Ab initio and semi-empirical Molecular Dynamics simulations of chemical reactions in isolated molecules and in clusters.

Recent progress in "on-the-fly" trajectory simulations of molecular reactions, using different electronic structure methods is discussed, with analysis of the insights that such calculations can provide and of the strengths and limitations of the algorithms available. New developments in the use of both ab initio and semi-empirical electronic structure algorithms are described. The emphasis is on: (i) calculations of electronic properties along the reactive trajectories and the unique insights this can contribute to the processes; (ii) electronic structure methods recently introduced to this topic to improve accuracy, extend applicability or enhance computational efficiency. The methods are presented with examples, including new results, of reactions of both isolated molecules and of molecules in media, mostly clusters. Possible future directions for this fast growing field are suggested.

Mixture effects of azole fungicides on the adrenal gland in a broad dose range.

Consumers are exposed to low concentrations of a variety of pesticide residues in or on food. Some of them might interfere with the endocrine system. While each individual active substance has been extensively tested for toxicity and safety, potential combination effects possibly resulting from combined exposure to different pesticides have seldomly been tested so far, especially in vivo. Since the adrenal gland is a key endocrine organ, we investigated if and how substances of a group of fungicides presumed to interfere with the biosynthesis of steroid hormones affect this organ when applied individually and in combination in a broad dose range. A 28-day feeding study was conducted in Wistar rats by using three (tri)azole fungicides considered to potentially affect the endocrine system (cyproconazole, epoxiconazole and prochloraz) individually at five dose levels, ranging from 0.9ppm to 2400ppm, and in combination at three dose levels. The parameters analysed included classical toxicology (pathology, histopathology, clinical chemistry) and molecular toxicology endpoints (gene expression arrays and quantitative real time PCR e.g. of Star, HSD3ß, Cyp11a1, Cyp11b1, Cyp11b2, Cyp 21, ApoE), as well as hormone analysis. A dose-dependent decrease in the adrenal gland weight of rats treated with epoxiconazole alone, which was accompanied by an atrophy of the adrenal gland as well as by an increase in the serum cholesterol level and which only became statistically significant at the top dose levels, was observed. These effects were attenuated in the combination experiments, although the same epoxiconazole concentration was used.

Higgs physics at the CLIC electron-positron linear collider.

The Compact Linear Collider (CLIC) is an option for a future [Formula: see text] collider operating at centre-of-mass energies up to [Formula: see text], providing sensitivity to a wide range of new physics phenomena and precision physics measurements at the energy frontier. This paper is the first comprehensive presentation of the Higgs physics reach of CLIC operating at three energy stages: [Formula: see text], 1.4 and [Formula: see text]. The initial stage of operation allows the study of Higgs boson production in Higgsstrahlung ([Formula: see text]) and [Formula: see text]-fusion ([Formula: see text]), resulting in precise measurements of the production cross sections, the Higgs total decay width [Formula: see text], and model-independent determinations of the Higgs couplings. Operation at [Formula: see text] provides high-statistics samples of Higgs bosons produced through [Formula: see text]-fusion, enabling tight constraints on the Higgs boson couplings. Studies of the rarer processes [Formula: see text] and [Formula: see text] allow measurements of the top Yukawa coupling and the Higgs boson self-coupling. This paper presents detailed studies of the precision achievable with Higgs measurements at CLIC and describes the interpretation of these measurements in a global fit.

General vulnerability to stuttering: The experience of stuttering and conditions bringing about invulnerability.

General vulnerability to stuttering is the broad awareness of stuttering and the ever-present, experiential sense of a person who stutters (PWS). It is defined by stuttering in all its forms and the awareness of its presence, both in moments of stuttering and moments of perceivably fluent speech. Under the heading of general vulnerability to stuttering is specific vulnerability to stuttering, which includes the actual events of stuttering (i.e., overt symptoms, covert symptoms, subperceptual stuttering, and anticipation of stuttering). The differentiation between the two is that specific vulnerability requires a specific moment of stuttering where general vulnerability does not.

Carry-over fluency induced by extreme prolongations: A new behavioral paradigm.

Extreme prolongations, which can be generated via extreme delayed auditory feedback (DAF) (e.g., 250-500 ms) or mediated cognitively with timing applications (e.g., analog stopwatch) at 2 s per syllable, have long been behavioral techniques used to inhibit stuttering. Some therapies have used this rate solely to establish initial fluency, while others use extremely slowed speech to establish fluency and add other strategic techniques such as easy onsets and diaphragmatic breathing. Extreme prolongations generate effective, efficient, and immediate forward flowing fluent speech, removing the signature behaviors of discrete stuttering (i.e., syllable repetitions and audible and inaudible postural fixations). Prolonged use of extreme prolongations establishes carry-over fluency, which is spontaneous, effortless speech absent of most, if not all, overt and covert manifestations of stuttering. The creation of this immediate fluency and the immense potential of extreme prolongations to generate long periods of carry-over fluency have been overlooked by researchers and clinicians alike. Clinicians depart from these longer prolongation durations as they attempt to achieve the same fluent results at a near normal rate of speech. Clinicians assume they are re-teaching fluency and slow rates will give rise to more normal rates with less control, but without carry-over fluency, controls and cognitive mediation are always needed for the inherently unstable speech systems of persons who stutter to experience fluent speech. The assumption being that the speech system is untenable without some level of cognitive and motoric monitoring that is always necessary. The goal is omnipresent "near normal rate sounding fluency" with continuous mediation via cognitive and motoric processes. This pursuit of "normal sounding fluency" continues despite ever-present relapse. Relapse has become so common that acceptance of stuttering is the new therapy modality because relapse has come to be understood as somewhat inevitable. Researchers and clinicians fail to recognize that immediate amelioration of stuttering and its attendant carry-over fluency are signs of a different pathway to fluency. In this path, clinicians focus on extreme prolongations and the extent of their carry-over. While fluency is automatically generated under these extreme prolongations, the realization is that communication at this rate in routine speaking tasks is not feasible. The perceived solution is a systematic reduction in the duration of these prolongations, which attempts to approximate "normal speech." Typically, the reintroduction of speech at a normalized rate precipitates a laborious style that is undesirable to the person who stutters (PWS) and is discontinued, once departed from the comforts of the clinical setting. The inevitable typically occurs; the well-intentioned therapist instructs the PWS to focus on the techniques while speaking at a rate that is nearest normal speech, but the overlooked extreme prolongations are unlikely to ever be revisited. The foundation of this hypothesis is that the departure from fluency generators (e.g. extreme prolongations) is the cause of regression to the stuttering set point. In turn, we postulate that the continued use of extreme prolongations, as a solitary practice method, will establish and nurture different neural pathways that will create a modality of fluent speech, able to be experienced without cognitive or motoric mediation. This would therefore result in fewer occurrences of stuttering due to a phenomenon called carry-over fluency. Thus, we hypothesize that the use of extreme prolongations fosters neural pathways for fluent speech, which will result in carry-over fluency that does not require mediation by the speaker.

Physics at the [Formula: see text] linear collider.

A comprehensive review of physics at an [Formula: see text] linear collider in the energy range of [Formula: see text] GeV-3 TeV is presented in view of recent and expected LHC results, experiments from low-energy as well as astroparticle physics. The report focusses in particular on Higgs-boson, top-quark and electroweak precision physics, but also discusses several models of beyond the standard model physics such as supersymmetry, little Higgs models and extra gauge bosons. The connection to cosmology has been analysed as well.

Production of leukemia inhibitory factor mRNA and protein by malignant and immortalized bone cells.

Leukemia inhibitory factor (LIF) is a recently characterized glycoprotein with complex biologic activities on bone cells. We tested various rodent and human immortalized and malignant bone cell lines and primary osteoblast-enriched cell cultures from fetal rat calvarial digests for expression of LIF mRNA and LIF protein. Both human and rodent immortalized and malignant cells expressed a single 4.4 kb mRNA transcript that hybridized to a human LIF cDNA probe in Northern blots. LIF mRNA was undetectable in unstimulated rodent osteoblast-like cells lines MC3T3-E1 and Py1a. However, treatment with LPS (10 micrograms/ml), TGF-beta (1 ng/ml), TNF-alpha (100 ng/ml) or inhibitors of protein synthesis (cycloheximide, emetine, puromycin, and anisomycin) induced the expression of LIF message in these cells. In contrast, primary osteoblast-enriched cells did not express LIF mRNA in Northern blot assays either constitutively or after treatment with TNF-alpha or cycloheximide. The human osteosarcoma cells lines U-2 OS and Saos-2 constitutively expressed LIF mRNA and did not respond to LPS treatment. However, phorbol myristate acetate (PMA), an activator of protein kinase C, was a potent stimulator of LIF message in Saos-2 but not U-2 OS cells. The effects of PMA (0.5 ng/ml) on LIF mRNA in Saos-2 cells were detectable at 1 h and maximal at 6 h. TNF-alpha (100 ng/ml) and inhibitors of protein synthesis also increased LIF mRNA in both Saos-2 and U-2 OS cells. LIF protein was also detected constitutively in the conditioned medium from both Saos and U-2 OS cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-6 expression and histomorphometry of bones from mice deficient in receptors for interleukin-1 or tumor necrosis factor.

We examined the roles of interleukin-1 Type I receptor (IL-1R1) and tumor necrosis factor receptor 1 (TNFR1) in bone metabolism using mice rendered deficient in these receptors by gene targeting. Sections of decalcified paraffin-embedded calvariae and humeri from 11- to 12-week-old mice deficient in IL-1 Type I receptor (IL-1R1-/-) or TNF receptor 1 (TNFR1-/-) were examined by histomorphometry. Wild-type mice (C57BL/6J x 129/J, WILD) served as controls. Interleukin-6 (IL-6) production in primary osteoblastic and bone marrow stromal cell cultures in response to parathyroid hormone (PTH, 100 ng/ml), IL-1 alpha (10 ng/ml), and TNF-alpha (10 ng/ml) was also examined. IL-1R1-/- and TNFR1-/- mice were viable and appeared phenotypically normal. However, the body weights of the IL-1R1-/- mice were 30% less than WILD, while the TNFR1-/- mice weighed 30% more than WILD mice of equivalent age. Calvariae and humeri of IL-1R1-/- and TNFR1-/- mice were normal with respect to trabecular bone volume, osteoclast number, osteoclast surface, growth plate widths, and cortical thickness. Receptor deficiency was confirmed by determining the ability of PTH, IL-1 alpha, and TNF-alpha to stimulate IL-6 in the media of primary calvaria-derived osteoblastic cell cultures from CD-1 and cytokine receptor-deficient mice. After 24 h of treatment, IL-1 alpha and TNF-alpha did not stimulate IL-6 production in osteoblasts from IL-1R1-/- and TNFR1-/- mice, respectively. In contrast, PTH increased IL-6 levels in the cells from all mice. IL-6 protein levels in bone marrow supernatants and conditioned media from untreated bone marrow stromal cells were undetectable in WILD, IL-1R1-/-, and TNFR1-/- mice. PTH, IL-1 alpha and TNF-alpha increased IL-6 mRNA and protein production in the WILD bone marrow stromal cells. In contrast, PTH and TNF-alpha increased IL-6 mRNA and protein levels in IL-1R1-/- bone marrow stromal cells while IL-1 alpha had no effect. These findings demonstrate that normal bone development in mice can occur in the absence of IL-1R1 or TNFR1 expression.

Regulation of NFIL-6 and IL-6 expression by basic fibroblast growth factor in osteoblasts.

We determined whether basic fibroblast growth factor (bFGF) regulated the expression of IL-6 and NFIL-6 in osteoblasts. In mouse osteoblastic MC3T3-E1 cells, bFGF (10(-8) M) increased NFIL-6 mRNA 2-fold at 30 minutes and 3-fold at 2 h. IL-6 mRNA was increased by bFGF 10(-8) M after 1 h. IL-6 protein was detectable in control cultures but was significantly increased by bFGF (10(-8) M) at 4 h. Immunofluorescence analysis of MC3T3-E1 cells showed primarily cytoplasmic and perinuclear NFIL-6 staining in control cultures while bFGF-treated cells showed increased NFIL-6 staining at 2 and 4 h. Western blotting revealed that bFGF increased NFIL-6 protein at 2 h. In calvarial mouse osteoblasts, bFGF 10(-8) M induced IL-6 mRNA as early as 1 h and significantly increased IL-6 protein levels as early as 2 h. In conclusion, bFGF stimulates IL-6 and NFIL-6 mRNA in osteoblasts. The increase in NFIL-6 mRNA was associated with increased NFIL-6 protein. The increase in IL-6 mRNA was also associated with increased IL-6 protein. We propose that activations of NFIL-6 and IL-6 may be important mediators of the effects of bFGF on bone cells.

Lack of evidence for an increase in interleukin-6 expression in adult murine bone, bone marrow, and marrow stromal cell cultures after ovariectomy.

Interleukin-6 (IL-6) has been implicated as a mediator of postmenopausal bone loss. In vitro studies of bone and bone marrow cells have suggested that estrogen regulates bone turnover by controlling the production of IL-6, a potent stimulator of osteoclastogenesis and bone resorption. To investigate this hypothesis in an in vivo model, we examined the effect of ovariectomy or estrogen replacement on IL-6 mRNA and protein expression in adult mouse bone and bone marrow in vivo and in marrow stromal cell cultures. Eight-week-old CD-1 mice were sham-operated (SHAM), ovariectomized (OVX), or ovariectomized and subcutaneously implanted with 21-day slow-release pellets containing 10 micrograms of 17 beta-estradiol (O + E). Placebo pellets were implanted in the SHAM and OVX mice. Uterine weights at 1, 2, or 3 weeks after surgery were significantly decreased (68-76%) in OVX animals compared with SHAM or O + E. In mice sacrificed at 1 or 3 weeks after surgery, we found by nonquantitative reverse transcribed polymerase chain reaction (RT-PCR), that SHAM, OVX, and O + E calvariae (CALV) constitutively expressed IL-6 mRNA. In contrast, IL-6 mRNA was either barely detectable or absent in the tibia (TIB) and bone marrow (BM). In the mice sacrificed 3 weeks after surgery, we determined by quantitative RT-PCR that IL-6 mRNA in the CALV from the OVX and O + E groups were decreased by 81 and 92%, respectively, compared with SHAM. IL-6 protein levels in the flushed bone marrow (BMSups) were detectable and were not significantly different among the groups. In bone marrow cells that were cultured for 1 week, basal levels of IL-6 protein were low and did not differ significantly among the SHAM, OVX, or O + E groups sacrificed 1, 2, or 3 weeks after surgery. After the addition of hrIL-1 alpha, IL-6 protein levels increased 100- to 1300-fold over control. IL-6 levels in cells from animals sacrificed 2 weeks after surgery were significantly lower in the hrIL-1 alpha-stimulated OVX and O + E groups than in hrIL-1 alpha-stimulated SHAM cell cultures. In conclusion, in this model we could find no increase in IL-6 production with in vivo estrogen withdrawal in calvaria, long bones, bone marrow, or marrow stromal cell cultures. If increases in IL-6 expression are involved in the effects of estrogen withdrawal on bone, the magnitude of these changes are relatively small and below the limits of detection of the assays that we employed.

Mice lacking the type I interleukin-1 receptor do not lose bone mass after ovariectomy.

We measured the effects of ovariectomy on the bone mass of mice that lacked type I interleukin-1 receptor (IL-I R1 -/- mice) in two genetic backgrounds (C57BL/6 x 129/Sv and C57BL/6) to investigate the role of interleukin-1 in the actions of estrogen on bone. At three weeks after surgery, ovariectomized wild-type mice decreased trabecular bone volume in the proximal humerus by 70% in a C57BL/6 x 129/Sv background and 48% in a C57BL/6 background compared to sham-operated controls. In contrast, there was no significant decrease in trabecular bone mass in IL-1 R1 -/- mice after ovariectomy. The estrogen status of all groups was confirmed by measurement of uterine wet weight. These results demonstrate that a functional IL-1 response pathway is required for mice to lose trabecular bone mass after ovariectomy in this model and they imply that IL-1 is an important mediator of the effects of ovariectomy on bone mass. Hence, therapeutic interventions that block the effects of IL-1 on bone may be beneficial for treating diseases of rapid bone loss such as post-menopausal osteoporosis.

The Corynebacterium glutamicum cglIM gene encoding a 5-cytosine methyltransferase enzyme confers a specific DNA methylation pattern in an McrBC-deficient Escherichia coli strain.

The cglIM gene of the coryneform soil bacterium Corynebacterium glutamicum ATCC 13032 has been cloned and characterized. The coding region comprises 1092 nucleotides and specifies a protein of 363 amino acid residues with a deduced Mr of 40700. The amino acid sequence showed striking similarities to methyltransferase enzymes generating 5-methylcytosine residues, especially to M x NgoVII from Neisseria gonorrhoeae recognizing the sequence GCSGC. The cglIM gene is organized in an unusual operon which contains, in addition, two genes encoding stress-sensitive restriction enzymes. Using PCR techniques the entire gene including the promoter region was amplified from the wild-type chromosome and cloned in Escherichia coli. Expression of the cglIM gene in E. coli under the control of its own promoter conferred the C. glutamicum-specific methylation pattern to co-resident shuttle plasmids and led to a 260-fold increase in the transformation rate of C. glutamicum. In addition, the methylation pattern produced by this methyltransferase enzyme is responsible for the sensitivity of DNA from C. glutamicum to the modified cytosine restriction (Mcr) system of E. coli.

Characterization of pGA1, a new plasmid from Corynebacterium glutamicum LP-6.

A new plasmid, pGA1, has been isolated from Corynebacterium glutamicum LP-6, and its detailed restriction map has been prepared. The 4.9-kb plasmid has a G + C content of 57%. It replicates in C. glutamicum ATCC13032 and is compatible with the three other plasmids, pCC1, pBL1 and pHM1519, commonly used for vector construction for amino acid-producing corynebacteria. Fusions of pGA1 with different Escherichia coli replicons (transferred from E. coli to Corynebacterium via transformation of spheroplasts or by filter mating experiments with intact cells) are shown to be suitable as shuttle plasmids; some of them are highly stable in C. glutamicum, even when propagated without any selection pressure.

Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum.

Here we describe small mobilizable vectors based on the Escherichia coli plasmids pK18 and pK19. We combined the useful properties of the pK plasmids (e.g., multiple cloning site, lacZ alpha fragment, sequencing with M13 primers) with the broad-host-range transfer machinery of plasmid RP4 and a modified sacB gene from Bacillus subtilis. The new pK derivatives can be transferred by RP4-mediated conjugation into a wide range of Gram- and Gram+ bacteria, and should facilitate gene disruption and allelic exchange by homologous recombination. As an application example, the generation of a defined deletion of the hom-thrB genes in the chromosome of the Gram+ bacterium Corynebacterium glutamicum is presented.

Isolation of insertion elements from gram-positive Brevibacterium, Corynebacterium and Rhodococcus strains using the Bacillus subtilis sacB gene as a positive selection marker.

The sacB gene of Bacillus subtilis was successfully applied in various Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains for the isolation of transposable elements. Three different insertion sequence (IS) elements entrapped in sacB were isolated. The IS elements IS-Bl and IS-Cg isolated from Brevibacterium lactofermentum and Corynebacterium glutamicum, respectively, were found to be similar in size (1.45 kb) and generated target duplications of 8 bp. Their inverted repeats showed homology. In contrast, the IS element IS-Rf isolated from Rhodococcus fascians was only 1.3 kb long and generated a 3-bp target duplication. IS-Cg and IS-Rf were not restricted to their original host strains, and we also found strains harbouring more than one element.

The tetAB genes of the Corynebacterium striatum R-plasmid pTP10 encode an ABC transporter and confer tetracycline, oxytetracycline and oxacillin resistance in Corynebacterium glutamicum.

The tetracycline resistance region of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium striatum M82B was analyzed in Corynebacterium glutamicum ATCC 13032 and confined to a 4.4-kb SphI-Sa/I DNA fragment. Nucleotide sequence analysis revealed two open reading frames, termed tetA and tetB, specifying proteins of 513 and 528 amino acids, respectively. The deduced amino acid sequences of tetAB displayed similarity to ATP-binding cassette transporters including StrV and StrW of Streptomyces glaucescens which are proposed to play a role in the export of streptomycin-like aminoglycosides. An antibiotic susceptibility screening in C. glutamicum showed that the tetAB genes confer resistance to tetracycline, oxytetracycline and to the structurally and functionally unrelated beta-lactam antibiotic oxacillin.

Corynebacterium glutamicum DNA is subjected to methylation-restriction in Escherichia coli.

Efficient electroporation of Escherichia coli with plasmid DNA isolated from Corynebacterium glutamicum depends on the use of Mcr-deficient E. coli strains. The transformation frequency increased nearly 800-fold when the Mcr-deficient E. coli DH5 alpha MCR was used instead of E. coli DH5 alpha. We used E. coli strains with different mutations in the methyl-specific restriction systems to show that McrBC-deficiency is sufficient to generate this effect. The results imply that C. glutamicum DNA contains methylcytosine in specific sequences recognized by the E. coli McrBC system.

Relaxed rrn expression and amino acid requirement of a Corynebacterium glutamicum rel mutant defective in (p)ppGpp metabolism.

The stringent response in Corynebacterium glutamicum was investigated. Sets of rrn-cat fusions were constructed in their native chromosomal position to examine the effects of amino acid starvation in a rel(+) strain and a Deltarel mutant defective in (p)ppGpp metabolism. The expression of the six rrn operons in the rel(+) control was stringently regulated and reduced to 79% upon induction of amino acid starvation. The Deltarel mutant displayed a relaxed regulation and was unable to reduce the rrn expression under amino acid depletion conditions. In addition, the Deltarel mutant grew more slowly in minimal medium than a rel(+) control. This growth effect was restored by a plasmid-encoded copy of rel or, alternatively, by supplementation of the minimal medium with the amino acid mixture casamino acids. In particular, the Deltarel strain of C. glutamicum displayed a requirement for the amino acids histidine and serine.

Inducement of fluent speech in persons who stutter via visual choral speech.

A novel phenomenon of fluency enhancement via visual gestures of speech in the absence of traditional auditory feedback is reported herein. The effect on visual choral speech on stuttering frequency was investigated. Ten participants who stuttered recited memorized text aloud under two conditions. In a visual choral speech (VCS) condition participants were instructed to focus their gaze on the face, lips and jaw of a research assistant who 'silently mouthed' the text in unison. In a control condition, participants recited memorized text to the research assistant who sat motionless. A statistically significant (P=0.0025) reduction of approximately 80% in stuttering frequency was observed in the VCS condition. As visual linguistic cues are sufficient to activate the auditory cortex, one may speculate that VCS induces fluency in a similar yet undetermined manner as altered auditory feedback does.

Stutter-free and stutter-filled speech signals and their role in stuttering amelioration for English speaking adults.

This study examined the power of an exogenously generated stuttered speech signal on stuttering frequency when compared to an exogenously generated normal speech signal. In addition, we examined the specific components of the second speech signal, which might be responsible for the inducement of fluency in people who stutter. Eight males and two females who stuttered participated in this study. Experiment I involved meaningful speech: normal continuous speech, normal interrupted speech, stuttered continuous speech, and stuttered interrupted speech, whereas Experiment II involved vowels and consonants: /a/, /a-i-u/, /s/, /s-sh-f/. The results indicated that stuttered and normal speech signals were equally effective in reducing stuttering frequency. Further, the vowels were more powerful than consonants in inducing fluency for people who stutter. It is suggested that acoustic manifestations of stuttering, rather than a problem, may be a natural compensatory mechanism to bypass or inhibit the 'involuntary block' at the neural level.