Imaging of biological macromolecules on mica in humid air by scanning electrochemical microscopy

Imaging of DNA, keyhole limpet hemocyanin, mouse monoclonal IgG, and glucose oxidase on a mica substrate has been accomplished by scanning electrochemical microscopy with a tungsten tip. The technique requires the use of a high relative humidity to form a thin film of water on the mica surface that allows electrochemical reactions to take place at the tip and produce a faradaic current (≈1 pA) that can be used to control tip position. The effect of relative humidity and surface pretreatment with buffer solutions on the ionic conductivity of a mica surface was investigated to find appropriate conditions for imaging. Resolution of the order of 1 nm was obtained.

Structural activity of a cloned potassium channel (ROMK1) monitored with the atomic force microscope: The “molecular-sandwich” technique

The atomic force microscope (AFM) was used to continuously follow height changes of individual protein molecules exposed to physiological stimuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial potassium channel known to be highly pH sensitive) and lowered onto atomically flat mica surface until the protein was sandwiched between AFM tip and mica. Because the AFM tip was an integral part of a highly flexible cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of the cantilever that was monitored by means of a laser beam. With this system it was possible to resolve vertical height changes in the ROMK1 protein of ≥0.2 nm (approximately 5% of the molecule’s height) with a time resolution of ≥1 msec. When bathed in electrolyte solution that contained the catalytic subunit of protein kinase A and 0.1 mM ATP (conditions that activate the native ion channel), we found stochastically occurring height fluctuations in the ROMK1 molecule. These changes in height were pH-dependent, being greatest at pH 7.6, and lowering the pH (either by titration or by the application of CO2) reduced their magnitude. The data show that overall changes in shape of proteins occur stochastically and increase in size and frequency when the proteins are active. This AFM “molecular-sandwich” technique, called MOST, measures structural activity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the molecular level.

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots1

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO2 evolution or as O2 uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 107 bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO2 production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO2, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.

Cotunneling theory of atomic spin inelastic electron tunneling spectroscopy

We propose cotunneling as the microscopic mechanism that makes possible inelastic electron tunneling spectroscopy of magnetic atoms in surfaces for a wide range of systems, including single magnetic adatoms, molecules, and molecular stacks. We describe electronic transport between the scanning tip and the conducting surface through the magnetic system (MS) with a generalized Anderson model, without making use of effective spin models. Transport and spin dynamics are described with an effective cotunneling Hamiltonian in which the correlations in the magnetic system are calculated exactly and the coupling to the electrodes is included up to second order in the tip MS and MS substrate. In the adequate limit our approach is equivalent to the phenomenological Kondo exchange model that successfully describes the experiments. We apply our method to study in detail inelastic transport in two systems, stacks of cobalt phthalocyanines and a single Mn atom on Cu2N. Our method accounts for both the large contribution of the inelastic spin exchange events to the conductance and the observed conductance asymmetry.

Inelastic electron tunneling spectroscopy of a Mn dimer

A scanning tunneling microscope can probe the inelastic spin excitations of single magnetic atoms in a surface via spin-flip assisted tunneling. A particular and intriguing case is the Mn dimer case. We show here that the existing theories for inelastic transport spectroscopy do not explain the observed spin transitions when both atoms are equally coupled to the scanning tunneling microscope tip and the substrate, the most likely experimental situation. The hyperfine coupling to the nuclear spins is shown to lead to a finite excitation amplitude, but the physical mechanism leading to the large inelastic signal observed is still unknown. We discuss some other alternatives that break the symmetry of the system and allow for larger excitation probabilities.

Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter. (c) 2006 Elsevier B.V. All rights reserved.

Evaluation of routine microbiological techniques for establishing the diagnosis of catheter-related bloodstream infection caused by coagulase-negative staphylococci

Microbiological diagnosis of catheter-related bloodstream infection (CR-BSI) is often based on isolation of indistinguishable micro-organisms from an explanted catheter tip and blood culture, confirmed by antibiograms. Whether phenotypic identification of coagulase-negative staphylococci (CoNS) allows an accurate diagnosis of CR-BSI to be established was evaluated. Eight patients with a diagnosis of CR-BSI had CoNS isolated from pure blood cultures and explanted catheter tips which were considered as indistinguishable strains by routine microbiological methods. For each patient, an additional three colonies of CoNS isolated from the blood and five from the catheter tip were subcultured and further characterized by antibiogram profiles, analytical profile index (API) biotyping and PFGE. PFGE distinguished more strains of CoNS compared to API biotyping or antibiograms (17, 10 and 11, respectively). By PFGE, indistinguishable micro-organisms were only isolated from pure blood and catheter tip cultures in four out of eight (50%) patients thus supporting the diagnosis of CR-BSI. In another patient, indistinguishable micro-organisms were identified in both cultures; however, other strains of CoNS were also present. The remaining three patients had multiple strains of CoNS, none of which were indistinguishable in the tip and blood cultures, thus questioning the diagnosis of CR-BSI. Phenotypic characterization of CoNS lacked discriminatory power. Current routine methods of characterizing a limited number of pooled colonies may generate misleading results as multiple strains may be present in the cultures. Multiple colonies should be studied using a rapid genotypic characterization method to confirm or refute the diagnosis of CR-BSI. © 2007 SGM.

The tribology of linear tape recording systems

This thesis is devoted to the tribology at the head~to~tape interface of linear tape recording systems, OnStream ADRTM system being used as an experimental platform, Combining experimental characterisation with computer modelling, a comprehensive picture of the mechanisms involved in a tape recording system is drawn. The work is designed to isolate the mechanisms responsible for the physical spacing between head and tape with the aim of minimising spacing losses and errors and optimising signal output. Standard heads-used in ADR current products-and prototype heads- DLC and SPL coated and dummy heads built from a AI203-TiC and alternative single-phase ceramics intended to constitute the head tape-bearing surface-are tested in controlled environment for up to 500 hours (exceptionally 1000 hours), Evidences of wear on the standard head are mainly observable as a preferential wear of the TiC phase of the AI203-TiC ceramic, The TiC grains are believed to delaminate due to a fatigue wear mechanism, a hypothesis further confirmed via modelling, locating the maximum von Mises equivalent stress at a depth equivalent to the TiC recession (20 to 30 nm). Debris of TiC delaminated residues is moreover found trapped within the pole-tip recession, assumed therefore to provide three~body abrasive particles, thus increasing the pole-tip recession. Iron rich stain is found over the cycled standard head surface (preferentially over the pole-tip and to a lesser extent over the TiC grains) at any environment condition except high temperature/humidity, where mainly organic stain was apparent, Temperature (locally or globally) affects staining rate and aspect; stain transfer is generally promoted at high temperature. Humidity affects transfer rate and quantity; low humidity produces, thinner stains at higher rate. Stain generally targets preferentially head materials with high electrical conductivity, i.e. Permalloy and TiC. Stains are found to decrease the friction at the head-to-tape interface, delay the TiC recession hollow-out and act as a protective soft coating reducing the pole-tip recession. This is obviously at the expense of an additional spacing at the head-to-tape interface of the order of 20 nm. Two kinds of wear resistant coating are tested: diamond like carbon (DLC) and superprotective layer (SPL), 10 nm and 20 to 40 nm thick, respectively. DLC coating disappears within 100 hours due possibly to abrasive and fatigue wear. SPL coatings are generally more resistant, particularly at high temperature and low humidity, possibly in relation with stain transfer. 20 nm coatings are found to rely on the substrate wear behaviour whereas 40 nm coatings are found to rely on the adhesive strength at the coating/substrate interface. These observations seem to locate the wear-driving forces 40 nm below the surface, hence indicate that for coatings in the 10 nm thickness range-· i,e. compatible with high-density recording-the substrate resistance must be taken into account. Single-phase ceramic as candidate for wear-resistant tape-bearing surface are tested in form of full-contour dummy-heads. The absence of a second phase eliminates the preferential wear observed at the AI203-TiC surface; very low wear rates and no evidence of brittle fracture are observed.

A novel actuated digit with tactile feedback for clinical applications

This thesis describes the work carried out on the development of a novel digit actuator system with tactile perception feedback to a user and demonstrated as a master-slave system. For the tactile surface of the digit, contrasting sensor elements of resistive strain gauges and optical fibre Bragg grating sensors were evaluated. A distributive tactile sensing system consisting of optimised neural networking schemes was developed, resulting in taxonomy of artificial touch. The device is suitable for use in minimal invasive surgical (MIS) procedures as a steerable tip and a digit constructed wholly from polymers makes it suitable for use in Magnetic Resonance Imaging (MRI) environments enabling active monitoring of the patient during a procedure. To provide a realistic template of the work the research responded to the needs of two contrasting procedures: palpation of the prostate and endotracheal intubation in anaesthesia where the application of touch sense can significantly assist navigation. The performance of the approach was demonstrated with an experimental digit constructed for use in the laboratory in phantom trials. The phantom unit was developed to resemble facets of the clinical applications and digit system is able to evaluate reactive force distributions acting over the surface of the digit as well as different descriptions of contact and motion relative to the surface of the lumen. Completing control of the digit is via an instrumented glove, such that the digit actuates in sympathy with finger gesture and tactile information feedback is achieved by a combination of the tactile and visual means.

Compressive strength of dental composites photo-activated with different light tips

The aim of this study was to evaluate the compressive strength of microhybrid (FiltekTM Z250) and nanofilled (FiltekTM Supreme XT) composite resins photo-activated with two different light guide tips, fiber optic and polymer, coupled with one LED. The power density was 653 mW cm−2 when using the fiber optic light tip and 596 mW cm−2 with the polymer. After storage in distilled water at 37 ± 2 "C for seven days, the samples were subjected to mechanical testing of compressive strength in an EMIC universal mechanical testing machine with a load cell of 5 kN and speed of 0.5 mm min−1. The statistical analysis was performed using ANOVA with a confidence interval of 95% and Tamhane’s test. The results showed that the mean values of compressive strength were not influenced by the different light tips (p > 0.05). However, a statistical difference was observed (p < 0.001) between the microhybrid composite resin photo-activated with the fiber optic light tip and the nanofilled composite resin. Based on these results, it can be concluded that microhybrid composite resin photo-activated with the fiber optic light tip showed better results than nanofilled, regardless of the tip used, and the type of the light tip did not influence the compressive strength of either composite. Thus, the presented results suggest that both the fiber optic and polymer light guide tips provide adequate compressive strength to be used to make restorations. However, the fiber optic light tip associated with microhybrid composite resin may be an interesting option for restorations mainly in posterior teeth.

Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida)

Background: Gene expression studies are a prerequisite for understanding the biological function of genes. Because of its high sensitivity and easy use, quantitative PCR (qPCR) has become the gold standard for gene expression quantification. To normalise qPCR measurements between samples, the most prominent technique is the use of stably expressed endogenous control genes, the so called reference genes. However, recent studies show there is no universal reference gene for all biological questions. Roses are important ornamental plants for which there has been no evaluation of useful reference genes for gene expression studies. Results: We used three different algorithms (BestKeeper, geNorm and NormFinder) to validate the expression stability of nine candidate reference genes in different rose tissues from three different genotypes of Rosa hybrida and in leaves treated with various stress factors. The candidate genes comprised the classical "housekeeping genes" (Actin, EF-1α, GAPDH, Tubulin and Ubiquitin), and genes showing stable expression in studies in Arabidopsis (PP2A, SAND, TIP and UBC). The programs identified no single gene that showed stable expression under all of the conditions tested, and the individual rankings of the genes differed between the algorithms. Nevertheless the new candidate genes, specifically, PP2A and UBC, were ranked higher as compared to the other traditional reference genes. In general, Tubulin showed the most variable expression and should be avoided as a reference gene. Conclusions: Reference genes evaluated as suitable in experiments with Arabidopsis thaliana were stably expressed in roses under various experimental conditions. In most cases, these genes outperformed conventional reference genes, such as EF1-α and Tubulin. We identified PP2A, SAND and UBC as suitable reference genes, which in different combinations may be used for normalisation in expression analyses via qPCR for different rose tissues and stress treatments. However, the vast genetic variation found within the genus Rosa, including differences in ploidy levels, might also influence expression stability of reference genes, so that future research should also consider different genotypes and ploidy levels.

Dual Roles of Diadenosine Polyphosphates in Corneal Epithelial Cell Migration

Purpose. To investigate the influence of diadenosine polyphosphates on the rate of corneal epithelial cell migration. Methods. Primary corneal epithelial cell cultures were obtained from New Zealand White rabbits. Immunocytochemical experiments were performed by fixing the cells with 4% paraformaldehyde (PFA) and incubated with cytokeratin 3 primary antibody, which was subsequently incubated with a secondary IgG mouse labeled with FITC, and the cells were observed under confocal microscopy. Migration studies were performed by taking confluent monolayers that were wounded with a pipette tip and challenged with different di- and mononucleotides with or without P2 antagonist (n = 8 each treatment). For concentration–response analysis, compounds were tested in doses ranging from 10−8 to 10−3 M (n = 8). The stability of the dinucleotides was assayed by HPLC, with an isocratic method (n = 4). Results. Cells under study were verified as corneal epithelial cells via the immunocytochemical analysis. Cell migration experiments showed that Ap4A, UTP, and ATP accelerated the rate of healing (5, 2.75, and 3 hours, respectively; P < 0.05; P < 0.001), whereas Ap3A, Ap5A, and UDP delayed it (6.5, 10, and 2 hours, respectively; P < 0.05). ADP did not modify the rate of migration. Antagonists demonstrated that Ap4A and Ap3A did activate different P2Y receptors mediating corneal wound-healing acceleration and delay. Concerning the possible degradation of the dinucleotides, it was almost impossible to detect any products resulting from their cleavage. Conclusions. Based on the pharmacological profile of all the compounds tested, the two main P2Y receptors that exist in these corneal cells are a P2Y2 receptor accelerating the rate of healing and a P2Y6 receptor that delays this process.

Skin microbiome before development of atopic dermatitis: Early colonization with commensal staphylococci at 2 months is associated with a lower risk of atopic dermatitis at 1 year

Background: Disease flares of established atopic dermatitis (AD) are generally associated with a low-diversity skin microbiota and Staphylococcus aureus dominance. The temporal transition of the skin microbiome between early infancy and the dysbiosis of established AD is unknown. Methods: We randomly selected 50 children from the Cork Babies After SCOPE: Evaluating the Longitudinal Impact Using Neurological and Nutritional Endpoints (BASELINE) longitudinal birth cohort for microbiome sampling at 3 points in the first 6 months of life at 4 skin sites relevant to AD: the antecubital and popliteal fossae, nasal tip, and cheek. We identified 10 infants with AD and compared them with 10 randomly selected control infants with no AD. We performed bacterial 16S ribosomal RNA sequencing and analysis directly from clinical samples. Results: Bacterial community structures and diversity shifted over time, suggesting that age strongly affects the skin microbiome in infants. Unlike established AD, these patients with infantile AD did not have noticeably dysbiotic communities before or with disease and were not colonized by S aureus. In comparing patients and control subjects, infants who had affected skin at month 12 had statistically significant differences in bacterial communities on the antecubital fossa at month 2 compared with infants who were unaffected at month 12. In particular, commensal staphylococci were significantly less abundant in infants affected at month 12, suggesting that this genus might protect against the later development of AD. Conclusions: This study suggests that 12-month-old infants with AD were not colonized with S aureus before having AD. Additional studies are needed to confirm whether colonization with commensal staphylococci modulates skin immunity and attenuates development of AD.

Analysis of the tip roundness effects on the micro- and macroindentation response of elastic-plastic materials

In this work, the effects of indenter tip roundness oil the load-depth indentation curves were analyzed using finite element modeling. The tip roundness level was Studied based on the ratio between tip radius and maximum penetration depth (R/h(max)), which varied from 0.02 to 1. The proportional Curvature constant (C), the exponent of depth during loading (alpha), the initial unloading slope (S), the correction factor (beta), the level of piling-up or sinking-in (h(c)/h(max)), and the ratio h(max)/h(f) are shown to be strongly influenced by the ratio R/h(max). The hardness (H) was found to be independent of R/h(max) in the range studied. The Oliver and Pharr method was successful in following the variation of h(c)/h(max) with the ratio R/h(max) through the variation of S with the ratio R/h(max). However, this work confirmed the differences between the hardness values calculated using the Oliver-Pharr method and those obtained directly from finite element calculations; differences which derive from the error in area calculation that Occurs when given combinations of indented material properties are present. The ratio of plastic work to total work (W(p)/W(t)) was found to be independent of the ratio R/h(max), which demonstrates that the methods for the Calculation of mechanical properties based on the *indentation energy are potentially not Susceptible to errors caused by tip roundness.

Effects of mechanical properties, residual stress and indenter tip geometry on instrumented indentation data in thin films

In this work, an axisymmetric two-dimensional finite element model was developed to simulate instrumented indentation testing of thin ceramic films deposited onto hard steel substrates. The level of film residual stress (sigma(r)), the film elastic modulus (E) and the film work hardening exponent (n) were varied to analyze their effects on indentation data. These numerical results were used to analyze experimental data that were obtained with titanium nitride coated specimens, in which the substrate bias applied during deposition was modified to obtain films with different levels of sigma(r). Good qualitative correlation was obtained when numerical and experimental results were compared, as long as all film properties are considered in the analyses, and not only sigma(r). The numerical analyses were also used to further understand the effect of sigma(r) on the mechanical properties calculated based on instrumented indentation data. In this case, the hardness values obtained based on real or calculated contact areas are similar only when sink-in occurs, i.e. with high n or high ratio VIE, where Y is the yield strength of the film. In an additional analysis, four ratios (R/h(max)) between indenter tip radius and maximum penetration depth were simulated to analyze the combined effects of R and sigma(r) on the indentation load-displacement curves. In this case, or did not significantly affect the load curve exponent, which was affected only by the indenter tip radius. On the other hand, the proportional curvature coefficient was significantly affected by sigma(r) and n. (C) 2010 Elsevier B.V. All rights reserved.