Performance of a rapid human metapneumovirus antigen test during an outbreak in a long-term care facility.

Using a newly developed rapid test, an outbreak of human metapneumovirus (HMPV) infection in a long-term care facility was detected within only 2 days after the onset of symptoms in a putative index case. The outbreak was almost under control within 8 days mainly by zoning patients, with the exception of two cases of HMPV that were diagnosed 16 and 17 days after the onset of the outbreak. According to an immunological diagnosis as well as the rapid test, it was eventually proven that 18 patients had HMPV infections. We suspected that even asymptomatic residents, who had not been completely separated from the facility population, were a source of infection. That suggested that all asymptomatic residents should be tested and that the separation of the infected patients should be absolute, if an outbreak of HMPV infection is suspected in such a facility.

Comprehensive safety management and assessment at rugby football competitions.

The present study aims to improve medical systems by designing objective safety assessment criteria for rugby competitions. We evaluated 195 competitions between 2002 and 2011 using an original safety scale comprising the following sections: 1) competence of staff such as referees, medical attendants and match day doctor; 2) environment such as weather, wet bulb globe temperature and field conditions; and 3) emergency medical care systems at the competitions. Each section was subdivided into groups A, B and C according to good, normal or fair degrees of safety determined by combinations of the results.Overall safety was assessed as A, B and C for 110, 78 and 7 competitions, respectively. The assessments of individual major factors were mostly favorable for staff, but the environment and medical care systems were assessed as C in 25 and 70, respectively, of the 195 competitions. Medical management involves not having a match day doctor, but also comprehensive management including preventive factors and responses from the staff, environment and medical-care systems. 6 cases of severe injuries and accidents occurred between 2002 and 2011, which were observed in Grade A competition. These cases revealed better prognosis without obvious impairment, thus confirming the value of the present assessment scale.

Tunable terahertz emission from the intrinsic Josephson junctions in acute isosceles triangular Bi2Sr2CaCu2O8+δ mesas.

In order to determine if the mesa geometry might affect the properties of the coherent terahertz (THz) radiation emitted from the intrinsic Josephson junctions in mesas constructed from single crystals of the high-temperature superconductor, Bi2Sr2CaCu2O8+δ, we studied triangular mesas. For equilateral triangular mesas, the observed emission was found to be limited to the single mesa TM(1,0) mode. However, tunable radiation over the range from 0.495 to 0.934 THz was found to arise from an acute isosceles triangular mesa. This 47% tunability is the widest yet observed from the outer current-voltage characteristic branch of such mesas of any geometry. Although the radiation at a few of the frequencies in the tunable range appear to have been enhanced by cavity resonances, most frequencies are far from such resonance frequencies, and can only be attributed to the ac-Josephson effect.

Restoration of innate and adaptive immune responses by HCV viral inhibition with an induction approach using natural interferon-beta in chronic hepatitis C.

Chronic hepatitis C (CHC) is a serious medical problem necessitating more effective treatment. This study investigated the hypothesis that an induction approach with nIFN-beta for 24 weeks followed by PEG-IFN-alpha+ribavirin (standard of care: SOC) for 48 weeks (novel combination treatment: NCT) would increase the initial virologic response rate and restore innate and adaptive immune responses in CHC. Seven CHC patients with a high viral load and genotype 1b were treated with NCT. Serum cytokine and chemokine levels were evaluated during NCT. NCT prevented viral escape and breakthrough resulting in persistent viral clearance of HCVRNA. IL-15 was increased at the end of induction therapy in both early virologic responders (EAVRs) and late virologic responders (LAVRs); CXCL-8, CXCL-10, and CCL-4 levels were significantly decreased (P < 0.05) in EAVR but not in LAVR during NCT, and IL-12 increased significantly (P < 0.05) and CXCL-8 decreased significantly (P < 0.05) after the end of NCT in EAVR but not in LAVR. NCT prevented viral breakthrough with viral clearance leading to improvement of innate and adaptive immunity resulting in a sustained virologic response (SVR). NCT (n = 8) achieved a higher SVR rate than SOC (n = 8) in difficult-to-treat CHC patients with genotype 1 and high viral loads.

Biology, ecology and conservation of the Mobulidae.

The Mobulidae are zooplanktivorous elasmobranchs comprising two recognized species of manta rays (Manta spp.) and nine recognized species of devil rays (Mobula spp.). They are found circumglobally in tropical, subtropical and temperate coastal waters. Although mobulids have been recorded for over 400 years, critical knowledge gaps still compromise the ability to assess the status of these species. On the basis of a review of 263 publications, a comparative synthesis of the biology and ecology of mobulids was conducted to examine their evolution, taxonomy, distribution, population trends, movements and aggregation, reproduction, growth and longevity, feeding, natural mortality and direct and indirect anthropogenic threats. There has been a marked increase in the number of published studies on mobulids since c. 1990, particularly for the genus Manta, although the genus Mobula remains poorly understood. Mobulid species have many common biological characteristics although their ecologies appear to be species-specific, and sometimes region-specific. Movement studies suggest that mobulids are highly mobile and have the potential to rapidly travel large distances. Fishing pressure is the major threat to many mobulid populations, with current levels of exploitation in target fisheries unlikely to be sustainable. Advances in the fields of population genetics, acoustic and satellite tracking, and stable-isotope and fatty-acid analyses will provide new insights into the biology and ecology of these species. Future research should focus on the uncertain taxonomy of mobulid species, the degree of overlap between their large-scale movement and human activities such as fisheries and pollution, and the need for management of inter-jurisdictional fisheries in developing nations to ensure their long-term sustainability. Closer collaboration among researchers worldwide is necessary to ensure standardized sampling and modelling methodologies to underpin global population estimates and status.

A review of the application of molecular genetics for fisheries management and conservation of sharks and rays.

Since the first investigation 25 years ago, the application of genetic tools to address ecological and evolutionary questions in elasmobranch studies has greatly expanded. Major developments in genetic theory as well as in the availability, cost effectiveness and resolution of genetic markers were instrumental for particularly rapid progress over the last 10 years. Genetic studies of elasmobranchs are of direct importance and have application to fisheries management and conservation issues such as the definition of management units and identification of species from fins. In the future, increased application of the most recent and emerging technologies will enable accelerated genetic data production and the development of new markers at reduced costs, paving the way for a paradigm shift from gene to genome-scale research, and more focus on adaptive rather than just neutral variation. Current literature is reviewed in six fields of elasmobranch molecular genetics relevant to fisheries and conservation management (species identification, phylogeography, philopatry, genetic effective population size, molecular evolutionary rate and emerging methods). Where possible, examples from the Indo-Pacific region, which has been underrepresented in previous reviews, are emphasized within a global perspective.

L1503R is a member of group I mutation and has dominant-negative effect on secretion of full-length VWF multimers: an analysis of two patients with type 2A von Willebrand disease.

Type 2A von Willebrand disease (VWD) is characterized by decreased platelet-dependent function of von Willebrand factor (VWF); this in turn is associated with an absence of high-molecular-weight multimers. Sequence analysis of the VWF gene from two unrelated type 2A VWD patients showed an identical, novel, heterozygous T-->G transversion at nucleotide 4508, resulting in the substitution of L1503R in the VWF A2 domain. This substitution, which was not found in 60 unrelated normal individuals, was introduced into a full-length VWF cDNA and subsequently expressed in 293T cells. Only trace amount of the mutant VWF protein was secreted but most of the same was retained in 293T cells. Co-transfection experiment of both wild-type and mutant plasmids indicated the dominant-negative mechanism of disease development; as more of mutant DNA was transfected, VWF secretion was impaired in the media, whereas more of VWF was stored in the cell lysates. Molecular dynamic simulations of structural changes induced by L1503R indicated that the mean value of all-atom root-mean-squared-deviation was shifted from those with wild type or another mutation L1503Q that has been reported to be a group II mutation, which is susceptible to ADAMTS13 proteolysis. Protein instability of L1503R may be responsible for its intracellular retention and perhaps the larger VWF multimers, containing more mutant VWF subunits, are likely to be mal-processed and retained within the cell.

Phase I/II study of irinotecan and UFT for advanced or metastatic colorectal cancer.

UNLABELLED: The aim of this study was to determine the recommended dose of irinotecan in combination with the fixed dose of oral UFT as first-line therapy in patients with advanced or recurrent colorectal cancer, and to evaluate the response rate and overall survival as a phase II study. PATIENTS AND METHODS: Thirteen patients were recruited into a phase I trial. Four doses of irinotecan ranging from 60 to 150 mg/m2/day were administered intravenously on day 1 and day 16 in combination with UFT given orally from day 2 to day 15. In a phase II study, 53 patients received at least one cycle of this therapy. RESULTS: The recommended dose of this combination was determined as irinotecan 120 mg/m2/day and UFT 400 mg/m2/day. Dose-limiting toxicities were neutropenia and prolonged leucopenia. On an intent-to-treat analysis, the response rate in the phase II study was 24.5% (95% confidence interval 13.8% to 38.2%). The median overall survival time was 20.3 months (95% confidence interval, 15.0-22.8 months). Out of 20 patients with stable disease, 17 who received more than 4 cycles of the regimen lived longer than the other 3 patients who received fewer than 3 cycles (p = 0.0353). Hematological adverse events were mainly grade 3/4 neutropenia observed in 6 out of 53 patients. Grade 3 non-hematological toxicities, such as diarrhea, anorexia, nausea/vomiting and alopecia were observed in 6 patients. CONCLUSION: Irinotecan combined with oral UFT was effective and well-tolerated. This regimen may be considered as a first-line therapy for advanced or metastatic colorectal cancer and may result in fairly long survival, even for patients with stable disease.

Engineering of Escherichia coli L-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by L-cysteine.

L-Serine O-acetyltransferase (SAT) from Escherichia coli catalyzes the first step of L-cysteine synthesis in E.coli and is strictly inhibited by the second step product, L-cysteine. To establish a fermentation process to produce L-cysteine, we embarked on a mutational study of E.coli SAT to desensitize the feedback inhibition by L-cysteine. The crystal structure and the reaction mechanism of SAT from E.coli have shown that the substrate L-serine and the inhibitor L-cysteine bind to the identical region in the SAT protein. To decrease the affinity for only L-cysteine, we first built the structure model of L-serine-binding SAT on the basis of the crystal structure with bound L-cysteine and compared these two structures. The comparison showed that the Calpha of Asp92 underwent a substantial positional change upon the replacement of L-cysteine by L-serine. We then introduced various amino acid substitutions at positions 89-96 around Asp92 by randomized, fragment-directed mutagenesis to change the position of the Asp92. As a result, we successfully obtained mutant SATs which have both extreme insensitivity to an inhibition by L-cysteine (the concentration that inhibits 50% activity; IC(50) = 1,100 micromol/l, the inhibition constant; K(i) = 950.0 micromol/l) and extremely high emzymatic activities.

The novel acidophilic structure of the killer toxin from halotolerant yeast demonstrates remarkable folding similarity with a fungal killer toxin.

BACKGROUND: Several strains of yeasts and fungi produce proteinous substances, termed killer toxins, which kill sensitive strains. The SMK toxin, secreted by the halotolerant yeast Pichia farinosa KK1 strain, uniquely exhibits its maximum killer activity under conditions of acidic pH and high salt concentration. The toxin is composed of two distinct subunits, alpha and beta, which tightly interact with each other under acidic conditions. However, they are easily dissociated under neutral conditions and lose the killer activity. The three-dimensional structure of the SMK toxin will provide a better understanding of the mechanism of toxicity of this protein and the cause of its unique pH-dependent stability. RESULTS: Two crystal structures of the SMK toxin have been determined at 1.8 A resolution in different ionic strength conditions. The two subunits, alpha and beta, are jointly folded into an ellipsoidal, single domain structure belonging to the alpha/beta-sandwich family. The folding topology of the SMK toxin is essentially the same as that of the fungal killer toxin, KP4. This shared topology contains two left-handed split betaalphabeta motifs, which are rare in the other proteins. Many acidic residues are clustered at the bottom of the SMK toxin molecule. Some of the carboxyl sidechains interact with each other through hydrogen bonds. The ionic strength difference induces no evident structural change of the SMK toxin except that, in the high ionic strength crystal, a number of sulfate ions are electrostatically bound near the basic residues which are also locally distributed at the bottom of the toxin molecule. CONCLUSIONS: The two killer toxins, SMK and KP4, share a unique folding topology which contains a rare structural motif. This observation may suggest that these toxins are evolutionally and/or functionally related. The pH-dependent stability of the SMK toxin is a result of the intensive interactions between the carboxyl groups. This finding is important for protein engineering, for instance, towards stabilization of the toxin molecule in a broader pH range. The present crystallographic study revealed that the structure of the SMK toxin itself is hardly affected by the ionic strength, implying that a high salt concentration affects the sensitivity of the cell against the toxin.

0.43 THz emission from high-T(c) superconducting emitters optimized at 77 K.

A liquid helium-free, compact and continuous sub-terahertz radiation system operating at 77 K has been developed using a rectangular mesa device made from a high T(c)-superconducting Bi2Sr2CaCu2O(8+δ) single crystal, based on a different design of a stand-alone mesa sandwich structure to reduce the dc-current Joule heating effects. The mesa was thermally connected to sapphire plates through thin thermal grease embedded with diamond nano-crystals. When immersed in liquid N 2, the device emits intense radiation at 0.437 THz, the highest frequency ever achieved at 77 K, due to excitation of the TM(1, 0) rectangular cavity mode. By varying the dc current-voltage bias and the bath temperature in a He-flow cryostat, the device's emission frequency is broadly tunable from 0.31 THz at 79 K to 1.31 THz at 30 K.

Antibody-scanning and epitope-tagging methods; molecular mapping of proteins using antibodies.

Because synthetic short peptides bearing critical binding residues, can chemically mimic the folded antigenic determinants on proteins, short synthetic peptides can generate antibodies that react with cognate sequences in intact folded proteins. According to this mimotope theory, we produced site-specific antibodies by immunization with short peptides which overlapped each other and covered the entire protein, and used them for domain mapping of influenza virus RNA polymerase (antibody-scanning method). We also used a tagged-epitope and its monoclonal antibodies for topology mapping of clathrin light chains in clathrin triskelions by electron microscopy. Both methods using specific epitopes in combination with their antibodies enable us to determine the domains of interesting proteins systematically without the need to generate monoclonal antibodies or mutant proteins.

Inhibition of influenza virus replication in cultured cells by RNA-cleaving DNA enzyme.

Influenza virus replication has been effectively inhibited by antisense phosphothioate oligonucleotides targeting the AUG initiation codon of PB2 mRNA. We designed RNA-cleaving DNA enzymes from 10-23 catalytic motif to target PB2-AUG initiation codon and measured their RNA-cleaving activity in vitro. Although the RNA-cleaving activity was not optimal under physiological conditions, DNA enzymes inhibited viral replication in cultured cells more effectively than antisense phosphothioate oligonucleotides. Our data indicated that DNA enzymes could be useful for the control of viral infection.

Improvement of deficits in mnemonic rhyme for multiplication in Japanese aphasics.

Since Japanese learn the multiplication tables by means of mnemonic rhymes called kuku, i.e., via the auditory-speaking route, it is hypothesized that aphasics who have a deficit in kuku might relearn it via the visual-writing route. After having ordinary language therapy for more than 3 months, eight patients were admitted to this special kuku training. The comparisons of scores on multiplication tests showed that the visual-writing route improved most after a 1- or 2-month training period while the auditory-speaking route improved least. These results, suggesting that the visual processor can take on the new role in conducting multiplication, can be considered a realization of functional reorganization in the brain.

Hemispheric asymmetry of processing temporal aspects of repetitive movement in two patients with infarction involving the corpus callosum.

Two right-handed patients with infarction involving the forebrain commissural fibres produced irregular but rapid repetitive movements with their left hands while producing normal movements with their right hands as well as having normal oral expression. The abnormality was more conspicuous when producing slow tapping patterns. When required to reproduce rapid tapping patterns of around 5 beats/sec, one of the patients produced a comparatively regular tapping pattern. Thus, we believe that both hemispheres are equipped with the faculty to produce rapid repetitive patterns and that only the left hemisphere is responsible for the temporal processes involved in producing required repetitive patterns.

Sensitivity to functors in Japanese aphasics.

Forty-two aphasic patients (11 amnesic, 11 Wernicke's, and 20 Broca's) and 10 controls were tested for sensitivity to functors in affirmative declarative sentences with sentence- verification tasks. The stimuli were constructed according to the following factors: Reversibility of Subject and Object, Voice, Word Order and Truth. The task required the subject to decide if a sentence presented auditorily was true or false with respect to a picture. The results indicated that functors differed among themselves in their understandability depending on sentence types in which they were embedded, and that there were no qualitative differences in sensitivity to functors among different clinical types of aphasia.

Evaluation of portosystemic collaterals by SPECT imaging after endoscopic variceal sclerotherapy: usefulness for predicting recurrence.

UNLABELLED: Bleeding from esophageal varices is a major cause of morbidity and mortality in cirrhotic patients. Identification of patients at high risk for bleeding is particularly important. The aim of this study was to determine whether detection of portosystemic collaterals by SPECT could predict the outcome of endoscopic injection sclerotherapy of esophageal varices and be useful for selecting appropriate therapy. METHODS: Sixty-two patients with liver cirrhosis who were considered at high risk of bleeding were treated with endoscopic injection sclerotherapy. Endoscopy was performed every 3 mo after therapy or until bleeding occurred. Before and within 2 wk after therapy, tomographic images of intra-abdominal blood pool were constructed by SPECT. RESULTS: Before therapy, the following portosystemic collateral routes were observed: coronary veins in 53 (85.5%) of 62 patients, short gastric veins in 8 patients (12.9%), splenorenal shunts in 10 patients (16.1%), and paraumbilical veins in 6 patients (9.7%). Patients positive for imaging of coronary veins were divided into 3 groups on the basis of changes in images after therapy: complete responders (n = 17), whose coronary vein images disappeared completely; partial responders (n = 18), whose images became smaller; and nonresponders (n = 18), whose images did not change significantly before or after therapy. The rates of recurrence after endoscopic injection sclerotherapy until 6 mo in complete responders (4/17, 23.5%) and partial responders (7/18, 38.9%) were significantly less (P < 0.05) than that in nonresponders (11/13, 84.6%). The rate of recurrence of esophageal varices until 6 mo in nonresponders treated with additional submucosal injection sclerotherapy (1/5, 20.0%) was significantly less (P < 0.05) than that in nonresponders without additional submucosal injection sclerotherapy (11/13, 84.6%). CONCLUSION: Abdominal blood-pool SPECT, a noninvasive method, is useful for evaluating the therapeutic effectiveness of endoscopic sclerotherapy, for predicting the recurrence of varices, and for selecting appropriate management after sclerotherapy.

Evaluation of effective portal venous flow in chronic liver diseases using echo-Doppler flowmetry combined with per jejunal portal scintigraphy.

Portal circulation changes due to the progression of chronic liver disease and portal venous flow are also affected by pharmacotherapy. Thus, noninvasive measurement of effective portal venous flow (EPVF) is highly desirable. We evaluated EPVF under steady-state conditions using echo-Doppler flowmetry combined with per jejunal portal scintigraphy in 32 patients with chronic liver disease. After introduodenal administration of 37 MBq (1 mCi) of 123I-iodoamphetamine, scintigraphy of the pulmonary and hepatic regions was performed and a portosystemic shunt index (SI) calculated. EPVF was calculated as follows: EPVF = PVFx (1-SI/100). EPVF in chronic hepatitis, compensated cirrhosis and decompensated cirrhosis was 12.0 +/- 1.8 ml/min/kg, 10.3 +/- 1.6 ml/min/kg and 8.0 +/- 2.5 ml/min/kg, respectively. There were significant differences in EPVF between all groups, although PVF was similar in each group. EPVF correlated with liver function tests and was a better indicator of liver function than PVF. Measurement of EPVF may provide useful information in the management of patients with chronic liver disease.

Quantitating nuclear opacification in color Scheimpflug photographs.

PURPOSE: To test the validity of the color component subtraction method, a new objective technique of measuring nuclear cataract, and to correlate the tangent values of the different degrees of nuclear opacities to their corresponding subjective nuclear cataract grading using the Lens Opacities Classification System II and to another objective method of measuring nuclear cataract. METHODS: Densitometries for red, green, and blue colors of the color Scheimpflug image were conducted simultaneously along the anteroposterior axis of the lens nucleus. The three color curves were subtracted from each other, giving rise to three subtracted curves: blue-red, green-blue, and red-green. This technique was applied to 99 color Scheimpflug photographs taken in 99 eyes of 51 patients with varying degrees of nuclear opacities. Using linear regression analysis, the regression coefficient obtained, called tangent value (TV), represents the degree of nuclear opacification. RESULTS: Higher tangent value indicated greater nuclear opacification and was associated with higher correlation coefficient. This was seen graphically as an increased steepness in the slope of the subtracted curve. Good correlation existed between the subjective nuclear opacity grading and the objective tangent value. When the tangent values were correlated to the mean density of the three original color curves, good correlation was present in red and green, but only fair for blue, where maximum light scattering occurs. CONCLUSIONS: These results support the validity of color component subtraction technique in measuring nuclear cataract. This technique provides quantitative measures of nuclear opacification, has good reproducibility, and is useful for monitoring nuclear cataract longitudinally.

An electrospray-ionization mass spectrometry analysis of the pH-dependent dissociation and denaturation processes of a heterodimeric protein.

Electrospray ionization mass spectrometry (ESI-MS) was applied to the analysis of the dissociation and denaturation processes of a heterodimeric yeast killer toxin SMKT. The two distinct subunits of SMKT noncovalently associate under acidic conditions, but become dissociated and denatured under neutral and basic conditions. In order to understand the unique pH-dependent denaturation mechanism of this protein, a pH titration was performed by utilizing ESI-MS. The molecular ions of the heterodimer which possesses the highly ordered structure, were mainly observed below pH 4.6. However, the two subunits immediately dissociated at this pH. The spectra measured with various settings of the mass spectrometer indirectly demonstrated that the pH-dependent dissociation occurs in the liquid phase. The current result as well as the three-dimensional structure of SMKT suggest that the deprotonation of a specific carboxyl group triggers a cooperative dissociation process of this protein. In conclusion, the pH titration of a protein by ESI-MS is particularly effective, when the unfolding process or the biological function of the protein is related to the interaction with other molecules.