser to provide external ion injection into the relativistic heavy ion collider EBIS.

Optimization of laser-target parameters for the production of stable lithium beam.

A laser ion source coupled with a radio frequency quadrupole linac accelerator is being proposed as a suitable system for the production of a low energy, high-current stable lithium beam. In order to maximize the lithium yield, plasmas generated by laser ablation of different materials based on lithium (Li, LiOH, and LiNbO3) have been characterized by using a Faraday cup and an electrostatic ion analyzer in the time of flight configuration. A wide range of laser power density has been investigated (109-1012 W/cm2) using two Nd:YAG lasers operating at different wavelengths (1064 nm and 532 nm), pulse durations (6 ns and 17 ns), and maximum energies (1400 mJ and 210 mJ). This paper outlines the pros and cons of the investigated materials by studying how the ion energy, yields, and charge state distributions are modified when the laser power density is changed. Considerable attention has been paid to the higher charge states of oxygen, which may occur with the same mass-to-charge ratio of Li3+. The analysis has evidenced that LiNbO3 represents a valid target since it allows minimizing the O6+/7Li3+ ratio down to 2.5% by using a laser power density of 1.8 × 1010 W/cm2. For such a condition, a Li3+ current of 1.4 mA/cm2 has been measured.

Feasibility study of a compact heavy ion source for investigation of laboratory magnetospheric plasma.

We are developing a laser ion source to provide a high brightness multi-charged heavy ion beam as a part of the heavy ion beam probe system, which will be used to diagnose plasma potential in the Ring Trap 1 device at the University of Tokyo. As a probe beam, Nb2+ was selected, and a detailed laser irradiation condition was explored. It was found that the laser power density of 1.2 × 109 W/cm2 gives the maximum particle number of Nb2+ per laser energy from a niobium foil target. Essential ablation plasma parameters to design the laser ion source were also obtained. The expected beam current was more than 12 mA/cm2, with a pulse width of 3.1 µs at 200 mm away from the target.

96Zr beam production for isobar experiment in relativistic heavy ion collider.

To investigate the chiral magnetic effect, 96Zr and 96Ru beams were accelerated at the relativistic heavy ion collider (RHIC) during Run-18 at Brookhaven National Laboratory. The 96Zr beam was provided from the electron beam ion source (EBIS) injector, which consists of a laser ion source, an EBIS high charge state ion breeder, a 300 keV/u radio frequency quadrupole, and a 2 MeV/u interdigital H type drift tube linear accelerator (IH-DTL). The natural abundance of 96Zr is only 2.8% with about 50% of 90Zr. To obtain a sufficient beam current, Zr material enriched to about 60% of 96Zr was used. The only available form of the enriched material was zirconium oxide (ZrO2) powder, which was not well suited for a laser ion source target. We studied and established a sintering technique of the ZrO2 powder to make a solid sample which could be installed into the laser ion source. The singly charged Zr was produced in a laser ablation plasma, extracted, and delivered to the EBIS to be ionized further to 96Zr16+. We optimized the laser irradiation condition, the EBIS confinement time, and transport through the RF linacs to maximize the performance of the injector. The total number of shots provided from the laser ion source for injection into the EBIS was 489 910. The EBIS facility provided a 192 MeV stable beam of 96Zr16+ ions to the booster ring of alternating gradient synchrotron (AGS) for further acceleration and stripping in the AGS/RHIC complex, allowing for successful data acquisition at the Solenoidal Tracker at the RHIC.

A novel orexin antagonist from a natural plant was discovered using zebrafish behavioural analysis.

OBJECTIVE: Phenotypic screening is one of the most practical approaches to the identification of mediators of behaviour, since it is difficult to model brain function in vitro, at a cellular level. We used a zebrafish (Danio rerio) behavioural assay to discover novel, natural, neuroactive compounds. MATERIALS AND METHODS: A zebrafish behavioural assay was performed for seven natural compounds, obtained from plants. The behavioural profiles were compared to those of known psychoactive drugs. We characterised a natural compound exhibiting a behaviour profile similar to that of suvorexant, using in silico, in vitro and microarray expression analysis. RESULTS: The behavioural analysis performed in this study classified central nervous system drugs according to their mechanism. Zebrafish treated with a natural compound, 8b-(4'-Hydroxytigloyloxy) costunolide (8b), showed behaviour profiles similar to those of zebrafish treated with suvorexant, a known orexin antagonist. This behavioural assay was validated using in silico and in vitro assays, which revealed that the new compound was a dual orexin receptor antagonist. In addition, transcriptome analysis suggested that 8b might regulate the nuclear factor-κB (NF-κB) related pathway. CONCLUSIONS: We conclude that zebrafish phenotypic screening, combined with in silico assays and gene expression profiling, is a useful strategy to discover and characterize novel therapeutic compounds, including natural products.

Cluster ion source for external injection and high capacity filling of light elements into the relativistic heavy ion collider electron beam ion source.

An advanced Electron Beam Ion Source (EBIS) is the primary ion source to supply highly charged ion beams of different elements to the Relativistic Heavy Ion Collider (RHIC) and to the NASA Space Radiation Laboratory (NSRL). Intense beams of highly charged ions of various elements of the periodic table, ranging from helium to uranium, have been demonstrated since EBIS became operational in 2010. EBIS routinely provides ion beams to RHIC and NSRL quasisimultaneously with about 1 s switching time between different ion species. Such unique flexibility and rapid switching between ion species are based on external injection of singly charged ions into the EBIS trap either in "fast" or "slow" injection modes. At present, a Laser Ion Source (LIS) provides most of the ion species of solid materials using the "fast" injection mode into the EBIS trap and a Hollow Cathode Ion Source (HCIS) provides most of the ion species of gaseous elements using the "slow" injection mode into the EBIS trap. Gas injection into the EBIS trap is also possible and has been used but imposes some restrictions for the simultaneous generation of highly charged ions such as Au32+ ions for RHIC and ions of gaseous species for NSRL. Because light ions have relatively high velocity inside the EBIS trap, efficient injection of hydrogen and helium ions and filling of the EBIS trap to high capacity is difficult from either LIS or HCIS. To overcome this restriction and enhance EBIS operational capability, we suggest injecting beams of hydrogen and helium cluster ions into the EBIS trap. Required parameters of cluster ion beam injection into the EBIS trap are estimated, and advantages of such an injection are highlighted. A cluster ion source with required high intensity is visible and will be designed, built, optimized, and tested.

Direct plasma injection scheme in accelerators.

The idea of direct plasma injection scheme (DPIS) was proposed in 2000. This new technique has been studied and proven to accelerate intense ion beams. To provide medium mass ions with highly charged states, small tabletop solid lasers were used for plasma production. Based on the measured plasma properties, aluminum and carbon ions were accelerated with more than 60 mA of current. The next experiments will use an radio frequency quadrupole designed for q/m=1/6 and explore beam productions using targets up to silver, and future work will explore production up to uranium. The DPIS has been established and is ready to be used with various accelerators which require pulsed high current, high charge state ion beams.

Blood pressure regulates platelet-derived growth factor A-chain gene expression in vascular smooth muscle cells in vivo. An autocrine mechanism promoting hypertensive vascular hypertrophy.

To clarify the role of PDGF A-chain in hypertensive vascular hypertrophy of spontaneously hypertensive rats (SHRs), we studied levels of PDGF A-chain gene expression and transcription factors related to the gene in vascular smooth muscle cells (VSMCs) of SHRs in vivo. RNase protection assay and in situ hybridization showed that PDGF A-chain mRNA levels in VSMCs of SHRs were twofold higher than in those of normotensive Wistar-Kyoto rats. Gel retardation assays showed that levels of Sp1 and AP-2 in VSMCs of SHRs were twofold more abundant than in those of Wistar-Kyoto rats. Treatment with four pharmacologically different species of antihypertensive drugs for 2 wk decreased the levels of both PDGF A-chain mRNA and Sp1, but not AP-2 level in VSMCs of SHRs with regression of aortic hypertrophy, indicating that increases in levels of both PDGF A-chain mRNA and Sp1 in VSMCs of SHRs were associated with high blood pressure. These results suggest that high blood pressure is a stimulus which upregulates PDGF A-chain gene expression in VSMCs of SHRs, resulting in an autocrine enhancement in hypertensive vascular hypertrophy, and that the activation of the gene may be mediated through increases in Sp1 in these cells.

Light induced EPR spectra of reaction centers from Rhodobacter sphaeroides at 80K: Evidence for reduction of Q(B) by B-branch electron transfer in native reaction centers.

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides capture solar energy by electron transfer from primary donor, D, to quinone acceptor, Q(B,) through the active A-branch of electron acceptors, but not the inactive B-branch. The light induced EPR spectrum from native RCs that had Fe(2+) replaced by Zn(2+) was investigated at cryogenic temperature (80K, 35 GHz). In addition to the light induced signal due to formation of D(+•)Q(A) (-•) observed previously, a small fraction (~5%) of the signal displayed very different characteristics: (1) The signal was absent in RCs in which the Q(B) was displaced by the inhibitor stigmatellin. (2) Its decay time (τ=6 s) was the same as observed for D(+•)Q(B) (-•) in mutant RCs lacking Q(A,) which is significantly slower than for D(+•)Q(A) (-•) (τ=30 ms). (3) Its EPR spectrum was identical to that of D(+•)Q(B) (-•). (4) The quantum efficiency for forming the major component of the signal was the same as that found for mutant RCs lacking Q(A) (Φ =0.2%) and was temperature independent. These results are explained by direct photochemical reduction of Q(B)via B-branch electron transfer in a small fraction of native RCs.

Chronic toxicity and carcinogenicity of dietary administered ammonium sulfate in F344 rats.

Chronic toxicity and carcinogenicity studies of ammonium sulfate, used as a food additive in fermentation, were performed in male and female Fisher 344 rats at dietary concentrations of 0%, 0.1%, 0.6% and 3.0% in a 52-week toxicity study and 0%, 1.5% and 3.0% in a 104-week carcinogenicity study. Treatment with ammonium sulfate caused significant increase in kidney and/or liver weights in males and females of the 3.0% diet group, but no effects were found on survival rate, body weights, and hematological, serum biochemical or histopathological parameters at any dose levels in the chronic toxicity study. Regarding carcinogenicity, ammonium sulfate did not exert any significant influence on the incidences of tumors in any of the organs and tissues examined. It was concluded that the no observed adverse effect level of ammonium sulfate was the 0.6% diet, which is equivalent to 256 and 284 mg/kg b.w./day in males and females, respectively, and the compound is non-carcinogenic under the conditions of the study.

Behavior of moving plasma in solenoidal magnetic field in a laser ion source.

In a laser ion source, a solenoidal magnetic field is useful to guide the plasma and to control the extracted beam current. However, the behavior of the plasma drifting in the magnetic field has not been well understood. Therefore, to investigate the behavior, we measured the plasma ion current and the total charge within a single pulse in the solenoid by changing the distance from the entrance of the solenoid to a detector. We observed that the decrease of the total charge along the distance became smaller as the magnetic field became larger and then the charge became almost constant with a certain magnetic flux density. The results indicate that the transverse spreading speed of the plasma decreased with increasing the field and the plasma was confined transversely with the magnetic flux density. We found that the reason of the confinement was not magnetization of ions but an influence induced by electrons.

Effect of the solenoid in various conditions of the laser ion source at Brookhaven National Laboratory.

In the laser ion source (LIS) at the Brookhaven National Laboratory (BNL), a solenoid is used to guide the laser ablation plasma and modulate the extracted beam current. Many types of ion species are guided. In some cases, the plasma plume is injected into the solenoid away from the solenoidal axis. To investigate the effects of the solenoid on the beam extracted from the plasma that has different properties, the beam current was measured in the setup of the LIS at the BNL. The beam current of Li, Al, Si, Fe, and Au increased when the magnetic field was applied. For most of the species the peak current and the total charge within a single beam pulse increased around 10 times with a magnetic field less than 100 G. In addition, for some species the rate of increase of the peak currents became smaller when the magnetic flux densities were larger than certain values depending on the species. In this case, the current waveforms were distorted. At the same magnetic field value, the field was more effective on lighter species than on heavier ones. When plasma was injected offset from the axis of the solenoid, peak current and total charge became half of those without offset. The experimental data are useful for the operation of the LIS at the BNL.

Low charge state heavy ion production with sub-nanosecond laser.

We have investigated laser ablation plasma of various species using nanosecond and sub-nanosecond lasers for both high and low charge state ion productions. We found that with sub-nanosecond laser, the generated plasma has a long tail which has low charge state ions determined by an electrostatic ion analyzer even under the laser irradiation condition for highly charged ion production. This can be caused by insufficient laser absorption in plasma plume. This property might be suitable for low charge state ion production. We used a nanosecond laser and a sub-nanosecond laser for low charge state ion production to investigate the difference of generated plasma using the Zirconium target.