Analysis of a newly discovered antigen of Bacillus cereus biovar anthracis for its suitability in specific serological antibody testing.

AIMS: The aim of this work was to identify a protein which can be used for specific detection of antibodies against Bacillus cereus biovar anthracis (Bcbva), an anthrax-causing pathogen that so far has been described in African rainforest areas. METHODS AND RESULTS: Culture supernatants of Bcbva and classic Bacillus anthracis (Ba) were analysed by gel electrophoresis, and a 35-kDa protein secreted only by Bcbva and not Ba was detected. The protein was identified as pXO2-60 by mass spectrometry. Sequence analysis showed that Ba is unable to secrete this protein due to a premature stop codon in the sequence for the signal peptide. Immunization of five outbred mice with sterile bacterial culture supernatants of Bcbva revealed an immune response in ELISA against pXO2-60 (three mice positive, one borderline) and the protective antigen (PA; four mice). When supernatants of classic Ba were injected into mice or human sera from anthrax patients were analysed, only antibodies against PA were detected. CONCLUSIONS: In combination with PA, the pXO2-60 protein can be used for the detection of antibodies specific against Bcbva and discriminating from Ba. SIGNIFICANCE AND IMPACT OF THE STUDY: After further validation, serological assays based on pXO2-60 can be used to perform seroprevalence studies to determine the epidemiology of B. cereus bv anthracis in affected countries and assess its impact on the human population.

WISP1 mediates IL-6-dependent proliferation in primary human lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. IPF is characterized by epithelial cell injury and reprogramming, increases in (myo)fibroblasts, and altered deposition of extracellular matrix. The Wnt1-inducible signaling protein 1 (WISP1) is involved in impaired epithelial-mesenchymal crosstalk in pulmonary fibrosis. Here, we aimed to further investigate WISP1 regulation and function in primary human lung fibroblasts (phLFs). We demonstrate that WISP1 is directly upregulated by Transforming growth factor ß1 (TGFß1) and Tumor necrosis factor α (TNFα) in phLFs, using a luciferase-based reporter system. WISP1 mRNA and protein secretion increased in a time- and concentration-dependent manner by TGFß1 and TNFα in phLFs, as analysed by qPCR and ELISA, respectively. Notably, WISP1 is required for TGFß1- and TNFα-dependent induction of interleukin 6 (IL-6), a mechanism that is conserved in IPF phLFs. The siRNA-mediated WISP1 knockdown led to a significant IL-6 reduction after TGFß1 or TNFα stimulation. Furthermore, siRNA-mediated downregulation or antibody-mediated neutralization of WISP1 reduced phLFs proliferation, a process that was in part rescued by IL-6. Taken together, these results strongly indicate that WISP1-induced IL-6 expression contributes to the pro-proliferative effect on fibroblasts, which is likely orchestrated by a variety of profibrotic mediators, including Wnts, TGFß1 and TNFα.

Analysis of the microwave, terahertz, and far infrared spectra of monodeuterated methanol CH2DOH up to J = 26, K = 11, and o(1).

The first theoretical approach aimed at accounting for the energy levels of a non-rigid molecule displaying asymmetric-top asymmetric-frame internal rotation is developed. It is applied to a line position analysis of the high-resolution spectrum of the non-rigid CH2DOH molecule and allows us to carry out a global analysis of a data set consisting of already available data and of microwave and far infrared transitions measured in this work. The analysis is restricted to the three lowest lying torsional levels (e0, e1, and o1), to K ⩽ 11, and to J ⩽ 26. For the 8211 fitted lines, the unitless standard deviation is 2.4 and 103 parameters are determined including kinetic energy, hindering potential, and distortion effects parameters.

Anthrax among heroin users in Europe possibly caused by same Bacillus anthracis strain since 2000.

Injection anthrax was described first in 2000 in a heroin-injecting drug user in Norway. New anthrax cases among heroin consumers were detected in the United Kingdom (52 cases) and Germany (3 cases) in 2009-10. In June 2012, a fatal case occurred in Regensburg, Bavaria. As of December 2012, 13 cases had been reported in this new outbreak from Germany, Denmark, France and the United Kingdom. We analysed isolates from 2009-10 and 2012 as well as from the first injection anthrax case in Norway in 2000 by comparative molecular typing using a high resolution 31 marker multilocus variable-number tandem repeat analysis (MLVA) and a broad single nucleotide polymorphism (SNP) analysis. Our results show that all cases may be traced back to the same outbreak strain. They also indicate the probability of a single source contaminating heroin and that the outbreak could have lasted for at least a decade. However, an additional serological pilot study in two German regions conducted in 2011 failed to discover additional anthrax cases among 288 heroin users.

Analysis of the torsional spectrum of monodeuterated methanol CH2DOH.

An analysis of the torsional spectrum of monodeuterated methanol CH(2)DOH is presented. Twenty nine torsional subbands have been assigned in the 20-800 cm(-1) region. The newly assigned subbands and those already available in the literature were analyzed with a theoretical approach accounting for internal rotation of an asymmetrical CH(2)D methyl group. Seventy six subband centers were reproduced with an rms value of 0.09 cm(-1). Spectroscopic parameters corresponding to the generalized inertia tensor and to the hindering potential were determined as well as rotation-torsion distortion constants.

[Investigation of a family cluster of influenza A/H1N1 infections in Germany, 2009]. / Untersuchungen zu einem familiären Cluster von Infektionen durch Influenza A/H1N1 in Deutschland, 2009.

INTRODUCTION: On May 3, 2009, a first case of influenza A/H1N1 infection occurred in the federal state of Saxony-Anhalt, Germany. In order to stop the possible spread of the virus and to study the epidemiological and clinical characteristics of the infection, an investigation was launched by the local health authorities and the RKI. METHODS: Standardised questionnaires were used to assess demographic and clinical data. Specimens were collected from case patients and close contacts and were analysed for influenza A/H1N1 using real-time PCR. RESULTS: The index patient showed fever and coughing 3.5 days after returning from a holiday in Mexico. The local health authorities were informed on May 3, and measures were rapidly implemented. These measures included a trace-back of possible contact persons, isolation of the case and close contacts, prophylactic treatment with Oseltamivir. Virological investigations showed that the case shedded viral genome up until the last day of antiviral therapy. Viral genome was also detected in the spouse and the son of the patient. Both showed no symptoms under a prophylactic treatment with antiviral medication. No viral genome was detected in three other family members, and in six other contact persons outside of the family. DISCUSSION: The spread of the virus was contained due to the fast response of the local health authorities. Two secondary cases occurred in the family. These cases remained asymptomatic, possibly due to antiviral prophylaxis. Epidemiological and virological results suggest that the influenza A/H1N1 virus has a longer incubation period and that viral shedding may probably be prolonged when compared with seasonal influenza.

Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group.

AIMS: To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores. METHODS AND RESULTS: Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis, e.g. lack of beta-haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis. PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis, but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an AluI restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different. CONCLUSIONS: Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.

[Efficient killing of anthrax spores using aqueous and alcoholic peracetic acid solutions]. / Effiziente Abtötung von Milzbrandsporen durch wässrige und alkoholische Peressigsäure-Lösungen.

We analysed the sporicidal effect of different concentrations of aqueous and alcoholic peracetic acid (PAA) solutions on anthrax spores in suspension and germ carrier tests. In activation of anthrax spores in suspension assays was achieved in less than 2 min using 1% PAA solution and in less than 3 min using 0.5% PAA solution, respectively. In contrast, in germ carrier as says, a test under practical conditions, spores on 38% of the germ carriers survived treatment with 1% PAA solution for 15 min. The use of PAA in 80% ethyl alcohol outclassed the sporicidal effect of aqueous PAA solutions in both suspension and germ carrier assays. Anthrax spores on 14% of germ carriers tested survived 30 min of treatment with a 1% aqueous PAA solution. In contrast anthrax spores were reliably inactivated under the same test procedure using a 1% alcoholic PAA solution for 30 min. The proven enhancement of the sporicidal effect of alcoholic PAA solutions should be kept in mind when using disinfectants in practice. In further surveys we will optimise the test conditions.

Evaluation of physiological standard pressures of the forearm flexor muscles during sport specific ergometry in sport climbers.

BACKGROUND: Chronic exertional compartment syndromes (CECS) are well known in sports medicine. Most commonly affected is the tibialis anterior muscle compartment in runners and walkers. Only a few cases of CECS of the forearm flexor muscles have been reported. OBJECTIVES: To determine pressure levels inside the deep flexor compartment of the forearms during a sport specific stress test. METHOD: Ten healthy, high level climbers were enrolled in a prospective study. All underwent climbing specific ergometry, using a rotating climbing wall (step test, total climbing time 9-15 minutes). Pressure was measured using a slit catheter placed in the deep flexor compartment of the forearm. Pressure, blood lactate, and heart rate were recorded every three minutes and during recovery. RESULTS: In all the subjects, physical exhaustion of the forearms defined the end point of the climbing ergometry. Blood lactate increased with physical stress, reaching a mean of 3.48 mmol/l. Compartment pressure was related to physical stress, exceeding 30 mm Hg in only three subjects. A critical pressure of more than 40 mm Hg was never observed. After the test, the pressure decreased to normal levels within three minutes in seven subjects. The three with higher pressure levels (>30 mm Hg) required a longer time to recover. CONCLUSIONS: For further clinical and therapeutic consequences, an algorithm was derived. Basic pressure below 15 mm Hg and stress pressure below 30 mm Hg as well as pressures during the 15 minute recovery period below 15 mm Hg are physiological. Pressures of 15-30 mm Hg during recovery suggest high risk of CECS, and pressures above 30 mm Hg confirm CECS.

A two-tiered mechanism by which Cdc42 controls the localization and activation of an Arp2/3-activating motor complex in yeast.

The establishment of cell polarity in budding yeast involves assembly of actin filaments at specified cortical domains. Elucidation of the underlying mechanism requires an understanding of the machinery that controls actin polymerization and how this machinery is in turn controlled by signaling proteins that respond to polarity cues. We showed previously that the yeast orthologue of the Wiskott-Aldrich Syndrome protein, Bee1/Las17p, and the type I myosins are key regulators of cortical actin polymerization. Here, we demonstrate further that these proteins together with Vrp1p form a multivalent Arp2/3-activating complex. During cell polarization, a bifurcated signaling pathway downstream of the Rho-type GTPase Cdc42p recruits and activates this complex, leading to local assembly of actin filaments. One branch, which requires formin homologues, mediates the recruitment of the Bee1p complex to the cortical site where the activated Cdc42p resides. The other is mediated by the p21-activated kinases, which activate the motor activity of myosin-I through phosphorylation. Together, these findings provide insights into the essential processes leading to polarization of the actin cytoskeleton.

Far-infrared spectroscopy of CH3OD in highly excited torsional states and the atlas of the Fourier transform spectra in the range 200-350 cm(-1).

The high resolution Fourier transform far-infrared (FIR) spectrum of the torsion rotation band of CH3OD has been analyzed for the highly excited torsion states (n > or = 2) in the vibrational ground state. The spectrum shows splitting of the lines due to strong torsional-rotational-vibrational interactions in the molecule. Assignments were possible for rotational sub-bands in the torsional state as high as n = 4 and for K values up to 8 and J values of up to approximately 30 in most cases, for all the symmetry species. For the third excited torsional state n = 3 assignments were possible to K = 10. The data were analyzed with the help of the energy expansion model, which has been proven very successful in methanol. The state dependent expansion parameters are presented. These molecular parameters were able to reproduce the observed wavenumbers almost to within experimental accuracy of 0.0002 cm(-1) for clean unblended lines. These expansion coefficients should prove valuable in the calculation of precise energy values for excited torsional states up to n = 4, which is way above the torsional barrier. The detailed high-resolution spectral atlas of CH3OD has been presented in the range 200-350 cm(-1). This atlas is an extension of our earlier atlas in the range 20-205 cm(-1). The availability of this atlas in the journal will be very valuable for spectroscopists and astrophysicists seeking information in the infrared (IR) region in the laboratory and in outer space.

Evidence for a trigger function of valproic acid in xenobiotic-induced hepatotoxicity.

The influence of the antiepileptic drug, valproic acid (2-n-propylpentanoic acid), on the hepatocellular capacity, to cope with an extrinsic oxidative stress was investigated. Freshly isolated rat hepatocytes exposed to therapeutic concentrations of valproic acid (0.25-1.0 mmol/l) were less resistant than controls, as evidenced by a significant cytotoxic response after challenge of the cells with a non-toxic dose of allyl alcohol (2-propen-1-ol). Valproic acid alone was not toxic to hepatocytes even at ten times higher concentrations (10 mmol/l), suggesting that cell damage was not a mere additive effect. Incubation with valproic acid plus allyl alcohol induced an irreversible depletion of hepatocellular glutathione, in contrast to allyl alcohol alone which induced a transient loss. Hepatocytes treated with valproic acid plus allyl alcohol were protected by N-acetylcysteine, a precursor of glutathione. These findings indicate that valproic acid affects hepatocellular defence mechanisms and suggest that a predisposition of hepatocytes to oxidative stress may play a role in the fatal hepatotoxicity of valproic acid in epileptic patients.

An investigation into the effect and mechanisms of action of nicotine in inflammatory bowel disease.

OBJECTIVE AND DESIGN: To determine the effect of nicotine on colonic inflammation in the trinitrobenzenesulphonic acid (TNBS) model of inflammatory bowel disease in comparison with sulphasalazine. MATERIALS: Male Wistar rats were used for the in-vivo and ex-vivo studies. In-vitro studies were performed using human leukemia peripheral blood monocyte cells (THP-1 cells) grown in continuous culture. TREATMENT: Rats were given access to either nicotine (5 or 100 microg/mL) or sulphasalazine (375 microg/mL) in their drinking water for 10 or 2 days respectively before and 3 days after TNBS administration. THP-1 cells were treated with nicotine (10(-14) to 10(-11) M) for 2 h before and after stimulation with 3 microg/mL lipopolysaccharide (LPS). METHODS: Inflammation in the TNBS model was assessed by measuring the tissue myeloperoxidase activity, leukotriene B4 concentration, inducible nitric oxide protein expression, the ex-vivo production of tumour necrosis factor alpha (TNFalpha), macroscopic damage score, plasma corticosterone levels and by a qualitative histological evolution. The effect of nicotine on TNFalpha production in LPS stimulated THP-1 monocyte cells in-vitro was also determined. Statistical comparisons were made using the Mann-Whitney U-test for the macroscopic damage score and an ANOVA for all other parameters. RESULTS: TNBS treated rats given access to 100 microg/mL nicotine in their drinking water had a marked reduction in several of the markers of inflammation compared to control TNBS treated rats, but a greater reduction was found at 5 microg/mL nicotine or 375 microg/mL sulphasalazine, the latter producing comparable reductions in inflammation to the low dose nicotine. Nicotine also caused a significant reduction in TNFalpha release from THP-1 cells. CONCLUSIONS: Nicotine reduced inflammation in the TNBS model of colonic damage confirming the use of nicotine in IBD although the choice of dose requires further investigation. The mechanism of action of nicotine does not involve increased corticosterone levels, but may be a consequence of a reduction in TNFalpha or leukotriene B4 production.

Molecular phylogenetic analysis of the Pneumoroidea (Orthoptera, Caelifera): molecular data resolve morphological character conflicts in the basal acridomorpha.

A key transition in the evolution of the insect suborder Caelifera (Orthoptera; Insecta) was from predominantly non-angiosperm-feeding basal lineages to the modern acridomorph fauna (grasshoppers and related insects). However, because of conflicts in the distribution of several complex morphological characters, the relationships of the presumed intermediates, and in particular of the superfamily Pneumoroidea, are presently unclear. We undertook a phylogenetic study of representatives of all of the transitional acridomorph families using mitochondrial and nuclear DNA sequences. No support for pneumoroid monophyly was obtained from nonparametric bootstrap analysis. Furthermore, adopting a maximum-likelihood approach, specific hypotheses of relationships within the Pneumoroidea were firmly rejected using parametric bootstrapping and Kishino-Hasegawa tests. The results indicate that the Pneumoroidea are at best a grade. This distinction implies that the evolution of the proposed pneumoroid synapomorphies, femoro-abdominal stridulation and simple male genital structure, might previously have been misinterpreted as cases of single character gains or losses within lineages. Reconstructions of character states for the femoro-abdominal stridulation indicate that, in fact, multiple losses or gains are equally likely. An important implication of our findings is that, in grasshoppers, auditory tympana may have evolved before stridulation, supporting the argument that the original function of tympana may have been related not to conspecific communication but to predator detection. Overall, the results of this study emphasize the high information content of these minor groups (in this case, the four intermediate families under consideration contain only 0.2% of extant orthopteran species diversity). Our analyses also demonstrate the advantages of model-based methods in analyzing systematic problems and, in particular, of the importance of testing specific phylogenetic hypotheses when a priori support for groupings (e.g., from nonparametric bootstrapping) is marginal.

Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491.

Neisseria meningitidis causes bacterial meningitis and is therefore responsible for considerable morbidity and mortality in both the developed and the developing world. Meningococci are opportunistic pathogens that colonize the nasopharynges and oropharynges of asymptomatic carriers. For reasons that are still mostly unknown, they occasionally gain access to the blood, and subsequently to the cerebrospinal fluid, to cause septicaemia and meningitis. N. meningitidis strains are divided into a number of serogroups on the basis of the immunochemistry of their capsular polysaccharides; serogroup A strains are responsible for major epidemics and pandemics of meningococcal disease, and therefore most of the morbidity and mortality associated with this disease. Here we have determined the complete genome sequence of a serogroup A strain of Neisseria meningitidis, Z2491. The sequence is 2,184,406 base pairs in length, with an overall G+C content of 51.8%, and contains 2,121 predicted coding sequences. The most notable feature of the genome is the presence of many hundreds of repetitive elements, ranging from short repeats, positioned either singly or in large multiple arrays, to insertion sequences and gene duplications of one kilobase or more. Many of these repeats appear to be involved in genome fluidity and antigenic variation in this important human pathogen.

Molecular and biological analysis of eight genetic islands that distinguish Neisseria meningitidis from the closely related pathogen Neisseria gonorrhoeae.

The pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae cause dramatically different diseases despite strong relatedness at the genetic and biochemical levels. N. meningitidis can cross the blood-brain barrier to cause meningitis and has a propensity for toxic septicemia unlike N. gonorrhoeae. We previously used subtractive hybridization to identify DNA sequences which might encode functions specific to bacteremia and invasion of the meninges because they are specific to N. meningitidis and absent from N. gonorrhoeae. In this report we show that these sequences mark eight genetic islands that range in size from 1.8 to 40 kb and whose chromosomal location is constant. Five of these genetic islands were conserved within a representative set of strains and/or carried genes with homologies to known virulence factors in other species. These were deleted, and the mutants were tested for correlates of virulence in vitro and in vivo. This strategy identified one island, region 8, which is needed to induce bacteremia in an infant rat model of meningococcal infection. Region 8 encodes a putative siderophore receptor and a disulfide oxidoreductase. None of the deleted mutants was modified in its resistance to the bactericidal effect of serum. Neither were the mutant strains altered in their ability to interact with endothelial cells, suggesting that such interactions are not encoded by large genetic islands in N. meningitidis.

Simultaneous Rovibrational Analysis of the nu(2), nu(3), nu(5), and nu(6) Bands of H(3)(12)CF.

A total of 4892 transition wavenumbers of the nu(2), nu(3), nu(5), and nu(6) fundamental bands of H(3)(12)CF and 1469 frequencies of the rotational transitions in the v(2) = 1, v(3) = 1, v(5) = 1, and v(6) = 1 vibrational states have been fitted simultaneously by taking explicitly into account various Coriolis interactions, l-type interactions, and alpha-resonance terms between and within the vibrational states. The standard deviation of the fit using 71 parameters was 8.10 x 10(-5) cm(-1) for the infrared data and 0.198 MHz for the rotational data. This study clearly shows that in order to obtain a really quantitative fit of the highly precise data of vibration-rotational and rotational spectroscopy, it is necessary to fit all the data simultaneously using a variational approach. The deperturbed values of the upper state rotational constants differ significantly from those obtained previously in fitting the nu(3) and nu(6) bands as isolated bands and the nu(2)/nu(5) bands by a variational approach. This is important for the precise determination of the equilibrium structure of methyl fluoride (J. Demaison, J. Breidung, W. Thiel, and D. Papousek, Struct. Chem., in press). Besides this, the results can be used in further studies of the reduced Hamiltonians for more complicated systems of interacting vibrational levels. Copyright 1999 Academic Press.

A contribution to safety assessment of veterinary drug residues: in vitro/ex vivo studies on the intestinal toxicity and transport of covalently bound residues.

1. The gastrointestinal fate of protein-bound residues of the model compound furazolidone (FZD) was investigated in vitro and ex vivo. Protein-bound residues were generated in rat liver microsomes, isolated by solvent extraction and digested with 0.5% hydrochloric acid and Pronase E. 2. During digestion, 3-amino-2-oxazolidinone (AOZ), the side chain of furazolidone, was partly released from bound residues. 3. The absorption of free AOZ and digested protein-bound residues was tested in isolated perfused rat gut segments (IPGS) and in the intestinal cell line Caco-2. Free AOZ was transfered both in the IPGS model and in Caco-2 monolayer cultures, while no indications for passage of bound residues were obtained. 4. No acute toxicity of AOZ or digested food residues respectively was observed in gut segments and Caco-2 cells at concentrations that were substantially above maximum residue levels to be expected in food of animal origin after administration of therapeutic doses. 5. The results demonstrate that digestive processes can alter the chemical nature of drug residues and yield degradation products that may be bioavailable for the consumer. Thus, the covalent binding of xenobiotics to macromolecular tissue constituents cannot necessarily be regarded as an irreversible endpoint of residue bioavailability and toxicity.

Perturbations Involving nu1 of NCCN.

From high-resolution infrared spectra of 14N12C12C14N, 14N13C13C14N, and 15N12C12C15N, we find that the levels 1000(0)0(0), 1000(0)1(1), 1000(0)2(0,2), and 1000(0)3(1,3) have very pronounced perturbations. Our analysis shows that these perturbations are due to a vibrational resonance among the levels 1000(0)0(0), 0102(0)2(0), and 0102(0)2(2) in the one case, and equivalent levels with one or more additional quanta of nu5 in the other three cases. The resonance constant for the perturbation involving nu1 is 0.25 cm-1. It has the dependence on v5 and l5 that is expected for the sextic potential constant, K124455, although it seems too large for such a high-order constant. The Deltal (or Deltak) = 2 interaction between, for instance, 1000(0)0(0) and 0102(0)2(2e) is shown to be primarily due to the l-type resonance mixing of the 0102(0)2(0) and 0102(0)2(2e) states. The resonance is nearly "turned off" for the 1000(0)2(0,2) and 1000(0)3(1,3) states of 14N13C13C14N because there are no level crossings between the interacting states and the band centers are too far away to have an obvious effect, although careful analysis shows that the perturbation can be seen in their effective centrifugal distortion constants. The spectrum of 15N12C12C15N shows level crossings only in the case of the 1000(0)1(1), 1000(0)2(0,2), and 1000(0)3(1,3) states. Copyright 1999 Academic Press.