Application of the specular and diffuse reflection analysis for in vitro diagnostics of dental erosion: correlation with enamel softening, roughness, and calcium release

We present assembly and application of an optical reflectometer for the analysis of dental erosion. The erosive procedure involved acid-induced softening and initial substance loss phases, which are considered to be difficult for visual diagnosis in a clinic. Change of the specular reflection signal showed the highest sensitivity for the detection of the early softening phase of erosion among tested methods. The exponential decrease of the specular reflection intensity with erosive duration was compared to the increase of enamel roughness. Surface roughness was measured by optical analysis, and the observed tendency was correlated with scanning electron microscopy images of eroded enamel. A high correlation between specular reflection intensity and measurement of enamel softening (r(2) ? -0.86) as well as calcium release (r(2) ? -0.86) was found during erosion progression. Measurement of diffuse reflection revealed higher tooth-to-tooth deviation in contrast to the analysis of specular reflection intensity and lower correlation with other applied methods (r(2) = 0.42-0.48). The proposed optical method allows simple and fast surface analysis and could be used for further optimization and construction of the first noncontact and cost-effective diagnostic tool for early erosion assessment in vivo.

Single- and double-electron transfer reactions of ground and metastable state Ar2+ ions

Electron transfer cross sections have been measured for reactions of Ar2+ ions with Ar, N2, O2, CO2, CH4 and C2H6. Time-of-flight techniques have been used to measure both fast neutral Ar0 and fast Ar+ products from single- and double-electron transfer processes involving Ar2+ ions with 4.0 to 7.0 keV impact energies. Incident Ar2+ ions have produced by controlled electron impact ionisation of argon atoms. Reactions have been examined as a function of ionising electron energy and cross sections determined for ground state Ar2+(3P) ions. Charge transfer cross sections have been determined to be in the range of 3*10-16 cm2 for the systems examined. Double-electron transfer cross sections are the same order of magnitude as those measured for the corresponding single-electron transfer reactions. The state distribution of the reactant ion beam has been estimated and electron transfer cross sections obtained for single- and double-electron transfer reactions of metastable Ar2+ions. The magnitudes of electron transfer cross sections in individual systems are similar for both ground and metastable state Ar2+ reactions.

Validation of MIPAS IMK/IAA temperature, water vapor, and ozone profiles with MOHAVE-2009 campaign measurements

MIPAS observations of temperature, water vapor, and ozone in October 2009 as derived with the scientific level-2 processor run by Karlsruhe Institute of Technology (KIT), Institute for Meteorology and Climate Research (IMK) and CSIC, Instituto de Astrofísica de Andalucía (IAA) and retrieved from version 4.67 level-1b data have been compared to co-located field campaign observations obtained during the MOHAVE-2009 campaign at the Table Mountain Facility near Pasadena, California in October 2009. The MIPAS measurements were validated regarding any potential biases of the profiles, and with respect to their precision estimates. The MOHAVE-2009 measurement campaign provided measurements of atmospheric profiles of temperature, water vapor/relative humidity, and ozone from the ground to the mesosphere by a suite of instruments including radiosondes, ozonesondes, frost point hygrometers, lidars, microwave radiometers and Fourier transform infra-red (FTIR) spectrometers. For MIPAS temperatures (version V4O_T_204), no significant bias was detected in the middle stratosphere; between 22 km and the tropopause MIPAS temperatures were found to be biased low by up to 2 K, while below the tropopause, they were found to be too high by the same amount. These findings confirm earlier comparisons of MIPAS temperatures to ECMWF data which revealed similar differences. Above 12 km up to 45 km, MIPAS water vapor (version V4O_H2O_203) is well within 10% of the data of all correlative instruments. The well-known dry bias of MIPAS water vapor above 50 km due to neglect of non-LTE effects in the current retrievals has been confirmed. Some instruments indicate that MIPAS water vapor might be biased high by 20 to 40% around 10 km (or 5 km below the tropopause), but a consistent picture from all comparisons could not be derived. MIPAS ozone (version V4O_O3_202) has a high bias of up to +0.9 ppmv around 37 km which is due to a non-identified continuum like radiance contribution. No further significant biases have been detected. Cross-comparison to co-located observations of other satellite instruments (Aura/MLS, ACE-FTS, AIRS) is provided as well.

Lateralized and frequency-dependent effects of prefrontal rTMS on regional cerebral blood flow

Repetitive transcranial magnetic stimulation (rTMS) is a means to study the function and connectivity of brain areas. The present study addressed the question of hemispheric asymmetry of frontal regions and aimed to further understand the acute effects of high- and low-frequency rTMS on regional cerebral blood flow (rCBF). Sixteen healthy right-handed men were imaged using H(2)(15)O positron emission tomography (PET) immediately after stimulation. High (10 Hz)- and low (1 Hz)-frequency suprathreshold short-duration rTMS was applied over either the left or right dorsolateral prefrontal cortex (DLPFC). Slow and fast rTMS applied over the left DLPFC significantly increased CBF in the stimulated area. Compared to baseline, slow rTMS induced a significant increase in CBF contralateral to the stimulation site, in the right caudate body and in the anterior cingulum. Furthermore, slow rTMS decreased CBF in the orbitofrontal cortex (OFC, ipsilateral to stimulation side). Fast rTMS applied over the right DLPFC was associated with increased activity at the stimulation site, in the bilateral orbitofrontal cortex and in the left medial thalamus compared to 1-Hz rTMS. These results show that rCBF changes induced by prefrontal rTMS differ upon hemisphere stimulated and vary with stimulation frequency. These differential neurophysiological effects of short-train rTMS with respect to side and frequency suggest hemisphere-dependent functional circuits of frontal cortico-subcortical areas.

A technique to detect and to quantify fasciocutaneous blood vessels in small laboratory animals ex vivo

PURPOSE: A microangiographical technique is described, which allows visualization of small and capillary blood vessels and quantification of fasciocutaneous blood vessels by means of digital computer analysis in very small laboratory animals. MATERIALS AND METHODS: The left carotid artery of 20 nu/nu mice was cannulated (26 gauge) and a mixture of gelatin, bariumsulfate, and green ink was injected according to standardized protocol. Fasciocutaneous blood vessels were visualized by digital mammography and analyzed for vessel length and vessel surface area as standardized units [SU] by computer program. RESULTS: With the described microangiography method, fasciocutaneous blood vessels down to capillary size level can be clearly visualized. Regions of interest (ROIs) can be defined and the containing vascular network quantified. Comparable results may be obtained by calculating the microvascular area index (MAI) and the microvascular length index (MLI), related to the ROIs size. Identical ROIs showed a high reproducibility for measured [SU] < 0.01 +/- 0.0012%. CONCLUSION: Combining microsurgical techniques, pharmacological knowledge, and modern digital image technology, we were able to visualize small and capillary blood vessels even in small laboratory animals. By using our own computer analytical program, quantification of vessels was reliable, highly reproducible, and fast.

FAST ADAPTIVE PENALIZED SPLINES

This paper proposes a numerically simple routine for locally adaptive smoothing. The locally heterogeneous regression function is modelled as a penalized spline with a smoothly varying smoothing parameter modelled as another penalized spline. This is being formulated as hierarchical mixed model, with spline coe±cients following a normal distribution, which by itself has a smooth structure over the variances. The modelling exercise is in line with Baladandayuthapani, Mallick & Carroll (2005) or Crainiceanu, Ruppert & Carroll (2006). But in contrast to these papers Laplace's method is used for estimation based on the marginal likelihood. This is numerically simple and fast and provides satisfactory results quickly. We also extend the idea to spatial smoothing and smoothing in the presence of non normal response.

Use of the Agilent 2100 bioanalyzer for rapid and reproducible molecular typing of Streptococcus pneumoniae

Restriction fragment length polymorphism (RFLP) analysis is an economic and fast technique for molecular typing but has the drawback of difficulties in accurately sizing DNA fragments and comparing banding patterns on agarose gels. We aimed to improve RFLP for typing of the important human pathogen Streptococcus pneumoniae and to compare the results with the commonly used typing techniques of pulsed-field gel electrophoresis and multilocus sequence typing. We designed primers to amplify a noncoding region adjacent to the pneumolysin gene. The PCR product was digested separately with six restriction endonucleases, and the DNA fragments were analyzed using an Agilent 2100 bioanalyzer for accurate sizing. The combined RFLP results for all enzymes allowed us to assign each of the 47 clinical isolates of S. pneumoniae tested to one of 33 RFLP types. RFLP analyzed using the bioanalyzer allowed discrimination between strains similar to that obtained by the more commonly used techniques of pulsed-field gel electrophoresis, which discriminated between 34 types, and multilocus sequence typing, which discriminated between 35 types, but more quickly and with less expense. RFLP of a noncoding region using the Agilent 2100 bioanalyzer could be a useful addition to the molecular typing techniques in current use for S. pneumoniae, especially as a first screen of a local population.

Local degradation of structural, mechanical, electrical and chemical properties of membrane electrode assembly in polymer electrolyte fuel cell

Polymer electrolyte fuel cell (PEMFC) is promising source of clean power in many applications ranging from portable electronics to automotive and land-based power generation. However, widespread commercialization of PEMFC is primarily challenged by degradation. The mechanisms of fuel cell degradation are not well understood. Even though the numbers of installed units around the world continue to increase and dominate the pre-markets, the present lifetime requirements for fuel cells cannot be guarantee, creating the need for a more comprehensive knowledge of material’s ageing mechanism. The objective of this project is to conduct experiments on membrane electrode assembly (MEA) components of PEMFC to study structural, mechanical, electrical and chemical changes during ageing and understanding failure/degradation mechanism. The first part of this project was devoted to surface roughness analysis on catalyst layer (CL) and gas diffusion layer (GDL) using surface mapping microscopy. This study was motivated by the need to have a quantitative understanding of the GDL and CL surface morphology at the submicron level to predict interfacial contact resistance. Nanoindentation studies using atomic force microscope (AFM) were introduced to investigate the effect of degradation on mechanical properties of CL. The elastic modulus was decreased by 45 % in end of life (EOL) CL as compare to beginning of life (BOL) CL. In another set of experiment, conductive AFM (cAFM) was used to probe the local electric current in CL. The conductivity drops by 62 % in EOL CL. The future task will include characterization of MEA degradation using Raman and Fourier transform infrared (FTIR) spectroscopy. Raman spectroscopy will help to detect degree of structural disorder in CL during degradation. FTIR will help to study the effect of CO in CL. XRD will be used to determine Pt particle size and its crystallinity. In-situ conductive AFM studies using electrochemical cell on CL to correlate its structure with oxygen reduction reaction (ORR) reactivity

Automation of shear-wave splitting parameter determination of local earthquakes at Yellowstone : application as indicator of crustal stress and temporal variation

Shear-wave splitting can be a useful technique for determining crustal stress fields in volcanic settings and temporal variations associated with activity. Splitting parameters were determined for a subset of local earthquakes recorded from 2000-2010 at Yellowstone. Analysis was automated using an unsupervised cluster analysis technique to determine optimum splitting parameters from 270 analysis windows for each event. Six stations clearly exhibit preferential fast polarization values sub-orthogonal to the direction of minimum horizontal compression. Yellowstone deformation results in a local crustal stress field differing from the regional field dominated by NE-SW extension, and fast directions reflect this difference rotating around the caldera maintaining perpendicularity to the rim. One station exhibits temporal variations concordant with identified periods of caldera subsidence and uplift. From splitting measurements, we calculated a crustal anisotropy of ~17-23% and crack density ~0.12-0.17 possibly resulting from stress-aligned fluid filled microcracks in the upper crust and an active hydrothermal system.

DESIGN AND DEVELOPMENT OF BODIPY-BASED FLUORESCENT PROBES FOR SENSING AND IMAGING OF CYANIDE, Zn (II) IONS, LYSOSOMAL pH AND CANCER CELLS

BODIPY (4,4-Difluoro-3a,4a-diaza-s-indacene) dyes have gained lots of attention in application of fluorescence sensing and imaging in recent years because they possess many distinctive and desirable properties such as high extinction coefficient, narrow absorption and emission bands, high quantum yield and low photobleaching effect. However, most of BODIPY-based fluorescent probes have very poor solubilities in aqueous solution, emit less than 650 nm fluorescence that can cause cell and tissue photodamages compared with bio-desirable near infrared (650-900 nm) light. These undesirable properties extremely limit the applications of BODIPY-based fluorescent probes in sensing and imaging applications. In order to overcome these drawbacks, we have developed a very effective strategy to prepare a series of neutral highly water- soluble BODIPY dyes by enhancing the water solubilities of BODIPY dyes via incorporation of tri(ethylene glycol)methyl ether (TEG) and branched oligo(ethylene glycol)methyl ether (BEG) residues onto BODIPY dyes at 1,7-, 2,6-, 3,5-, 4- and meso- positions. We also have effectively tuned absorptions and emissions of BOIDPY dyes to red, deep red and near infrared regions via significant extension of π-conjugation of BODIPY dyes by condensation reactions of aromatic aldehydes with 2,6-diformyl BODIPY dyes at 1,3,5,7-positions. Based on the foundation that we built for enhancing water solubility and tuning wavelength, we have designed and developed a series of water-soluble, BODIPY-based fluorescent probes for sensitive and selective sensing and imaging of cyanide, Zn (II) ions, lysosomal pH and cancer cells. We have developed three BODIPY-based fluorescent probes for sensing of cyanide ions by incorporating indolium moieties onto the 6-position of TEG- or BEG-modified BOIDPY dyes. Two of them are highly water-soluble. These fluorescent probes showed selective and fast ratiometric fluorescent responses to cyanide ions with a dramatic fluorescence color change from red to green accompanying a significant increase in fluorescent intensity. The detection limit was measured as 0.5 mM of cyanide ions. We also have prepared three highly water-soluble fluorescent probes for sensing of Zn (II) ions by introducing dipicoylamine (DPA, Zn ion chelator) onto 2- and/or 6-positions of BEG-modified BODIPY dyes. These probes showed selective and sensitive responses to Zn (II) ion in the range from 0.5 mM to 24 mM in aqueous solution at pH 7.0. Particularly, one of the probes displayed ratiometric responses to Zn (II) ions with fluorescence quenching at 661 nm and fluorescence enhancement at 521 nm. This probe has been successfully applied to the detection of intracellular Zn (II) ions inside the living cells. Then, we have further developed three acidotropic, near infrared emissive BODIPY- based fluorescent probes for detection of lysosomal pH by incorporating piperazine moiety at 3,5-positions of TEG- or BEG-modified BODIPY dyes as parts of conjugation. The probes have low auto-fluorescence at physiological neutral condition while their fluorescence intensities will significant increase at 715 nm when pH shift to acidic condition. These three probes have been successfully applied to the in vitro imaging of lysosomes inside two types of living cells. At the end, we have synthesized one water- soluble, near infrared emissive cancer cell targetable BODIPY-based fluorescent polymer bearing cancer homing peptide (cRGD) residues for cancer cell imaging applications. This polymer exhibited excellent water-solubility, near infrared emission (712 nm), good biocompatibility. It also showed low nonspecific interactions to normal endothelial cells and can effectively detect breast tumor cells.

Experimental dissection and characterization of the functional domains in cardiac troponin C

Contraction of vertebrate cardiac muscle is regulated by the binding of Ca$\sp{2+}$ to the troponin C (cTnC) subunit of the troponin complex. In this study, we have used site-directed mutagenesis and a variety of assay techniques to explore the functional roles of regions in cTnC, including Ca$\sp{2+}$/Mg$\sp{2+}$-binding sites III and IV, the functionally inactive site I, the N-terminal helix, the N-terminal hydrophobic pocket and the two cysteine residues with regard to their ability to form disulfide bonds. Conversion of the first Ca$\sp{2+}$ ligand from Asp to Ala inactivated sites III and IV and decreased the apparent affinity of cTnC for the thin filament. Conversion of the second ligand from Asn to Ala also inactivated these sites in the free protein but Ca$\sp{2+}$-binding was recovered upon association with troponin I and troponin T. The Ca$\sp{2+}$-concentrations required for tight thin filament-binding by proteins containing second-ligand mutations were significantly greater than that required for the wild-type protein. Mutation of site I such that the primary sequence was that of an active site with the first Ca$\sp{2+}$ ligand changed from Asp to Ala resulted in a 70% decrease in maximal Ca$\sp{2\sp+}$ dependent ATPase activity in both cardiac and fast skeletal myofibrils. Thus, the primary sequence of the inactive site I in cTnC is functionally important. Major changes in the sequence of the N-terminus had little effect on the ability of cTnC to recover maximal activity but deletion of the first nine residues resulted in a 60 to 80% decrease in maximal activity with only a minor decrease in the pCa$\sb{50}$ of activation, suggesting that the N-terminal helix must be present but that a specific sequence is not required. The formation of an inter- or intramolecular disulfide bonds caused the exposure of hydrophobic surfaces on cTnC and rendered the protein Ca$\sp{2+}$ independent. Finally, elution patterns from a hydrophobic interactions column suggest that cTnC undergoes a significant change in hydrophobicity upon Ca$\sp{2+}$ binding, the majority of which is caused by site II. These latter data show an interesting correlation between exposure of hydrophobic surfaces on and activation of cTnC. Overall, these results represent significant progress toward the elucidation of the functional roles of a variety of structural regions in cTnC. ^

THE ISOLATION AND IDENTIFICATION OF O-ACETYLATED SIALIC ACIDS ON HUMAN CELLS (NEURAMINIC ACIDS, PAPER CHROMATOGRAPHY, HUMAN MALIGNANCY, BIOSYNTHETIC LABELING)

Unlike most carbohydrates, sialic acids have a restricted distribution in nature, being present in higher animals and in certain bacteriae. Unfortunately, most studies have not taken into account the fact that the parent sialic acid molecules, N-acetyl(or N-glycolyl)-neuraminic acid can be O-substituted at the 4, 7, 8 and 9 positions, generating many compounds and isomers. The approach and results of this research study demonstrates that proportions of non-, mono-, di-, and tri-O-acetylated sialic acids can be identified and quantitated on normal and malignant human cells. This was accomplished using a paper chromatographic technique to isolate and resolve individual species of non and O-substituted sialic acids. The chemical nature of these O-substituents, as an acetyl ester, was determined on the basis of chemical degradation, enzymatic and fast atom bombardment-mass spectrometry analysis.^ The working hypothesis of this study, that O-acetylated sialic acids are expressed in a restricted manner on normal and malignant cells, was confirmed using the above experimental approach; which identified mono-, di-, and tri-O-acetylated sialic acids on a variety of normal and malignant human cells. These O-acetylated sialic acids were expressed in restricted manner on subpopulations and subcellular fractions of PHL melanoma cells. Aberrant expression of O-acetylated sialic acids was associated with adenocarcinoma of the colon, leading to a nearly complete loss of di- and tri-O-acetylated sialic acids.^ Thus, the ability to isolate and identify biosynthetically radiolabeled O-acetylated sialic acids offers an efficient method of monitoring the expression of O-acetylated sialic acids in biochemical and cellular interactions. Furthermore, the ability to identify abnormal ratios of O-acetylated sialic acids in the human colon, represents a possible diagnostic tool to evaluate and identify patients who may be genetically or culturally predisposed to the development of adenocarcinoma of the colon. ^

Structure and inferred dynamics of a large grove of Microberlinia bisulcata trees in central African rain forest: the possible role of periods of multiple disturbance events

A 272-ha grove of dominant Microberlinia bisulcata (Caesalpinioideae) adult trees greater than or equal to 50 cm stem diameter was mapped in its entirety in the southern part of Korup National Park, Cameroon. The approach used an earlier-established 82.5-ha permanent plot with a new surrounding 50-m grid of transect lines. Tree diameters were available from the plot but trees on the grid were recorded as being greater than or equal to 50 cm. The grove consisted of 1028 trees in 2000. Other species occurred within the grove. including the associated subdominants Tetraberlinia bifoliolata and T. korupensis. Microberlinia bisulcata becomes adult at a stein diameter of c. 50 cm and at an estimated age of 50 y. Three oval-shaped subgroves with dimensions c. 8 50 in x 13 50 in (90 ha) were defined. For two of them (within the plot) tree diameters were available. Subgroves differed in their scales and intensities of spatial tree patterns, and in their size frequency distributions, these suggesting differing past dynamics. The modal scale of clumping was 40-50 m. Seed dispersal by pod ejection (to c. 50 in) was evident from the semi-circles of trees at the grove's edge and from the many internal circles (100-200 m diameter). The grove has the capacity. therefore, to increase at c. 100 m per century. To form its present extent and structure. it is inferred that it expanded and infilled from a possibly smaller area of lower adult-tree density. This possibly happened in three waves of recruitment, each one determined by a period of several intense disturbances. Climate records for Africa show that 1740-50 and 1820-30 were periods of drought, and that 1870-1895 was also regionally very dry. Canopy openings allow the light-demanding and fast-growing ectomycorrhizal M. bisulcata to establish, but successive releases are thought to be required to achieve effective recruitment. Nevertheless, in the last 50 y there were no major events and recruitment in the grove was very poor. This present study leads to a new hypothesis of the role of periods of multiple extreme events being the driving factor for the population dynamics of many large African tree species such as M. bisulcata.

Polysomnographic record and successful management of augmentation in restless legs syndrome/Willis-Ekbom disease

BACKGROUND: Dopamine agonists (DAs) represent the first-line treatment in restless legs syndrome (RLS); however, in the long term, a substantial proportion of patients will develop augmentation, which is a severe drug-related exacerbation of symptoms and the main reason for late DA withdrawal. Polysomnographic features and mechanisms underlining augmentation are unknown. No practice guidelines for management of augmentation are available. METHODS: A clinical case series of 24 consecutive outpatients affected by RLS with clinically significant augmentation during treatment with immediate-release DA was performed. All patients underwent a full-night polysomnographic recording during augmentation. A switchover from immediate-release DAs (l-dopa, pramipexole, ropinirole, rotigotine) to the long-acting, extended-release formula of pramipexole was performed. RESULTS: Fifty percent of patients presented more than 15 periodic limb movements per hour of sleep during augmentation, showing longer sleep latency and shorter total sleep time than subjects without periodic limb movements. In all patients, resolution of augmentation was observed within two to four weeks during which immediate-release dopamine agonists could be completely withdrawn. Treatment efficacy of extended-release pramipexole has persisted, thus far, over a mean follow-up interval of 13 months. CONCLUSIONS: Pramipexole extended release could be an easy, safe, and fast pharmacological option to treat augmentation in patients with restless legs syndrome. As such it warrants further prospective and controlled investigations. This observation supports the hypothesis that the duration of action of the drug plays a key role in the mechanism of augmentation.

Tracking individual membrane proteins and their biochemistry: The power of direct observation

The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit.