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Sample records for pathway direct afm

  1. Mapping individual cosmid DNAs by direct AFM imaging.

    PubMed

    Allison, D P; Kerper, P S; Doktycz, M J; Thundat, T; Modrich, P; Larimer, F W; Johnson, D K; Hoyt, P R; Mucenski, M L; Warmack, R J

    1997-05-01

    Individual cosmid clones have been restriction mapped by directly imaging, with the atomic force microscope (AFM), a mutant EcoRI endonuclease site-specifically bound to DNA. Images and data are presented that locate six restriction sites, predicted from gel electrophoresis, on a 35-kb cosmid isolated from mouse chromosome 7. Measured distances between endonuclease molecules bound to lambda DNA, when compared to known values, demonstrate the accuracy of AFM mapping to better than 1%. These results may be extended to identify other important site-specific protein-DNA interactions, such as transcription factor and mismatch repair enzyme binding, difficult to resolve by current techniques.

  2. Direct Measurement of Optical Force Induced by Near-Field Plasmonic Cavity Using Dynamic Mode AFM

    PubMed Central

    Guan, Dongshi; Hang, Zhi Hong; Marcet, Zsolt; Liu, Hui; Kravchenko, I. I.; Chan, C. T.; Chan, H. B.; Tong, Penger

    2015-01-01

    Plasmonic nanostructures have attracted much attention in recent years because of their potential applications in optical manipulation through near-field enhancement. Continuing experimental efforts have been made to develop accurate techniques to directly measure the near-field optical force induced by the plasmonic nanostructures in the visible frequency range. In this work, we report a new application of dynamic mode atomic force microscopy (DM-AFM) in the measurement of the enhanced optical force acting on a nano-structured plasmonic resonant cavity. The plasmonic cavity is made of an upper gold-coated glass sphere and a lower quartz substrate patterned with an array of subwavelength gold disks. In the near-field when the sphere is positioned close to the disk array, plasmonic resonance is excited in the cavity and the induced force by a 1550 nm infrared laser is found to be increased by an order of magnitude compared with the photon pressure generated by the same laser light. The experiment demonstrates that DM-AFM is a powerful tool for the study of light induced forces and their enhancement in plasmonic nanostructures. PMID:26586455

  3. Direct measurement of optical force induced by near-field plasmonic cavity using dynamic mode AFM

    DOE PAGES

    Guan, Dongshi; Hang, Zhi Hong; Marset, Zsolt; ...

    2015-11-20

    Plasmonic nanostructures have attracted much attention in recent years because of their potential applications in optical manipulation through near-field enhancement. Continuing experimental efforts have been made to develop accurate techniques to directly measure the near-field optical force induced by the plasmonic nanostructures in the visible frequency range. In this work, we report a new application of dynamic mode atomic force microscopy (DM-AFM) in the measurement of the enhanced optical force acting on a nano-structured plasmonic resonant cavity. The plasmonic cavity is made of an upper gold-coated glass sphere and a lower quartz substrate patterned with an array of subwavelength goldmore » disks. In the near-field when the sphere is positioned close to the disk array, plasmonic resonance is excited in the cavity and the induced force by a 1550 nm infrared laser is found to be increased by an order of magnitude compared with the photon pressure generated by the same laser light. Lastly, the experiment demonstrates that DM-AFM is a powerful tool for the study of light induced forces and their enhancement in plasmonic nanostructures.« less

  4. Direct measurement of optical force induced by near-field plasmonic cavity using dynamic mode AFM

    SciTech Connect

    Guan, Dongshi; Hang, Zhi Hong; Marset, Zsolt; Liu, Hui; Kravchenko, Ivan I.; Chan, Ho Bun; Chan, C. T.; Tong, Penger

    2015-11-20

    Plasmonic nanostructures have attracted much attention in recent years because of their potential applications in optical manipulation through near-field enhancement. Continuing experimental efforts have been made to develop accurate techniques to directly measure the near-field optical force induced by the plasmonic nanostructures in the visible frequency range. In this work, we report a new application of dynamic mode atomic force microscopy (DM-AFM) in the measurement of the enhanced optical force acting on a nano-structured plasmonic resonant cavity. The plasmonic cavity is made of an upper gold-coated glass sphere and a lower quartz substrate patterned with an array of subwavelength gold disks. In the near-field when the sphere is positioned close to the disk array, plasmonic resonance is excited in the cavity and the induced force by a 1550 nm infrared laser is found to be increased by an order of magnitude compared with the photon pressure generated by the same laser light. Lastly, the experiment demonstrates that DM-AFM is a powerful tool for the study of light induced forces and their enhancement in plasmonic nanostructures.

  5. Direct electrochemical and AFM detection of amyloid-β peptide aggregation on basal plane HOPG

    NASA Astrophysics Data System (ADS)

    Lopes, Paula; Xu, Meng; Zhang, Min; Zhou, Ting; Yang, Yanlian; Wang, Chen; Ferapontova, Elena E.

    2014-06-01

    Amyloidogenesis is associated with more than 30 human diseases, including Alzheimer's which is related to aggregation of β-amyloid peptide (Aβ). Here, consecutive stages of Aβ42 aggregation and amyloid fibril formation were followed electrochemically via oxidation of tyrosines in Aβ42 adsorbed on the basal plane graphite electrode and directly correlated with Aβ42 morphological changes observed by atomic force microscopy of the same substrate. The results offer new tools for analysis of mechanisms of Aβ aggregation.Amyloidogenesis is associated with more than 30 human diseases, including Alzheimer's which is related to aggregation of β-amyloid peptide (Aβ). Here, consecutive stages of Aβ42 aggregation and amyloid fibril formation were followed electrochemically via oxidation of tyrosines in Aβ42 adsorbed on the basal plane graphite electrode and directly correlated with Aβ42 morphological changes observed by atomic force microscopy of the same substrate. The results offer new tools for analysis of mechanisms of Aβ aggregation. Electronic supplementary information (ESI) available: Experimental details: procedures for Aβ42 aggregation and electrode modification, DPV/AFM measurements and analysis. See DOI: 10.1039/c4nr02413c

  6. Non-reciprocal directional dichroism in the AFM phase of BiFeO3 at THz frequencies

    NASA Astrophysics Data System (ADS)

    Nagel, Urmas; Rõõm, T.; Farkas, D.; Szaller, D.; Bordács, S.; Kézsmárki, I.; Engelkamp, H.; Ozaki, Y.; Tomiaki, Y.; Ito, T.; Fishman, Randy S.

    We did THz absorption spectroscopy of BiFeO3 single crystals in the AFM phase, where the spin cycloid is destroyed in magnetic fields between 18 T and 32 T in Voigt geometry at 1.6 K. If B0 ∥ [ 1 1 0 ] , we see strong directional dichroism (DD) of absorption of the magnon mode with light propagating along the direction of the ferroelectric polarization k ∥ P ∥ [ 111 ] and eω ∥ [ 1 1 0 ] , bω ∥ [ 1 1 2 ] . The sign of DD can be reversed (i) by reversing the direction of B0 or (ii) by flipping the sample, thus reversing the propagation direction of light. The observed effect is caused by the strong magneto-electric coupling in the collinear AFM phase. Research sponsored by the Estonian Ministry of Education and Research (IUT23-3).

  7. Useful oriented immobilization of antibodies on chimeric magnetic particles: direct correlation of biomacromolecule orientation with biological activity by AFM studies.

    PubMed

    Marciello, Marzia; Filice, Marco; Olea, David; Velez, Marisela; Guisan, José M; Mateo, Cesar

    2014-12-16

    The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in a covalent way without any previous site-selective chemical modification of biomacromolecules. Different key parameters attending the process were studied and optimized. The optimal preparation was performed using a controlled loading (1 nmol Ab g(-1) chimeric support) at pH 9 and a short reaction time to recover a biological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and the final achievement of a highly active and recyclable chimeric immune sensor. This direct technique was demonstrated to be a powerful alternative to the indirect immunoactivity assay methods for the study of biomacromolecule-oriented immobilizations.

  8. Germ direct observation by AFM under crystallization of self-organized assemblies of mono-protonated meso-tetraphenylporphine dimers

    NASA Astrophysics Data System (ADS)

    Udal'tsov, Alexander V.

    2016-08-01

    Assemblies consisting of mono-protonated meso-tetraphenylporphine dimers and water have been investigated by UV-vis spectroscopy in solution and atomic force microscopy (AFM) in thin layers. These assemblies self-organized into domains produce microcrystals in thin layer. Morphology of the microcrystals and characteristic features of crystallization germ on the top found by contact AFM indicate that surface tension of an aqueous layer on the domain generates the crystallization process. Estimations of the pressure producing the germ and bulk modulus (Bm) of microcrystals give 26.3±2.6 MPa and 3.72 GPa and Bm=12.7 GPa obtained for dried thin films. The former modulus is comparable with bulk modulus of water (2.174 GPa) that implies liquid crystals formation. Absorptions of longitudinal optical (LO) phonons with ћωLO=0.3761 and 0.3577 eV, which are arisen because of hole polaron moving through water, are found in the electronic spectra of the assemblies. The crystallization is suggested to occur due to Zundel cation (H5O2+) operation like the water-porphyrin matrix self-organization found earlier.

  9. Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

    PubMed Central

    Umeno, Daisuke; Tobias, Alexander V.; Arnold, Frances H.

    2005-01-01

    Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products—those that could be made biosynthetically—remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these “evolved” pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed. PMID:15755953

  10. The planar cell polarity pathway directs parietal endoderm migration.

    PubMed

    LaMonica, Kristi; Bass, Maya; Grabel, Laura

    2009-06-01

    Parietal endoderm (PE) contributes to the yolk sac and is the first migratory cell type in the mammalian embryo. We can visualize PE migration in vitro using the F9 teratocarcinoma derived embryoid body outgrowth system and, show here that PE migration is directed by the non-canonical Wnt planar cell polarity (PCP) pathway via Rho/ROCK. Based on golgi apparatus localization and microtubule orientation, 68.6% of cells in control outgrowths are oriented in the direction of migration. Perturbation of Wnt signaling via sFRP treatment results in a loss of orientation coupled with an increase in cell migration. Inhibition of the PCP pathway at the level of Daam1 also results in a loss of cell orientation along with an increase in cell migration, as seen with sFRP treatment. Constitutively active Daam can inhibit the loss of orientation that occurs with sFRP treatment. We previously demonstrated that ROCK inhibition leads to an increase in cell migration, and we now show that these cells also lack oriented migration. Canonical Wnt signaling or the Rac arm of the PCP pathway does not appear to play a role in PE oriented migration. These data suggest the PCP pathway via Rho/ROCK modulates migration of PE.

  11. Chemical Shifts to Metabolic Pathways: Identifying Metabolic Pathways Directly from a Single 2D NMR Spectrum.

    PubMed

    Dubey, Abhinav; Rangarajan, Annapoorni; Pal, Debnath; Atreya, Hanudatta S

    2015-12-15

    Identifying cellular processes in terms of metabolic pathways is one of the avowed goals of metabolomics studies. Currently, this is done after relevant metabolites are identified to allow their mapping onto specific pathways. This task is daunting due to the complex nature of cellular processes and the difficulty in establishing the identity of individual metabolites. We propose here a new method: ChemSMP (Chemical Shifts to Metabolic Pathways), which facilitates rapid analysis by identifying the active metabolic pathways directly from chemical shifts obtained from a single two-dimensional (2D) [(13)C-(1)H] correlation NMR spectrum without the need for identification and assignment of individual metabolites. ChemSMP uses a novel indexing and scoring system comprised of a "uniqueness score" and a "coverage score". Our method is demonstrated on metabolic pathways data from the Small Molecule Pathway Database (SMPDB) and chemical shifts from the Human Metabolome Database (HMDB). Benchmarks show that ChemSMP has a positive prediction rate of >90% in the presence of decluttered data and can sustain the same at 60-70% even in the presence of noise, such as deletions of peaks and chemical shift deviations. The method tested on NMR data acquired for a mixture of 20 amino acids shows a success rate of 93% in correct recovery of pathways. When used on data obtained from the cell lysate of an unexplored oncogenic cell line, it revealed active metabolic pathways responsible for regulating energy homeostasis of cancer cells. Our unique tool is thus expected to significantly enhance analysis of NMR-based metabolomics data by reducing existing impediments.

  12. Direct and indirect dorsolateral striatum pathways reinforce different action strategies.

    PubMed

    Vicente, Ana M; Galvão-Ferreira, Pedro; Tecuapetla, Fatuel; Costa, Rui M

    2016-04-04

    The basal ganglia, and the striatum in particular, are critical for action reinforcement [1,2]. The dorsal striatum, which can be further subdivided into dorsomedial (DMS) and dorsolateral (DLS) striatum, is mainly composed of two subpopulations of striatal medium spiny projection neurons (MSNs): dopamine D1 receptor-expressing MSNs that constitute the striatonigral or direct pathway (dMSNs); and dopamine D2 receptor-expressing MSNs that constitute the striatopallidal or indirect pathway (iMSNs) [3]. It has been suggested that each pathway has opposing roles in reinforcement, with dMSNs being important to learn positive reinforcement and iMSNs to learn to avoid undesired actions (Go/No-Go) [1]. Furthermore, optogenetic self-stimulation of dMSNs in DMS leads to reinforcement of actions, while self-stimulation of iMSNs leads to avoidance of actions [2]. However, in DLS, which has been implicated in the consolidation of well-trained actions and habits in mice [4,5], both pathways are active during lever-pressing for reward [6]. Furthermore, extensive skill training leads to long-lasting potentiation of glutamatergic inputs into both dMSNs and iMSNs [4]. We report here that, in DLS, both dMSNs and iMSNs are involved in positive reinforcement, but support different action strategies.

  13. Nanoscale Nucleosome Dynamics Assessed with Time-lapse AFM

    PubMed Central

    Lyubchenko, Yuri L.

    2013-01-01

    A fundamental challenge associated with chromosomal gene regulation is accessibility of DNA within nucleosomes. Recent studies performed by various techniques, including single-molecule approaches, led to the realization that nucleosomes are dynamic structures rather than static systems, as it was once believed. Direct data is required in order to understand the dynamics of nucleosomes more clearly and answer fundamental questions, including: What is the range of nucleosome dynamics? Does a non-ATP dependent unwrapping process of nucleosomes exist? What are the factors facilitating the large scale opening and unwrapping of nucleosomes? This review summarizes the results of nucleosome dynamics obtained with time-lapse AFM, including a high-speed version (HS-AFM) capable of visualizing molecular dynamics on the millisecond time scale. With HS-AFM, the dynamics of nucleosomes at a sub-second time scale was observed allowing one to visualize various pathways of nucleosome dynamics, such as sliding and unwrapping, including complete dissociation. Overall, these findings reveal new insights into the dynamics of nucleosomes and the novel mechanisms controlling spontaneous chromatin dynamics. PMID:24839467

  14. Molecular pathways for defect annihilation in directed self-assembly.

    DOE PAGES

    Hur, Su-Mi; Thapar, Vikram; Ramirez-Hernandez, Abelardo; ...

    2015-11-17

    Over the last few years, the directed self-assembly of block copolymers by surface patterns has transitioned from academic curiosity to viable contender for commercial fabrication of next-generation nanocircuits by lithography. Recently, it has become apparent that kinetics, and not only thermodynamics, plays a key role for the ability of a polymeric material to self-assemble into a perfect, defect-free ordered state. Perfection, in this context, implies not more than one defect, with characteristic dimensions on the order of 5 nm, over a sample area as large as 100 cm2. In this work, we identify the key pathways and the corresponding free-energymore » barriers for eliminating defects, and we demonstrate that an extraordinarily large thermodynamic driving force is not necessarily sufficient for their removal. By adopting a concerted computational and experimental approach, we explain the molecular origins of these barriers, how they depend on material characteristics, and we propose strategies designed to over-come them. The validity of our conclusions for industrially-relevant patterning processes is established by relying on instruments and assembly lines that are only available at state-of-the-art fabrication facilities and, through this confluence of fundamental and applied research, we are able to discern the evolution of morphology at the smallest relevant length scales - a handful of nanometers -, and present a view of defect annihilation in directed self-assembly at an unprecedented level of detail.« less

  15. Molecular pathways for defect annihilation in directed self-assembly.

    SciTech Connect

    Hur, Su-Mi; Thapar, Vikram; Ramirez-Hernandez, Abelardo; Khaira, Gurdaman S.; Segal-Peretz, Tamar; Rincon-Delgadillo, Paulina A.; Li, Weihua; Muller, Marcus; Nealey, Paul F.; de Pablo, Juan J.

    2015-11-17

    Over the last few years, the directed self-assembly of block copolymers by surface patterns has transitioned from academic curiosity to viable contender for commercial fabrication of next-generation nanocircuits by lithography. Recently, it has become apparent that kinetics, and not only thermodynamics, plays a key role for the ability of a polymeric material to self-assemble into a perfect, defect-free ordered state. Perfection, in this context, implies not more than one defect, with characteristic dimensions on the order of 5 nm, over a sample area as large as 100 cm2. In this work, we identify the key pathways and the corresponding free-energy barriers for eliminating defects, and we demonstrate that an extraordinarily large thermodynamic driving force is not necessarily sufficient for their removal. By adopting a concerted computational and experimental approach, we explain the molecular origins of these barriers, how they depend on material characteristics, and we propose strategies designed to over-come them. The validity of our conclusions for industrially-relevant patterning processes is established by relying on instruments and assembly lines that are only available at state-of-the-art fabrication facilities and, through this confluence of fundamental and applied research, we are able to discern the evolution of morphology at the smallest relevant length scales - a handful of nanometers -, and present a view of defect annihilation in directed self-assembly at an unprecedented level of detail.

  16. Molecular pathways for defect annihilation in directed self-assembly.

    PubMed

    Hur, Su-Mi; Thapar, Vikram; Ramírez-Hernández, Abelardo; Khaira, Gurdaman; Segal-Peretz, Tamar; Rincon-Delgadillo, Paulina A; Li, Weihua; Müller, Marcus; Nealey, Paul F; de Pablo, Juan J

    2015-11-17

    Over the last few years, the directed self-assembly of block copolymers by surface patterns has transitioned from academic curiosity to viable contender for commercial fabrication of next-generation nanocircuits by lithography. Recently, it has become apparent that kinetics, and not only thermodynamics, plays a key role for the ability of a polymeric material to self-assemble into a perfect, defect-free ordered state. Perfection, in this context, implies not more than one defect, with characteristic dimensions on the order of 5 nm, over a sample area as large as 100 cm(2). In this work, we identify the key pathways and the corresponding free energy barriers for eliminating defects, and we demonstrate that an extraordinarily large thermodynamic driving force is not necessarily sufficient for their removal. By adopting a concerted computational and experimental approach, we explain the molecular origins of these barriers and how they depend on material characteristics, and we propose strategies designed to overcome them. The validity of our conclusions for industrially relevant patterning processes is established by relying on instruments and assembly lines that are only available at state-of-the-art fabrication facilities, and, through this confluence of fundamental and applied research, we are able to discern the evolution of morphology at the smallest relevant length scales-a handful of nanometers-and present a view of defect annihilation in directed self-assembly at an unprecedented level of detail.

  17. Determination of the aflatoxin M1 (AFM1) from milk by direct analysis in real time - mass spectrometry (DART-MS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Certain fungi that grow on crops can produce aflatoxins, which are highly carcinogenic. One of these, aflatoxin B1 can be metabolized by mammals to aflatoxin M1, a form that retains potent carcinogenicity and which can be excreted into milk. Direct analysis in real time (DART) ionization coupled to ...

  18. Contributions of Rod and Cone Pathways to Retinal Direction Selectivity Through Development

    PubMed Central

    Rosa, Juliana M.; Morrie, Ryan D.; Baertsch, Hans C.

    2016-01-01

    Direction selectivity is a robust computation across a broad stimulus space that is mediated by activity of both rod and cone photoreceptors through the ON and OFF pathways. However, rods, S-cones, and M-cones activate the ON and OFF circuits via distinct pathways and the relative contribution of each to direction selectivity is unknown. Using a variety of stimulation paradigms, pharmacological agents, and knockout mice that lack rod transduction, we found that inputs from the ON pathway were critical for strong direction-selective (DS) tuning in the OFF pathway. For UV light stimulation, the ON pathway inputs to the OFF pathway originated with rod signaling, whereas for visible stimulation, the ON pathway inputs to the OFF pathway originated with both rod and M-cone signaling. Whole-cell voltage-clamp recordings revealed that blocking the ON pathway reduced directional tuning in the OFF pathway via a reduction in null-side inhibition, which is provided by OFF starburst amacrine cells (SACs). Consistent with this, our recordings from OFF SACs confirmed that signals originating in the ON pathway contribute to their excitation. Finally, we observed that, for UV stimulation, ON contributions to OFF DS tuning matured earlier than direct signaling via the OFF pathway. These data indicate that the retina uses multiple strategies for computing DS responses across different colors and stages of development. SIGNIFICANCE STATEMENT The retina uses parallel pathways to encode different features of the visual scene. In some cases, these distinct pathways converge on circuits that mediate a distinct computation. For example, rod and cone pathways enable direction-selective (DS) ganglion cells to encode motion over a wide range of light intensities. Here, we show that although direction selectivity is robust across light intensities, motion discrimination for OFF signals is dependent upon ON signaling. At eye opening, ON directional tuning is mature, whereas OFF DS tuning is

  19. Control of Basal Ganglia Output by Direct and Indirect Pathway Projection Neurons

    PubMed Central

    Freeze, Benjamin S.; Kravitz, Alexxai V.; Hammack, Nora; Berke, Joshua D.

    2013-01-01

    The direct and indirect efferent pathways from striatum ultimately reconverge to influence basal ganglia output nuclei, which in turn regulate behavior via thalamocortical and brainstem motor circuits. However, the distinct contributions of these two efferent pathways in shaping basal ganglia output are not well understood. We investigated these processes using selective optogenetic control of the direct and indirect pathways, in combination with single-unit recording in the basal ganglia output nucleus substantia nigra pars reticulata (SNr) in mice. Optogenetic activation of striatal direct and indirect pathway projection neurons produced diverse cellular responses in SNr neurons, with stimulation of each pathway eliciting both excitations and inhibitions. Despite this response heterogeneity, the effectiveness of direct pathway stimulation in producing movement initiation correlated selectively with the subpopulation of inhibited SNr neurons. In contrast, effective indirect pathway-mediated motor suppression was most strongly influenced by excited SNr neurons. Our results support the theory that key basal ganglia output neurons serve as an inhibitory gate over motor output that can be opened or closed by striatal direct and indirect pathways, respectively. PMID:24259575

  20. Direct evidence for a xylose metabolic pathway in Saccharomyces cerevisiae

    SciTech Connect

    Batt, C.A.; Carvallo, S.; Easson, D.D.; Akedo, M.; Sinskey, A.J.

    1986-04-01

    Xylose transport, xylose reductase, and xylitol dehydrogenase activities are demonstrated in Saccharomyces cerevisiae. The enzymes in the xylose catabolic pathway necessary for the conversion of xylose xylulose are present, although S. cerevisiae cannot grow on xylose as a sole carbon source. Xylose transport is less efficient than glucose transport, and its rate is dependent upon aeration. Xylose reductase appears to be a xylose inducible enzyme and xylitol dehydrogenase activity is constitutive, although both are repressed by glucose. Both xylose reductase and xylitol dehydrogenase activities are five- to tenfold lower in S. cerevisie as compared to Candida utilis. In vivo conversion of /sup 14/C-xylose in S. cerevisiage is demonstrated and xylitol is detected, although no significant levels of any other /sup 14/C-labeled metabolites (e.g., ethanol) are observed. 22 references.

  1. Bidirectional Plasticity in Striatonigral Synapses: A Switch to Balance Direct and Indirect Basal Ganglia Pathways

    ERIC Educational Resources Information Center

    Aceves, Jose J.; Rueda-Orozco, Pavel E.; Hernandez-Martinez, Ricardo; Galarraga, Elvira; Bargas, Jose

    2011-01-01

    There is no hypothesis to explain how direct and indirect basal ganglia (BG) pathways interact to reach a balance during the learning of motor procedures. Both pathways converge in the substantia nigra pars reticulata (SNr) carrying the result of striatal processing. Unfortunately, the mechanisms that regulate synaptic plasticity in striatonigral…

  2. Directed evolution of a cellobiose utilization pathway in Saccharomyces cerevisiae by simultaneously engineering multiple proteins

    PubMed Central

    2013-01-01

    Background The optimization of metabolic pathways is critical for efficient and economical production of biofuels and specialty chemicals. One such significant pathway is the cellobiose utilization pathway, identified as a promising route in biomass utilization. Here we describe the optimization of cellobiose consumption and ethanol productivity by simultaneously engineering both proteins of the pathway, the β-glucosidase (gh1-1) and the cellodextrin transporter (cdt-1), in an example of pathway engineering through directed evolution. Results The improved pathway was assessed based on the strain specific growth rate on cellobiose, with the final mutant exhibiting a 47% increase over the wild-type pathway. Metabolite analysis of the engineered pathway identified a 49% increase in cellobiose consumption (1.78 to 2.65 g cellobiose/(L · h)) and a 64% increase in ethanol productivity (0.611 to 1.00 g ethanol/(L · h)). Conclusions By simultaneously engineering multiple proteins in the pathway, cellobiose utilization in S. cerevisiae was improved. This optimization can be generally applied to other metabolic pathways, provided a selection/screening method is available for the desired phenotype. The improved in vivo cellobiose utilization demonstrated here could help to decrease the in vitro enzyme load in biomass pretreatment, ultimately contributing to a reduction in the high cost of biofuel production. PMID:23802545

  3. Direct sampling of multiple single-molecular rupture dominant pathways involving a multistep transition.

    PubMed

    Jiang, Huijun; Ding, Huai; Hou, Zhonghuai

    2014-12-14

    We report a novel single-molecular rupture mechanism revealed by direct sampling of the dominant pathway using a self-optimized path sampling method. Multiple dominant pathways involving multistep transitions are identified. The rupture may take place via a direct unfolding from the native state to the unfolding state, or through a two-step pathway bypassing a distinct intermediate metastable state (IMS). This scenario facilitates us to propose a three-state kinetic model, which can produce a nonlinear dependence of the rupture time on pulling forces similar to the ones reported in the literature. In particular, molecule conformations in the IMS maintain an elongation of the tail at one terminal, by which external pulling will enhance the relative stability of IMS. Consequently, even though the overall transition rate of the multistep pathway is relatively small, the molecule still has to be ruptured via the multistep pathway rather than the direct pathway. Thus, our work demonstrates an IMS trapping effect induced rupture mechanism involving an abnormal switching from a fast dominant pathway to a slow one.

  4. PREFACE: Non-contact AFM Non-contact AFM

    NASA Astrophysics Data System (ADS)

    Giessibl, Franz J.; Morita, Seizo

    2012-02-01

    This special issue is focussed on high resolution non-contact atomic force microscopy (AFM). Non-contact atomic force microscopy was established approximately 15 years ago as a tool to image conducting and insulating surfaces with atomic resolution. Since 1998, an annual international conference has taken place, and although the proceedings of these conferences are a useful source of information, several key developments warrant devoting a special issue to this subject. In the theoretic field, the possibility of supplementing established techniques such as scanning tunneling microscopy (STM) and Kelvin probe microscopy with atomically resolved force micrsoscopy poses many challenges in the calculation of contrast and contrast reversal. The surface science of insulators, self-assembled monolayers and adsorbates on insulators is a fruitful field for the application of non-contact AFM: several articles in this issue are devoted to these subjects. Atomic imaging and manipulation have been pioneered using STM, but because AFM allows the measurement of forces, AFM has had a profound impact in this field as well. Three-dimensional force spectroscopy has allowed many important insights into surface science. In this issue a combined 3D tunneling and force microscopy is introduced. Non-contact AFM typically uses frequency modulation to measure force gradients and was initially used mainly in a vacuum. As can be seen in this issue, frequency modulation is now also used in ambient conditions, allowing better spatial and force resolution. We thank all of the contributors for their time and efforts in making this special issue possible. We are also very grateful to the staff of IOP Publishing for handling the administrative aspects and for steering the refereeing process. Non-contact AFM contents Relation between the chemical force and the tunnelling current in atomic point contacts: a simple model Pavel Jelínek, Martin Ondrácek and Fernando Flores Theoretical simulation of

  5. AFM Structural Characterization of Drinking Water Biofilm ...

    EPA Pesticide Factsheets

    Due to the complexity of mixed culture drinking water biofilm, direct visual observation under in situ conditions has been challenging. In this study, atomic force microscopy (AFM) revealed the three dimensional morphology and arrangement of drinking water relevant biofilm in air and aqueous solution. Operating parameters were optimized to improve imaging of structural details for a mature biofilm in liquid. By using a soft cantilever (0.03 N/m) and slow scan rate (0.5 Hz), biofilm and individual bacterial cell’s structural topography were resolved and continuously imaged in liquid without loss of spatial resolution or sample damage. The developed methodology will allow future in situ investigations to temporally monitor mixed culture drinking water biofilm structural changes during disinfection treatments. Due to the complexity of mixed culture drinking water biofilm, direct visual observation under in situ conditions has been challenging. In this study, atomic force microscopy (AFM) revealed the three dimensional morphology and arrangement of drinking water relevant biofilm in air and aqueous solution. Operating parameters were optimized to improve imaging of structural details for a mature biofilm in liquid. By using a soft cantilever (0.03 N/m) and slow scan rate (0.5 Hz), biofilm and individual bacterial cell’s structural topography were resolved and continuously imaged in liquid without loss of spatial resolution or sample damage. The developed methodo

  6. Direct and Indirect Pathways between Parental Constructive Behavior and Adolescent Affiliation with Achievement-Oriented Peers

    ERIC Educational Resources Information Center

    Chen, Zeng-yin; Dornbusch, Sanford M.; Liu, Ruth X.

    2007-01-01

    We investigated the direct and indirect pathways through which parental constructive behavior may influence the adolescent's affiliation with achievement-oriented peers. Using a longitudinal survey data set from nine California and Wisconsin high schools (from 9th through 12th grades, with an approximate age range from 14 through 18) structural…

  7. Identification of a Specific Maleate Hydratase in the Direct Hydrolysis Route of the Gentisate Pathway

    PubMed Central

    Liu, Kun; Xu, Ying

    2015-01-01

    In contrast to the well-characterized and more common maleylpyruvate isomerization route of the gentisate pathway, the direct hydrolysis route occurs rarely and remains unsolved. In Pseudomonas alcaligenes NCIMB 9867, two gene clusters, xln and hbz, were previously proposed to be involved in gentisate catabolism, and HbzF was characterized as a maleylpyruvate hydrolase converting maleylpyruvate to maleate and pyruvate. However, the complete degradation pathway of gentisate through direct hydrolysis has not been characterized. In this study, we obtained from the NCIMB culture collection a Pseudomonas alcaligenes spontaneous mutant strain that lacked the xln cluster and designated the mutant strain SponMu. The hbz cluster in strain SponMu was resequenced, revealing the correct location of the stop codon for hbzI and identifying a new gene, hbzG. HbzIJ was demonstrated to be a maleate hydratase consisting of large and small subunits, stoichiometrically converting maleate to enantiomerically pure d-malate. HbzG is a glutathione-dependent maleylpyruvate isomerase, indicating the possible presence of two alternative pathways of maleylpyruvate catabolism. However, the hbzF-disrupted mutant could still grow on gentisate, while disruption of hbzG prevented this ability, indicating that the direct hydrolysis route was not a complete pathway in strain SponMu. Subsequently, a d-malate dehydrogenase gene was introduced into the hbzG-disrupted mutant, and the engineered strain was able to grow on gentisate via the direct hydrolysis route. This fills a gap in our understanding of the direct hydrolysis route of the gentisate pathway and provides an explanation for the high yield of d-malate from maleate by this d-malate dehydrogenase-deficient natural mutant. PMID:26070679

  8. Identification of a Specific Maleate Hydratase in the Direct Hydrolysis Route of the Gentisate Pathway.

    PubMed

    Liu, Kun; Xu, Ying; Zhou, Ning-Yi

    2015-09-01

    In contrast to the well-characterized and more common maleylpyruvate isomerization route of the gentisate pathway, the direct hydrolysis route occurs rarely and remains unsolved. In Pseudomonas alcaligenes NCIMB 9867, two gene clusters, xln and hbz, were previously proposed to be involved in gentisate catabolism, and HbzF was characterized as a maleylpyruvate hydrolase converting maleylpyruvate to maleate and pyruvate. However, the complete degradation pathway of gentisate through direct hydrolysis has not been characterized. In this study, we obtained from the NCIMB culture collection a Pseudomonas alcaligenes spontaneous mutant strain that lacked the xln cluster and designated the mutant strain SponMu. The hbz cluster in strain SponMu was resequenced, revealing the correct location of the stop codon for hbzI and identifying a new gene, hbzG. HbzIJ was demonstrated to be a maleate hydratase consisting of large and small subunits, stoichiometrically converting maleate to enantiomerically pure d-malate. HbzG is a glutathione-dependent maleylpyruvate isomerase, indicating the possible presence of two alternative pathways of maleylpyruvate catabolism. However, the hbzF-disrupted mutant could still grow on gentisate, while disruption of hbzG prevented this ability, indicating that the direct hydrolysis route was not a complete pathway in strain SponMu. Subsequently, a d-malate dehydrogenase gene was introduced into the hbzG-disrupted mutant, and the engineered strain was able to grow on gentisate via the direct hydrolysis route. This fills a gap in our understanding of the direct hydrolysis route of the gentisate pathway and provides an explanation for the high yield of d-malate from maleate by this d-malate dehydrogenase-deficient natural mutant.

  9. Critical Roles of the Direct GABAergic Pallido-cortical Pathway in Controlling Absence Seizures

    PubMed Central

    Li, Min; Ma, Tao; Wu, Shengdun; Ma, Jingling; Cui, Yan; Xia, Yang; Xu, Peng; Yao, Dezhong

    2015-01-01

    The basal ganglia (BG), serving as an intermediate bridge between the cerebral cortex and thalamus, are believed to play crucial roles in controlling absence seizure activities generated by the pathological corticothalamic system. Inspired by recent experiments, here we systematically investigate the contribution of a novel identified GABAergic pallido-cortical pathway, projecting from the globus pallidus externa (GPe) in the BG to the cerebral cortex, to the control of absence seizures. By computational modelling, we find that both increasing the activation of GPe neurons and enhancing the coupling strength of the inhibitory pallido-cortical pathway can suppress the bilaterally synchronous 2–4 Hz spike and wave discharges (SWDs) during absence seizures. Appropriate tuning of several GPe-related pathways may also trigger the SWD suppression, through modulating the activation level of GPe neurons. Furthermore, we show that the previously discovered bidirectional control of absence seizures due to the competition between other two BG output pathways also exists in our established model. Importantly, such bidirectional control is shaped by the coupling strength of this direct GABAergic pallido-cortical pathway. Our work suggests that the novel identified pallido-cortical pathway has a functional role in controlling absence seizures and the presented results might provide testable hypotheses for future experimental studies. PMID:26496656

  10. Developmental pathways to conduct disorder: implications for future directions in research, assessment, and treatment.

    PubMed

    Frick, Paul J

    2012-01-01

    Research has indicated that there are several common pathways through which children and adolescents develop conduct disorder, each with different risk factors and each with different underlying developmental mechanisms leading to the child's aggressive and antisocial behavior. The current article briefly summarizes research on these pathways, including one that onsets in adolescence and seems to be an exaggeration of normal adolescent rebellion against authority. The other two pathways typically involve conduct problems that onset early in childhood but differ on whether the child shows significant levels of callous-unemotional traits or whether the child shows significant problems in emotional and behavioral regulation. Important directions for future research on these pathways are highlighted, as well as implications of these pathways for assessing and diagnosing children and adolescents with conduct disorder. In particular, diagnostic criteria should recognize the importance of callous-unemotional traits for distinguishing a distinct subgroup of youths with the disorder. Finally, implications for the prevention and treatment of conduct disorder are discussed, especially the need for interventions that are comprehensive and individualized to the characteristics of children and adolescents in the various developmental pathways.

  11. Prenatal alcohol and other early childhood adverse exposures: Direct and indirect pathways to adolescent drinking

    PubMed Central

    Cornelius, Marie D.; De Genna, Natacha M.; Goldschmidt, Lidush; Larkby, Cynthia; Day, Nancy L.

    2016-01-01

    We examined direct and indirect pathways between adverse environmental exposures during gestation and childhood and drinking in mid-adolescence. Mothers and their offspring (n = 917 mother/child dyads) were followed prospectively from second trimester to a 16-year follow-up assessment. Interim assessments occurred at delivery, 6, 10, and 14 years. Adverse environmental factors included gestational exposures to alcohol, tobacco, and marijuana, exposures to childhood maltreatment and violence, maternal psychological symptoms, parenting practices, economic and home environments, and demographic characteristics of the mother and child. Indirect effects of early child behavioral characteristics including externalizing, internalizing activity, attention, and impulsivity were also examined. Polytomous logistic regression analyses were used to evaluate direct effects of adverse environmental exposures with level of adolescent drinking. Structural equation modeling (SEM) was applied to simultaneously estimate the relation between early adversity variables, childhood characteristics, and drinking level at age 16 while controlling for significant covariates. Level of drinking among the adolescent offspring was directly predicted by prenatal exposure to alcohol, less parental strictness, and exposures to maltreatment and violence during childhood. Whites and offspring with older mothers were more likely to drink at higher levels. There was a significant indirect effect between childhood exposure to violence and adolescent drinking via childhood externalizing behavior problems. All other hypothesized indirect pathways were not significant. Thus most of the early adversity measures directly predicted adolescent drinking and did not operate via childhood behavioral dysregulation characteristics. These results highlight the importance of adverse environmental exposures on pathways to adolescent drinking. PMID:26994529

  12. GPIM AF-M315E Propulsion System

    NASA Technical Reports Server (NTRS)

    Spores, Ronald A.; Masse, Robert; Kimbrel, Scott; McLean, Chris

    2014-01-01

    The NASA Space Technology mission Directorate's (STMD) Green Propellant Infusion Mission (GPIM) Technology Demonstration Mission (TDM) will demonstrate an operational AF-M315E green propellant propulsion system. Aerojet-Rocketdyne is responsible for the development of the propulsion system payload. This paper statuses the propulsion system module development, including thruster design and system design; Initial test results for the 1N engineering model thruster are presented. The culmination of this program will be high-performance, green AF-M315E propulsion system technology at TRL 7+, with components demonstrated to TRL 9, ready for direct infusion to a wide range of applications for the space user community.

  13. AFM Structural Characterization of Drinking Water Biofilm under Physiological Conditions

    EPA Science Inventory

    Due to the complexity of mixed culture drinking water biofilm, direct visual observation under in situ conditions has been challenging. In this study, atomic force microscopy (AFM) revealed the three dimensional morphology and arrangement of drinking water relevant biofilm in air...

  14. Direct biosynthesis of adipic acid from a synthetic pathway in recombinant Escherichia coli.

    PubMed

    Yu, Jia-Le; Xia, Xiao-Xia; Zhong, Jian-Jiang; Qian, Zhi-Gang

    2014-12-01

    The C6 dicarboxylic acid, adipic acid, is an important platform chemical in industry. Biobased production of adipic acid is a promising alternative to the current petrochemical route. Here, we report biosynthesis of adipic acid using an artificial pathway inspired by the reversal of beta-oxidation of dicarboxylic acids. The biosynthetic pathway comprises condensation of acetyl-CoA and succinyl-CoA to form the C6 backbone and subsequent reduction, dehydration, hydrogenation, and release of adipic acid from its thioester. The pathway was first tested in vitro with reconstituted pathway enzymes and then functionally introduced into Escherichia coli for the biosynthesis and excretion of adipic acid into the culture medium. The production titer was increased by approximately 20-fold through the combination of recruiting enzymes that were more suitable to catalyze the synthetic reactions and increasing availability of the condensation substrates. This work demonstrates direct biosynthesis of adipic acid via non-natural synthetic pathway, which may enable its renewable production.

  15. AFM-Based Mechanical Nanomanipulation

    NASA Astrophysics Data System (ADS)

    Landolsi, Fakhreddine

    2011-12-01

    Advances in several research areas increase the need for more sophisticated fabrication techniques and better performing materials. Tackling this problem from a bottom-up perspective is currently an active field of research. The bottom-up fabrication procedure offers sub-nanometer accurate manipulation. At this time, candidates to achieve nanomanipulation include chemical (self-assembly), biotechnology methods (DNA-based), or using controllable physical forces (e.g. electrokinetic forces, mechanical forces). In this thesis, new methods and techniques for mechanical nanomanipulation using probe force interaction are developed. The considered probes are commonly used in Atomic Force Microscopes (AFMs) for high resolution imaging. AFM-based mechanical nanomanipulation will enable arranging nanoscale entities such as nanotubes and molecules in a precise and controlled manner to assemble and produce novel devices and systems at the nanoscale. The novelty of this research stems from the development of new modeling of the physics and mechanics of the tip interaction with nanoscale entities, coupled with the development of new smart cantilevers with multiple degrees of freedom. The gained knowledge from the conducted simulations and analysis is expected to enable true precision and repeatability of nanomanipulation tasks which is not feasible with existing methods and technologies.

  16. Direct vs. indirect pathway of hepatic glycogen synthesis as a function of glucose infusion rate

    SciTech Connect

    Bagby, G.J.; Lang, C.H.; Johnson, J.L.; Blakesly, H.L.; Spitzer, J.J.

    1986-03-05

    This study was initiated to determine the influence of the rate of exogenous glucose administration on liver glycogen synthesis by the direct (glucose uptake and incorporation into glycogen) vs the indirect pathway (glucose degradation to 3-carbon intermediates, e.g., lactate, prior to incorporation into glycogen). Catheterized rats were fasted 2 days prior to receiving a 3 hr infusion of glucose at rates of 0 to 230 ..mu..mol/min/kg containing tracer (6-/sup 3/H)- and (U-/sup 14/C)-glucose. Plasma glucose (r = 0.80), insulin (r = 0.90) and lactate (r = 0.84) were correlated with glucose infusion rate. The rate of liver glycogen deposition (0.46 +/- 0.03 ..mu..mol/min/g) did not differ between a glucose infusion rate of 20 and 230 ..mu..mol/min/kg. At the lowest and highest glucose infusion rates hepatic glycogenesis accounted for 87 +/- 6 and 9 +/- 1% of the total glucose load, respectively. The percent contribution of the direct pathways to glycogen deposition ((/sup 3/H) specific activity in hepatic glycogen/(/sup 3/H) specific activity in plasma glucose) increased from 16 +/- 3 to 83 +/- 5% from lowest to highest glucose infusion rates (prevailing plasma glucose concentrations: 9 +/- 1 and 21 +/- 2 mM, respectively). The results indicate that the relative contribution of the direct and indirect pathways of glucogen synthesis are dependent upon the glucose load or plasma glucose concentration.

  17. BOREAS AFM-06 Mean Wind Profile Data

    NASA Technical Reports Server (NTRS)

    Wilczak, James; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-6 team from the National Oceanic and Atmospheric Administration/Environment Technology Laboratory (NOAA/ETL) operated a 915-MHz wind/Radio Acoustic Sounding System (RASS) profiler system in the Southern Study Area (SSA) near the Old Jack Pine (OJP) tower from 21 May 1994 to 20 Sep 1994. The data set provides wind profiles at 38 heights, containing the variables of wind speed; wind direction; and the u-, v-, and w-components of the total wind. The data are stored in tabular ASCII files. The mean wind profile data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  18. Differential and directional estrogenic signaling pathways induced by enterolignans and their precursors

    PubMed Central

    Zhu, Yun; Kawaguchi, Kayoko; Kiyama, Ryoiti

    2017-01-01

    Mammalian lignans or enterolignans are metabolites of plant lignans, an important category of phytochemicals. Although they are known to be associated with estrogenic activity, cell signaling pathways leading to specific cell functions, and especially the differences among lignans, have not been explored. We examined the estrogenic activity of enterolignans and their precursor plant lignans and cell signaling pathways for some cell functions, cell cycle and chemokine secretion. We used DNA microarray-based gene expression profiling in human breast cancer MCF-7 cells to examine the similarities, as well as the differences, among enterolignans, enterolactone and enterodiol, and their precursors, matairesinol, pinoresinol and sesamin. The profiles showed moderate to high levels of correlation (R values: 0.44 to 0.81) with that of estrogen (17β-estradiol or E2). Significant correlations were observed among lignans (R values: 0.77 to 0.97), and the correlations were higher for cell functions related to enzymes, signaling, proliferation and transport. All the enterolignans/precursors examined showed activation of the Erk1/2 and PI3K/Akt pathways, indicating the involvement of rapid signaling through the non-genomic estrogen signaling pathway. However, when their effects on specific cell functions, cell cycle progression and chemokine (MCP-1) secretion were examined, positive effects were observed only for enterolactone, suggesting that signals are given in certain directions at a position closer to cell functions. We hypothesized that, while estrogen signaling is initiated by the enterolignans/precursors examined, their signals are differentially and directionally modulated later in the pathways, resulting in the differences at the cell function level. PMID:28152041

  19. Exploration of molecular pathways mediating electric field-directed Schwann cell migration by RNA-Seq

    PubMed Central

    Yao, Li; Li, Yongchao; Knapp, Jennifer; Smith, Peter

    2015-01-01

    In peripheral nervous systems, Schwann cells wrap around axons of motor and sensory neurons to form the myelin sheath. Following spinal cord injury, Schwann cells regenerate and migrate to the lesion and are involved in the spinal cord regeneration process. Transplantation of Schwann cells into injured neural tissue results in enhanced spinal axonal regeneration. Effective directional migration of Schwann cells is critical in the neural regeneration process. In this study, we report that Schwann cells migrate anodally in an applied electric field (EF). The directedness and displacement of anodal migration increased significantly when the strength of the EF increased from 50 mV/mm to 200 mV/mm. The EF did not significantly affect the cell migration speed. To explore the genes and signaling pathways that regulate cell migration in EFs, we performed a comparative analysis of differential gene expression between cells stimulated with an EF (100 mV/mm) and those without using next-generation RNA sequencing, verified by RT-qPCR. Based on the cut-off criteria (FC > 1.2, q < 0.05), we identified 1,045 up-regulated and 1,636 down-regulated genes in control cells versus EF-stimulated cells. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that compared to the control group, 21 pathways are down-regulated, while 10 pathways are up-regulated. Differentially expressed genes participate in multiple cellular signaling pathways involved in the regulation of cell migration, including pathways of regulation of actin cytoskeleton, focal adhesion, and PI3K-Akt. PMID:25557037

  20. Formation of sensor array on the AFM chip surface by magnetron sputtering

    NASA Astrophysics Data System (ADS)

    Shumov, I. D.; Kanashenko, S. L.; Ziborov, V. S.; Ivanov, Yu D.; Archakov, A. I.; Pleshakova, T. O.

    2017-01-01

    Development of atomic force microscopy (AFM)-based nanotechnological approaches to highly sensitive detection of proteins is a perspective direction in biomedical research. These approaches use AFM chips to concentrate the target proteins from the test solution volume (buffer solution, diluted biological fluid) onto the chip surface for their subsequent registration on the chip surface by AFM. Atomic force microscope is a molecular detector that enables protein detection at ultra-low (subfemtomolar) concentrations in single-molecule counting mode. Due to extremely high sensitivity of AFM, its application for multiplexed protein detection is of great interest for use in proteomics and diagnostic applications. In this study, AFM chips containing an array of sensor areas have been fabricated. Magnetron sputtering of chromium and tungsten nanolayers has been used to form optically visible metallic marks on the AFM chip surface to provide necessary precision of AFM probe positioning against each sensor area for scanning. It has been demonstrated that the marks formed by magnetron sputtering of Cr and W are stable on the surface of the AFM chips during the following activation and intensive washing of this surface. The results obtained in our present study allow application of the developed chips for multiplexed protein analysis by AFM.

  1. RNA-Directed DNA Methylation: The Evolution of a Complex Epigenetic Pathway in Flowering Plants.

    PubMed

    Matzke, Marjori A; Kanno, Tatsuo; Matzke, Antonius J M

    2015-01-01

    RNA-directed DNA methylation (RdDM) is an epigenetic process in plants that involves both short and long noncoding RNAs. The generation of these RNAs and the induction of RdDM rely on complex transcriptional machineries comprising two plant-specific, RNA polymerase II (Pol II)-related RNA polymerases known as Pol IV and Pol V, as well as a host of auxiliary factors that include both novel and refashioned proteins. We present current views on the mechanism of RdDM with a focus on evolutionary innovations that occurred during the transition from a Pol II transcriptional pathway, which produces mRNA precursors and numerous noncoding RNAs, to the Pol IV and Pol V pathways, which are specialized for RdDM and gene silencing. We describe recently recognized deviations from the canonical RdDM pathway, discuss unresolved issues, and speculate on the biological significance of RdDM for flowering plants, which have a highly developed Pol V pathway.

  2. Exploring the transfer of recent plant photosynthates to soil microbes: mycorrhizal pathway vs direct root exudation.

    PubMed

    Kaiser, Christina; Kilburn, Matt R; Clode, Peta L; Fuchslueger, Lucia; Koranda, Marianne; Cliff, John B; Solaiman, Zakaria M; Murphy, Daniel V

    2015-03-01

    Plants rapidly release photoassimilated carbon (C) to the soil via direct root exudation and associated mycorrhizal fungi, with both pathways promoting plant nutrient availability. This study aimed to explore these pathways from the root's vascular bundle to soil microbial communities. Using nanoscale secondary ion mass spectrometry (NanoSIMS) imaging and (13) C-phospho- and neutral lipid fatty acids, we traced in-situ flows of recently photoassimilated C of (13) CO2 -exposed wheat (Triticum aestivum) through arbuscular mycorrhiza (AM) into root- and hyphae-associated soil microbial communities. Intraradical hyphae of AM fungi were significantly (13) C-enriched compared to other root-cortex areas after 8 h of labelling. Immature fine root areas close to the root tip, where AM features were absent, showed signs of passive C loss and co-location of photoassimilates with nitrogen taken up from the soil solution. A significant and exclusively fresh proportion of (13) C-photosynthates was delivered through the AM pathway and was utilised by different microbial groups compared to C directly released by roots. Our results indicate that a major release of recent photosynthates into soil leave plant roots via AM intraradical hyphae already upstream of passive root exudations. AM fungi may act as a rapid hub for translocating fresh plant C to soil microbes.

  3. Direct Hospital Cost of Outcome Pathways in Implant-Based Reconstruction with Acellular Dermal Matrices

    PubMed Central

    Qureshi, Ali A.; Broderick, Kristen; Funk, Susan; Reaven, Nancy; Tenenbaum, Marissa M.

    2016-01-01

    Background: Current cost data on tissue expansion followed by exchange for permanent implant (TE/I) reconstruction lack a necessary assessment of the experience of a heterogenous breast cancer patient population and their multiple outcome pathways. We extend our previous analysis to that of direct hospital cost as bundling of payments is likely to follow the changing centralization of cancer care at the hospital level. Methods: We performed a retrospective analysis (2003–2009) of TE/I reconstructions with or without an acellular dermal matrix (ADM), namely Alloderm RTM. Postreconstructive events were analyzed and organized into outcome pathways as previously described. Aggregated and normalized inpatient and outpatient hospital direct costs and physician reimbursement were generated for each outcome pathway with or without ADM. Results: Three hundred sixty-seven patients were analyzed. The average 2-year hospital direct cost per TE/I breast reconstruction patient was $11,862 in the +ADM and $12,319 in the −ADM groups (P > 0.05). Initial reconstructions were costlier in the +ADM ($6,868) than in the −ADM ($5,615) group, but the average cost of subsequent postreconstructive events within 2 years was significantly lower in +ADM ($5,176) than −ADM ($6,704) patients (P < 0.05). When a complication occurred, but reconstruction was still completed within 2 years, greater costs were incurred in the −ADM than in the +ADM group for most scenarios, leading to a net equalization of cost between study groups. Conclusion: Although direct hospital cost is an important factor for resource and fund allocation, it should not remain the sole factor when deciding to use ADM in TE/I reconstruction. PMID:27622099

  4. A Direct Cortico-Nigral Pathway as Revealed by Constrained Spherical Deconvolution Tractography in Humans

    PubMed Central

    Cacciola, Alberto; Milardi, Demetrio; Anastasi, Giuseppe P.; Basile, Gianpaolo A.; Ciolli, Pietro; Irrera, Mariangela; Cutroneo, Giuseppina; Bruschetta, Daniele; Rizzo, Giuseppina; Mondello, Stefania; Bramanti, Placido; Quartarone, Angelo

    2016-01-01

    Substantia nigra is an important neuronal structure, located in the ventral midbrain, that exerts a regulatory function within the basal ganglia circuitry through the nigro-striatal pathway. Although its subcortical connections are relatively well-known in human brain, little is known about its cortical connections. The existence of a direct cortico-nigral pathway has been demonstrated in rodents and primates but only hypothesized in humans. In this study, we aimed at evaluating cortical connections of substantia nigra in vivo in human brain by using probabilistic constrained spherical deconvolution (CSD) tractography on magnetic resonance diffusion weighted imaging data. We found that substantia nigra is connected with cerebral cortex as a whole, with the most representative connections involving prefrontal cortex, precentral and postcentral gyri and superior parietal lobule. These results may be relevant for the comprehension of the pathophysiology of several neurological disorders involving substantia nigra, such as parkinson's disease, schizophrenia, and pathological addictions. PMID:27507940

  5. A Direct Cortico-Nigral Pathway as Revealed by Constrained Spherical Deconvolution Tractography in Humans.

    PubMed

    Cacciola, Alberto; Milardi, Demetrio; Anastasi, Giuseppe P; Basile, Gianpaolo A; Ciolli, Pietro; Irrera, Mariangela; Cutroneo, Giuseppina; Bruschetta, Daniele; Rizzo, Giuseppina; Mondello, Stefania; Bramanti, Placido; Quartarone, Angelo

    2016-01-01

    Substantia nigra is an important neuronal structure, located in the ventral midbrain, that exerts a regulatory function within the basal ganglia circuitry through the nigro-striatal pathway. Although its subcortical connections are relatively well-known in human brain, little is known about its cortical connections. The existence of a direct cortico-nigral pathway has been demonstrated in rodents and primates but only hypothesized in humans. In this study, we aimed at evaluating cortical connections of substantia nigra in vivo in human brain by using probabilistic constrained spherical deconvolution (CSD) tractography on magnetic resonance diffusion weighted imaging data. We found that substantia nigra is connected with cerebral cortex as a whole, with the most representative connections involving prefrontal cortex, precentral and postcentral gyri and superior parietal lobule. These results may be relevant for the comprehension of the pathophysiology of several neurological disorders involving substantia nigra, such as parkinson's disease, schizophrenia, and pathological addictions.

  6. Keratocyte fragments and cells utilize competing pathways to move in opposite directions in an electric field.

    PubMed

    Sun, Yaohui; Do, Hao; Gao, Jing; Zhao, Ren; Zhao, Min; Mogilner, Alex

    2013-04-08

    Sensing of an electric field (EF) by cells-galvanotaxis-is important in wound healing [1], development [2], cell division, nerve growth, and angiogenesis [3]. Different cell types migrate in opposite directions in EFs [4], and the same cell can switch the directionality depending on conditions [5]. A tug-of-war mechanism between multiple signaling pathways [6] can direct Dictyostelium cells to either cathode or anode. Mechanics of motility is simplest in fish keratocytes, so we turned to keratocytes to investigate their migration in EFs. Keratocytes sense electric fields and migrate to the cathode [7, 8]. Keratocyte fragments [9, 10] are the simplest motile units. Cell fragments from leukocytes are able to respond to chemotactic signals [11], but whether cell fragments are galvanotactic was unknown. We found that keratocyte fragments are the smallest motile electric field-sensing unit: they migrate to the anode, in the opposite direction of whole cells. Myosin II was essential for the direction sensing of fragments but not for parental cells, while PI3 kinase was essential for the direction sensing of whole cells but not for fragments. Thus, two signal transduction pathways, one depending on PI3K, another on myosin, compete to orient motile cells in the electric field. Galvanotaxis is not due to EF force and does not depend on cell or fragment size. We propose a "compass" model according to which protrusive and contractile actomyosin networks self-polarize to the front and rear of the motile cell, respectively, and the electric signal orients both networks toward cathode with different strengths.

  7. New developments at PTB in 3D-AFM with tapping and torsion AFM mode and vector approach probing strategy

    NASA Astrophysics Data System (ADS)

    Dai, G.; Hässler-Grohne, W.; Hüser, D.; Wolff, H.; Fluegge, J.; Bosse, H.

    2011-06-01

    A new 3D-AFM for true 3D measurements of nano structures has been developed at Physikalisch Technische-Bundesanstalt, the national metrology institute of Germany. In its configuration, two piezo actuators are applied to drive the AFM cantilever near its vertical and torsional resonant frequencies. In such a way, the AFM tip can probe the surface with a vertical and/or a lateral oscillation, offering high 3D probing sensitivity. For enhancing measurement flexibility as well as reducing tip wear, a so called "vector approach probing" (VAP) method has been applied. The sample is measured point by point using this method. At each probing point, the tip is approached towards the surface in its normal direction until the desired tip-sample interaction is detected and then immediately withdrawn from the surface. Preliminary experimental results show promising performance of the developed system. The measurement of a line structure of 800 nm height employing a super sharp AFM tip is performed, showing a repeatability of its 3D profiles of better than 1 nm (p-v). A single crystal critical dimension reference material (SCCDRM) having features with almost vertical sidewall is measured using a flared AFM tip. Results show that the feature has averaged left and right sidewall angles of 88.64° and 88.67deg;, respectively. However, the feature width non-uniformity may reach 10 nm within the measurement range of 1 μm. The standard deviation of the averaged middle CD values of 7 repeated measurements reaches 0.35 nm. In addition, an investigation of long term measurement stability is performed on a PTB photomask. The results shows that the 3D-AFM has a drift rate of about 0.00033 nm per line, which confirms the high measurement stability and the very low tip wear.

  8. Nano-Wilhelmy investigation of dynamic wetting properties of AFM tips through tip-nanobubble interaction.

    PubMed

    Wang, Yuliang; Wang, Huimin; Bi, Shusheng; Guo, Bin

    2016-07-25

    The dynamic wetting properties of atomic force microscopy (AFM) tips are of much concern in many AFM-related measurement, fabrication, and manipulation applications. In this study, the wetting properties of silicon and silicon nitride AFM tips are investigated through dynamic contact angle measurement using a nano-Wilhelmy balance based method. This is done by capillary force measurement during extension and retraction motion of AFM tips relative to interfacial nanobubbles. The working principle of the proposed method and mathematic models for dynamic contact angle measurement are presented. Geometric models of AFM tips were constructed using scanning electronic microscopy (SEM) images taken from different view directions. The detailed process of tip-nanobubble interaction was investigated using force-distance curves of AFM on nanobubbles. Several parameters including nanobubble height, adhesion and capillary force between tip and nanobubbles are extracted. The variation of these parameters was studied over nanobubble surfaces. The dynamic contact angles of the AFM tips were calculated from the capillary force measurements. The proposed method provides direct measurement of dynamic contact angles for AFM tips and can also be taken as a general approach for nanoscale dynamic wetting property investigation.

  9. Nano-Wilhelmy investigation of dynamic wetting properties of AFM tips through tip-nanobubble interaction

    NASA Astrophysics Data System (ADS)

    Wang, Yuliang; Wang, Huimin; Bi, Shusheng; Guo, Bin

    2016-07-01

    The dynamic wetting properties of atomic force microscopy (AFM) tips are of much concern in many AFM-related measurement, fabrication, and manipulation applications. In this study, the wetting properties of silicon and silicon nitride AFM tips are investigated through dynamic contact angle measurement using a nano-Wilhelmy balance based method. This is done by capillary force measurement during extension and retraction motion of AFM tips relative to interfacial nanobubbles. The working principle of the proposed method and mathematic models for dynamic contact angle measurement are presented. Geometric models of AFM tips were constructed using scanning electronic microscopy (SEM) images taken from different view directions. The detailed process of tip-nanobubble interaction was investigated using force-distance curves of AFM on nanobubbles. Several parameters including nanobubble height, adhesion and capillary force between tip and nanobubbles are extracted. The variation of these parameters was studied over nanobubble surfaces. The dynamic contact angles of the AFM tips were calculated from the capillary force measurements. The proposed method provides direct measurement of dynamic contact angles for AFM tips and can also be taken as a general approach for nanoscale dynamic wetting property investigation.

  10. Nano-Wilhelmy investigation of dynamic wetting properties of AFM tips through tip-nanobubble interaction

    PubMed Central

    Wang, Yuliang; Wang, Huimin; Bi, Shusheng; Guo, Bin

    2016-01-01

    The dynamic wetting properties of atomic force microscopy (AFM) tips are of much concern in many AFM-related measurement, fabrication, and manipulation applications. In this study, the wetting properties of silicon and silicon nitride AFM tips are investigated through dynamic contact angle measurement using a nano-Wilhelmy balance based method. This is done by capillary force measurement during extension and retraction motion of AFM tips relative to interfacial nanobubbles. The working principle of the proposed method and mathematic models for dynamic contact angle measurement are presented. Geometric models of AFM tips were constructed using scanning electronic microscopy (SEM) images taken from different view directions. The detailed process of tip-nanobubble interaction was investigated using force-distance curves of AFM on nanobubbles. Several parameters including nanobubble height, adhesion and capillary force between tip and nanobubbles are extracted. The variation of these parameters was studied over nanobubble surfaces. The dynamic contact angles of the AFM tips were calculated from the capillary force measurements. The proposed method provides direct measurement of dynamic contact angles for AFM tips and can also be taken as a general approach for nanoscale dynamic wetting property investigation. PMID:27452115

  11. Decreasing Striatopallidal Pathway Function Enhances Motivation by Energizing the Initiation of Goal-Directed Action

    PubMed Central

    Carvalho Poyraz, Fernanda; Holzner, Eva; Bailey, Matthew R.; Meszaros, Jozsef; Kenney, Lindsay; Kheirbek, Mazen A.

    2016-01-01

    Altered dopamine D2 receptor (D2R) binding in the striatum has been associated with abnormal motivation in neuropsychiatric disorders, including schizophrenia. Here, we tested whether motivational deficits observed in mice with upregulated D2Rs (D2R-OEdev mice) are reversed by decreasing function of the striatopallidal “no-go” pathway. To this end, we expressed the Gαi-coupled designer receptor hM4D in adult striatopallidal neurons and activated the receptor with clozapine-N-oxide (CNO). Using a head-mounted miniature microscope we confirmed with calcium imaging in awake mice that hM4D activation by CNO inhibits striatopallidal function measured as disinhibited downstream activity in the globus pallidus. Mice were then tested in three operant tasks that address motivated behavior, the progressive ratio task, the progressive hold-down task, and outcome devaluation. Decreasing striatopallidal function in the dorsomedial striatum or nucleus accumbens core enhanced motivation in D2R-OEdev mice and control littermates. This effect was due to increased response initiation but came at the cost of goal-directed efficiency. Moreover, response vigor and the sensitivity to changes in reward value were not altered. Chronic activation of hM4D by administering CNO for 2 weeks in drinking water did not affect motivation due to a tolerance effect. However, the acute effect of CNO on motivation was reinstated after discontinuing chronic treatment for 48 h. Used as a therapeutic approach, striatopallidal inhibition should consider the risk of impairing goal-directed efficiency and behavioral desensitization. SIGNIFICANCE STATEMENT Motivation involves a directional component that allows subjects to efficiently select the behavior that will lead to an optimal outcome and an activational component that initiates and maintains the vigor and persistence of actions. Striatal output pathways modulate motivated behavior, but it remains unknown how these pathways regulate specific

  12. Cyanobacterial production of 1,3-propanediol directly from carbon dioxide using a synthetic metabolic pathway.

    PubMed

    Hirokawa, Yasutaka; Maki, Yuki; Tatsuke, Tsuneyuki; Hanai, Taizo

    2016-03-01

    Production of chemicals directly from carbon dioxide using light energy is an attractive option for a sustainable future. The 1,3-propanediol (1,3-PDO) production directly from carbon dioxide was achieved by engineered Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. Glycerol dehydratase catalyzing the conversion of glycerol to 3-hydroxypropionaldehyde in a coenzyme B12-dependent manner worked in S. elongatus PCC 7942 without addition of vitamin B12, suggesting that the intrinsic pseudovitamin B12 served as a substitute of coenzyme B12. The highest titers of 1,3-PDO (3.79±0.23 mM; 288±17.7 mg/L) and glycerol (12.62±1.55 mM; 1.16±0.14 g/L), precursor of 1,3-PDO, were reached after 14 days of culture under optimized conditions in this study.

  13. Direct pathway for hepatic glycogenesis predominates in meal-fed rats

    SciTech Connect

    Huang, M.T.; Veech, R.L.

    1987-05-01

    The pathway for hepatic glycogen synthesis in the postprandial state was studied in meal-fed rats chronically cannulated in the portal vein. The rate of glycogen synthesis in livers of rats meal-fed for seven days was found to be about 1 umol/g/min. Plasma glucose concentration in the portal vein was generally below 8 mM before meal-feeding and could reach up to 12 mM at the end of the meal-feeding. Studies on the hepatic-portal (H-P) difference of plasma glucose showed that liver released glucose in the fasted state and could either extract or release glucose after feeding, depending on plasma glucose concentration in the portal vein. The cross-over concentration for the transition was found to be 8 mM. The relative importance of the direct vs indirect pathway for the replenishment of hepatic glycogen was determined by injecting (3-/sup 3/H,U-/sup 14/C)-glucose into the portal vein at the end of meal-feeding. Six minutes after the injection, the ratio of /sup 3/H//sup 14/C in glycogen-glucose was found to be 83-92% of the ratio in liver free glucose. The H-P differences of glucose, lactate, pyruvate, and alanine during feeding were determined. It was found that the H-P difference of (glc) was about 9 times greater than the combined total of ..delta.. (lac), ..delta.. (pyr), and ..delta.. (ala) as early as 10 minutes after the onset of feeding. It is concluded that the direct pathway for the replenishment of hepatic glycogen is predominant and can account for more than 80% of the total glycogen synthesized in vivo in the postprandial state, in contrast to the result of < 30% reported previously by Newgard et al in acute traumatized rats.

  14. BPA Directly Decreases GnRH Neuronal Activity via Noncanonical Pathway.

    PubMed

    Klenke, Ulrike; Constantin, Stephanie; Wray, Susan

    2016-05-01

    Peripheral feedback of gonadal estrogen to the hypothalamus is critical for reproduction. Bisphenol A (BPA), an environmental pollutant with estrogenic actions, can disrupt this feedback and lead to infertility in both humans and animals. GnRH neurons are essential for reproduction, serving as an important link between brain, pituitary, and gonads. Because GnRH neurons express several receptors that bind estrogen, they are potential targets for endocrine disruptors. However, to date, direct effects of BPA on GnRH neurons have not been shown. This study investigated the effects of BPA on GnRH neuronal activity using an explant model in which large numbers of primary GnRH neurons are maintained and express many of the receptors found in vivo. Because oscillations in intracellular calcium have been shown to correlate with electrical activity in GnRH neurons, calcium imaging was used to assay the effects of BPA. Exposure to 50μM BPA significantly decreased GnRH calcium activity. Blockage of γ-aminobutyric acid ergic and glutamatergic input did not abrogate the inhibitory BPA effect, suggesting direct regulation of GnRH neurons by BPA. In addition to estrogen receptor-β, single-cell RT-PCR analysis confirmed that GnRH neurons express G protein-coupled receptor 30 (G protein-coupled estrogen receptor 1) and estrogen-related receptor-γ, all potential targets for BPA. Perturbation studies of the signaling pathway revealed that the BPA-mediated inhibition of GnRH neuronal activity occurred independent of estrogen receptors, GPER, or estrogen-related receptor-γ, via a noncanonical pathway. These results provide the first evidence of a direct effect of BPA on GnRH neurons.

  15. DNA damage response pathway and replication fork stress during oligonucleotide directed gene editing.

    PubMed

    Bonner, Melissa; Strouse, Bryan; Applegate, Mindy; Livingston, Paula; Kmiec, Eric B

    2012-04-03

    Single-stranded DNA oligonucleotides (ODNs) can be used to direct the exchange of nucleotides in the genome of mammalian cells in a process known as gene editing. Once refined, gene editing should become a viable option for gene therapy and molecular medicine. Gene editing is regulated by a number of DNA recombination and repair pathways whose natural activities often lead to single- and double-stranded DNA breaks. It has been previously shown that introduction of a phosphorotioated ODN, designed to direct a gene-editing event, into cells results in the activation of γH2AX, a well-recognized protein biomarker for double-stranded DNA breakage. Using a single copy, integrated mutant enhanced green fluorescent protein (eGFP) gene as our target, we now demonstrate that several types of ODNs, capable of directing gene editing, also activate the DNA damage response and the post-translational modification of proliferating cell nuclear antigen (PCNA), a signature modification of replication stress. We find that the gene editing reaction itself leads to transient DNA breakage, perhaps through replication fork collapse. Unmodified specific ODNs elicit a lesser degree of replication stress than their chemically modified counterparts, but are also less active in gene editing. Modified phosphothioate oligonucleotides (PTOs) are detrimental irrespective of the DNA sequence. Such collateral damage may prove problematic for proliferation of human cells genetically modified by gene editing.

  16. Direct photolysis rates and transformation pathways of the lampricides TFM and niclosamide in simulated sunlight

    USGS Publications Warehouse

    McConville, Megan B.; Hubert, Terrance D.; Remucal, Christina K.

    2016-01-01

    The lampricides 3-trifluoromethyl-4-nitrophenol (TFM) and 2′,5-dichloro-4′-nitrosalicylanilide (niclosamide) are directly added to many tributaries of the Great Lakes that harbor the invasive parasitic sea lamprey. Despite their long history of use, the fate of lampricides is not well understood. This study evaluates the rate and pathway of direct photodegradation of both lampricides under simulated sunlight. The estimated half-lives of TFM range from 16.6 ± 0.2 h (pH 9) to 32.9 ± 1.0 h (pH 6), while the half-lives of niclosamide range from 8.88 ± 0.52 days (pH 6) to 382 ± 83 days (pH 9) assuming continuous irradiation over a water depth of 55 cm. Both compounds degrade to form a series of aromatic intermediates, simple organic acids, ring cleavage products, and inorganic ions. Experimental data were used to construct a kinetic model which demonstrates that the aromatic products of TFM undergo rapid photolysis and emphasizes that niclosamide degradation is the rate-limiting step to dehalogenation and mineralization of the lampricide. This study demonstrates that TFM photodegradation is likely to occur on the time scale of lampricide applications (2–5 days), while niclosamide, the less selective lampricide, will undergo minimal direct photodegradation during its passage to the Great Lakes.

  17. Directing folding pathways for multi-component DNA origami nanostructures with complex topology

    NASA Astrophysics Data System (ADS)

    Marras, A. E.; Zhou, L.; Kolliopoulos, V.; Su, H.-J.; Castro, C. E.

    2016-05-01

    Molecular self-assembly has become a well-established technique to design complex nanostructures and hierarchical mesoscale assemblies. The typical approach is to design binding complementarity into nucleotide or amino acid sequences to achieve the desired final geometry. However, with an increasing interest in dynamic nanodevices, the need to design structures with motion has necessitated the development of multi-component structures. While this has been achieved through hierarchical assembly of similar structural units, here we focus on the assembly of topologically complex structures, specifically with concentric components, where post-folding assembly is not feasible. We exploit the ability to direct folding pathways to program the sequence of assembly and present a novel approach of designing the strand topology of intermediate folding states to program the topology of the final structure, in this case a DNA origami slider structure that functions much like a piston-cylinder assembly in an engine. The ability to program the sequence and control orientation and topology of multi-component DNA origami nanostructures provides a foundation for a new class of structures with internal and external moving parts and complex scaffold topology. Furthermore, this work provides critical insight to guide the design of intermediate states along a DNA origami folding pathway and to further understand the details of DNA origami self-assembly to more broadly control folding states and landscapes.

  18. Direct molecular interactions between Beclin 1 and the canonical NFκB activation pathway.

    PubMed

    Niso-Santano, Mireia; Criollo, Alfredo; Malik, Shoaib Ahmad; Michaud, Michael; Morselli, Eugenia; Mariño, Guillermo; Lachkar, Sylvie; Galluzzi, Lorenzo; Maiuri, Maria Chaira; Kroemer, Guido

    2012-02-01

    General (macro)autophagy and the activation of NFκB constitute prominent responses to a large array of intracellular and extracellular stress conditions. The depletion of any of the three subunits of the inhibitor of NFκB (IκB) kinase (IKKα, IKKβ, IKKγ/NEMO), each of which is essential for the canonical NFκB activation pathway, limits autophagy induction by physiological or pharmacological triggers, while constitutive active IKK subunits suffice to stimulate autophagy. The activation of IKK usually relies on TGFβ-activated kinase 1 (TAK1), which is also necessary for the optimal induction of autophagy in multiple settings. TAK1 interacts with two structurally similar co-activators, TAK1-binding proteins 2 and 3 (TAB2 and TAB3). Importantly, in resting conditions both TAB2 and TAB3 bind the essential autophagic factor Beclin 1, but not TAK1. In response to pro-autophagic stimuli, TAB2 and TAB3 dissociate from Beclin 1 and engage in stimulatory interactions with TAK1. The inhibitory interaction between TABs and Beclin 1 is mediated by their coiled-coil domains (CCDs). Accordingly, the overexpression of either TAB2 or TAB3 CCD stimulates Beclin 1- and TAK1-dependent autophagy. These results point to the existence of a direct molecular crosstalk between the canonical NFκB activation pathway and the autophagic core machinery that guarantees the coordinated induction of these processes in response to stress.

  19. A direct anterior cingulate pathway to the primate primary olfactory cortex may control attention to olfaction

    PubMed Central

    García-Cabezas, Miguel Á.; Barbas, Helen

    2016-01-01

    Behavioral and functional studies in humans suggest that attention plays a key role in activating the primary olfactory cortex through an unknown circuit mechanism. We report that a novel pathway from the anterior cingulate cortex, an area which has a key role in attention, projects directly to the primary olfactory cortex in rhesus monkeys, innervating mostly the anterior olfactory nucleus. Axons from the anterior cingulate cortex formed synapses mostly with spines of putative excitatory pyramidal neurons and with a small proportion of a neurochemical class of inhibitory neurons that are thought to have disinhibitory effect on excitatory neurons. This novel pathway from the anterior cingulate is poised to exert a powerful excitatory effect on the anterior olfactory nucleus, which is a critical hub for odorant processing via extensive bilateral connections with primary olfactory cortices and the olfactory bulb. Acting on the anterior olfactory nucleus, the anterior cingulate may activate the entire primary olfactory cortex to mediate the process of rapid attention to olfactory stimuli. PMID:23797208

  20. Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes

    PubMed Central

    Rocio Servin-Vences, M; Moroni, Mirko; Lewin, Gary R; Poole, Kate

    2017-01-01

    The joints of mammals are lined with cartilage, comprised of individual chondrocytes embedded in a specialized extracellular matrix. Chondrocytes experience a complex mechanical environment and respond to changing mechanical loads in order to maintain cartilage homeostasis. It has been proposed that mechanically gated ion channels are of functional importance in chondrocyte mechanotransduction; however, direct evidence of mechanical current activation in these cells has been lacking. We have used high-speed pressure clamp and elastomeric pillar arrays to apply distinct mechanical stimuli to primary murine chondrocytes, stretch of the membrane and deflection of cell-substrate contacts points, respectively. Both TRPV4 and PIEZO1 channels contribute to currents activated by stimuli applied at cell-substrate contacts but only PIEZO1 mediates stretch-activated currents. These data demonstrate that there are separate, but overlapping, mechanoelectrical transduction pathways in chondrocytes. DOI: http://dx.doi.org/10.7554/eLife.21074.001 PMID:28135189

  1. Heading in the right direction: thermodynamics-based network analysis and pathway engineering.

    PubMed

    Ataman, Meric; Hatzimanikatis, Vassily

    2015-12-01

    Thermodynamics-based network analysis through the introduction of thermodynamic constraints in metabolic models allows a deeper analysis of metabolism and guides pathway engineering. The number and the areas of applications of thermodynamics-based network analysis methods have been increasing in the last ten years. We review recent applications of these methods and we identify the areas that such analysis can contribute significantly, and the needs for future developments. We find that organisms with multiple compartments and extremophiles present challenges for modeling and thermodynamics-based flux analysis. The evolution of current and new methods must also address the issues of the multiple alternatives in flux directionalities and the uncertainties and partial information from analytical methods.

  2. A direct view of the complex multi-pathway folding of telomeric G-quadruplexes

    PubMed Central

    Aznauryan, Mikayel; Søndergaard, Siri; Noer, Sofie L.; Schiøtt, Birgit; Birkedal, Victoria

    2016-01-01

    G-quadruplexes (G4s) are DNA secondary structures that are capable of forming and function in vivo. The propensity of G4s to exhibit extreme polymorphism and complex dynamics is likely to influence their cellular function, yet a clear microscopic picture of their folding process is lacking. Here we employed single-molecule FRET microscopy to obtain a direct view of the folding and underlying conformational dynamics of G4s formed by the human telomeric sequence in potassium containing solutions. Our experiments allowed detecting several folded states that are populated in the course of G4 folding and determining their folding energetics and timescales. Combining the single-molecule data with molecular dynamics simulations enabled obtaining a structural description of the experimentally observed folded states. Our work thus provides a comprehensive thermodynamic and kinetic description of the folding of G4s that proceeds through a complex multi-route pathway, involving several marginally stable conformational states. PMID:27799468

  3. Modulation of direct pathway striatal projection neurons by muscarinic M₄-type receptors.

    PubMed

    Hernández-Flores, Teresa; Hernández-González, Omar; Pérez-Ramírez, María B; Lara-González, Esther; Arias-García, Mario A; Duhne, Mariana; Pérez-Burgos, Azucena; Prieto, G Aleph; Figueroa, Alejandra; Galarraga, Elvira; Bargas, José

    2015-02-01

    Models of basal ganglia (BG) function posit a dynamic balance between two classes of striatal projection neurons (SPNs): direct pathway neurons (dSPNs) that facilitate movements, and indirect pathway neurons (iSPNs) that repress movement execution. Two main modulatory transmitters regulate the output of these neurons: dopamine (DA) and acetylcholine (ACh). dSPNs express D1-type DA, M1-and M4-type ACh receptors, while iSPNs express D2-type DA and M1-type ACh receptors. Actions of M1-, D1-, and D2-receptors have been extensively reported, but we still ignore most actions of muscarinic M4-type receptors. Here, we used whole-cell recordings in acutely dissociated neurons, pharmacological tools such as mamba-toxins, and BAC D(1 or 2)-eGFP transgenic mice to show that activation of M4-type receptors with bath applied muscarine enhances Ca(2+)-currents through CaV1-channels in dSPNs and not in iSPNs. This action increases excitability of dSPNs after both direct current injection and synaptically driven stimulation. The increases in Ca(2+)-current and excitability were blocked specifically by mamba toxin-3, suggesting mediation via M4-type receptors. M4-receptor activation also increased network activity of dSPNs but not of iSPNs as seen with calcium-imaging techniques. Moreover, actions of D1-type and M4-type receptors may add to produce a larger enhancement of excitability of dSPNs or, paradoxically, oppose each other depending on the order of their activation. Possible implications of these findings are discussed.

  4. Degradation pathways of lamotrigine under advanced treatment by direct UV photolysis, hydroxyl radicals, and ozone.

    PubMed

    Keen, Olya S; Ferrer, Imma; Michael Thurman, E; Linden, Karl G

    2014-12-01

    Lamotrigine is recently recognized as a persistent pharmaceutical in the water environment and wastewater effluents. Its degradation was studied under UV and ozone advanced oxidation treatments with reaction kinetics of lamotrigine with ozone (≈4 M(-1)s(-1)), hydroxyl radical [(2.1 ± 0.3) × 10(9)M(-1)s(-1)] and by UV photolysis with low and medium pressure mercury vapor lamps [quantum yields ≈0 and (2.7 ± 0.4)× 10(-4) respectively] determined. All constants were measured at pH 6 and at temperature ≈20°C. The results indicate that lamotrigine is slow to respond to direct photolysis or oxidation by ozone and no attenuation of the contaminant is expected in UV or ozone disinfection applications. The compound reacts rapidly with hydroxyl radicals indicating that advanced oxidation processes would be effective for its treatment. Degradation products were identified under each treatment process using accurate mass time-of-flight spectrometry and pathways of decay were proposed. The main transformation pathways in each process were: dechlorination of the benzene ring during direct photolysis; hydroxyl group addition to the benzene ring during the reaction with hydroxyl radicals; and triazine ring opening after reaction with ozone. Different products that form in each process may be to a varying degree less environmentally stable than the parent lamotrigine. In addition, a novel method of ozone quenching without addition of salts is presented. The new quenching method would allow subsequent mass spectrometry analysis without a solid phase extraction clean-up step. The method involves raising the pH of the sample to approximately 10 for a few seconds and lowering it back and is therefore limited to applications for which temporary pH change is not expected to affect the outcome of the analysis.

  5. Local and distal effects of arbuscular mycorrhizal colonization on direct pathway Pi uptake and root growth in Medicago truncatula.

    PubMed

    Watts-Williams, Stephanie J; Jakobsen, Iver; Cavagnaro, Timothy R; Grønlund, Mette

    2015-07-01

    Two pathways exist for plant Pi uptake from soil: via root epidermal cells (direct pathway) or via associations with arbuscular mycorrhizal (AM) fungi, and the two pathways interact in a complex manner. This study investigated distal and local effects of AM colonization on direct root Pi uptake and root growth, at different soil P levels. Medicago truncatula was grown at three soil P levels in split-pots with or without AM fungal inoculation and where one root half grew into soil labelled with (33)P. Plant genotypes included the A17 wild type and the mtpt4 mutant. The mtpt4 mutant, colonized by AM fungi, but with no functional mycorrhizal pathway for Pi uptake, was included to better understand effects of AM colonization per se. Colonization by AM fungi decreased expression of direct Pi transporter genes locally, but not distally in the wild type. In mtpt4 mutant plants, direct Pi transporter genes and the Pi starvation-induced gene Mt4 were more highly expressed than in wild-type roots. In wild-type plants, less Pi was taken up via the direct pathway by non-colonized roots when the other root half was colonized by AM fungi, compared with non-mycorrhizal plants. Colonization by AM fungi strongly influenced root growth locally and distally, and direct root Pi uptake activity locally, but had only a weak influence on distal direct pathway activity. The responses to AM colonization in the mtpt4 mutant suggested that in the wild type, the increased P concentration of colonized roots was a major factor driving the effects of AM colonization on direct root Pi uptake.

  6. Nano Mechanical Machining Using AFM Probe

    NASA Astrophysics Data System (ADS)

    Mostofa, Md. Golam

    Complex miniaturized components with high form accuracy will play key roles in the future development of many products, as they provide portability, disposability, lower material consumption in production, low power consumption during operation, lower sample requirements for testing, and higher heat transfer due to their very high surface-to-volume ratio. Given the high market demand for such micro and nano featured components, different manufacturing methods have been developed for their fabrication. Some of the common technologies in micro/nano fabrication are photolithography, electron beam lithography, X-ray lithography and other semiconductor processing techniques. Although these methods are capable of fabricating micro/nano structures with a resolution of less than a few nanometers, some of the shortcomings associated with these methods, such as high production costs for customized products, limited material choices, necessitate the development of other fabricating techniques. Micro/nano mechanical machining, such an atomic force microscope (AFM) probe based nano fabrication, has, therefore, been used to overcome some the major restrictions of the traditional processes. This technique removes material from the workpiece by engaging micro/nano size cutting tool (i.e. AFM probe) and is applicable on a wider range of materials compared to the photolithographic process. In spite of the unique benefits of nano mechanical machining, there are also some challenges with this technique, since the scale is reduced, such as size effects, burr formations, chip adhesions, fragility of tools and tool wear. Moreover, AFM based machining does not have any rotational movement, which makes fabrication of 3D features more difficult. Thus, vibration-assisted machining is introduced into AFM probe based nano mechanical machining to overcome the limitations associated with the conventional AFM probe based scratching method. Vibration-assisted machining reduced the cutting forces

  7. Carbamazepine directly inhibits adipocyte differentiation through activation of the ERK 1/2 pathway

    PubMed Central

    Turpin, E; Muscat, A; Vatier, C; Chetrite, G; Corruble, E; Moldes, M; Fève, B

    2013-01-01

    Background and Purpose Carbamazepine (CBZ), known for its anti-epileptic, analgesic and mood-stabilizing properties, is also known to induce weight gain but the pathophysiology of this adverse effect is still largely unknown. We tested the hypothesis that CBZ could have a direct effect on adipocyte development and metabolism. Experimental Research We studied the effects of CBZ on morphological biochemical and molecular markers of adipogenesis, using several pre-adipocyte murine cell lines (3T3-L1, 3T3-F442A and T37i cells) and primary cultures of human pre-adipocytes. To delineate the mechanisms underlying the effect of CBZ, clonal expansion of pre-adipocytes, pro-adipogenic transcription factors, glucose uptake and lipolysis were also examined. Key Results CBZ strongly inhibited pre-adipocyte differentiation and triglyceride accumulation in a time- and dose-dependent manner in all models. Pleiotropic mechanisms were at the basis of the inhibitory effects of CBZ on adipogenesis and cell lipid accumulation. They included suppression of both clonal expansion and major adipogenic transcription factors such as PPAR-γ and CCAAT/enhancer binding protein-α, activation of basal lipolysis and decrease in insulin-stimulated glucose transport. Conclusions and Implications The effect of CBZ on adipogenesis involves activation of the ERK1/2 pathway. Our results show that CBZ acts directly on pre-adipocytes and adipocytes to alter adipose tissue development and metabolism. PMID:22889231

  8. Towards direct synthesis of alane: A predicted defect-mediated pathway confirmed experimentally

    SciTech Connect

    Wang, Lin -Lin; Herwadkar, Aditi; Reich, Jason M.; Johnson, Duane D.; House, Stephen D.; Pena-Martin, Pamela; Rockett, Angus A.; Robertson, Ian M.; Gupta, Shalabh; Pecharsky, Vitalij K.

    2016-08-18

    Here, alane (AlH3) is a unique energetic material that has not found a broad practical use for over 70 years because it is difficult to synthesize directly from its elements. Using density functional theory, we examine the defect-mediated formation of alane monomers on Al(111) in a two-step process: (1) dissociative adsorption of H2 and (2) alane formation, which are both endothermic on a clean surface. Only with Ti dopant to facilitate H2 dissociation and vacancies to provide Al adatoms, both processes become exothermic. In agreement, in situ scanning tunneling microscopy showed that during H2 exposure, alane monomers and clusters form primarily in the vicinity of Al vacancies and Ti atoms. Moreover, ball milling of the Al samples with Ti (providing necessary defects) showed a 10 % conversion of Al into AlH3 or closely related species at 344 bar H2, indicating that the predicted pathway may lead to the direct synthesis of alane from elements at pressures much lower than the 104 bar expected from bulk thermodynamics.

  9. Towards direct synthesis of alane: A predicted defect-mediated pathway confirmed experimentally

    DOE PAGES

    Wang, Lin -Lin; Herwadkar, Aditi; Reich, Jason M.; ...

    2016-08-18

    Here, alane (AlH3) is a unique energetic material that has not found a broad practical use for over 70 years because it is difficult to synthesize directly from its elements. Using density functional theory, we examine the defect-mediated formation of alane monomers on Al(111) in a two-step process: (1) dissociative adsorption of H2 and (2) alane formation, which are both endothermic on a clean surface. Only with Ti dopant to facilitate H2 dissociation and vacancies to provide Al adatoms, both processes become exothermic. In agreement, in situ scanning tunneling microscopy showed that during H2 exposure, alane monomers and clusters formmore » primarily in the vicinity of Al vacancies and Ti atoms. Moreover, ball milling of the Al samples with Ti (providing necessary defects) showed a 10 % conversion of Al into AlH3 or closely related species at 344 bar H2, indicating that the predicted pathway may lead to the direct synthesis of alane from elements at pressures much lower than the 104 bar expected from bulk thermodynamics.« less

  10. Combination STM/AFM and AFM Images of Magnetic Domains

    NASA Astrophysics Data System (ADS)

    Yi, L.; Gallagher, M.; Howells, S.; Chen, T.; Sarid, D.

    1991-12-01

    By employing a cantilevered tip in a scanning tunneling microscope, one obtains images that show an enhancement of features associated with forces whose derivatives vary along the direction of scanning. The theory of this process is described together with experimental results showing magnetic domains on a gold coated floppy disk. Also shown are atomic force microscopy images of a ferrofluid-developed magnetic tape.

  11. Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining

    PubMed Central

    Millán-Aguiñaga, Natalie; Zhang, Jia Jia; O'Neill, Ellis C.; Ugalde, Juan A.; Jensen, Paul R.; Mantovani, Simone M.; Moore, Bradley S.

    2016-01-01

    Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or “orphaned” secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs, and (ii) how to rapidly connect genes to biosynthesized small molecules. Here we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes co-localized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty acid synthase resistance gene. We employed a new synthetic double-stranded DNA-mediated cloning strategy based on transformation-associated recombination to efficiently capture tlm and the related ttm BGCs directly from genomic DNA and to heterologously express them in Streptomyces hosts. We show the production of a group of unusual thiotetronic acid natural products, including the well-known fatty acid synthase inhibitor thiolactomycin that was first described over 30 years ago, yet never at the genetic level in regards to biosynthesis and auto-resistance. This finding not only validates the target-directed genome mining strategy for the discovery of antibiotic producing gene clusters without a priori knowledge of the molecule synthesized, but also paves the way for the investigation of novel enzymology involved in thiotetronic acid natural product biosynthesis. PMID:26458099

  12. Developmental Pathway of the MPER-Directed HIV-1-Neutralizing Antibody 10E8

    PubMed Central

    Zhang, Baoshan; McKee, Krisha; Longo, Nancy S.; Yang, Yongping; Huang, Jinghe; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Alam, S. Munir; Haynes, Barton F.; Mullikin, James C.; Connors, Mark; Mascola, John R.; Shapiro, Lawrence; Kwong, Peter D.

    2016-01-01

    Antibody 10E8 targets the membrane-proximal external region (MPER) of HIV-1 gp41, neutralizes >97% of HIV-1 isolates, and lacks the auto-reactivity often associated with MPER-directed antibodies. The developmental pathway of 10E8 might therefore serve as a promising template for vaccine design, but samples from time-of-infection—often used to infer the B cell record—are unavailable. In this study, we used crystallography, next-generation sequencing (NGS), and functional assessments to infer the 10E8 developmental pathway from a single time point. Mutational analysis indicated somatic hypermutation of the 2nd-heavy chain-complementarity determining region (CDR H2) to be critical for neutralization, and structures of 10E8 variants with V-gene regions reverted to genomic origin for heavy-and-light chains or heavy chain-only showed structural differences >2 Å relative to mature 10E8 in the CDR H2 and H3. To understand these developmental changes, we used bioinformatic sieving, maximum likelihood, and parsimony analyses of immunoglobulin transcripts to identify 10E8-lineage members, to infer the 10E8-unmutated common ancestor (UCA), and to calculate 10E8-developmental intermediates. We were assisted in this analysis by the preservation of a critical D-gene segment, which was unmutated in most 10E8-lineage sequences. UCA and early intermediates weakly bound a 26-residue-MPER peptide, whereas HIV-1 neutralization and epitope recognition in liposomes were only observed with late intermediates. Antibody 10E8 thus develops from a UCA with weak MPER affinity and substantial differences in CDR H2 and H3 from the mature 10E8; only after extensive somatic hypermutation do 10E8-lineage members gain recognition in the context of membrane and HIV-1 neutralization. PMID:27299673

  13. Fsh and Lh direct conserved and specific pathways during flatfish semicystic spermatogenesis.

    PubMed

    Chauvigné, François; Zapater, Cinta; Crespo, Diego; Planas, Josep V; Cerdà, Joan

    2014-10-01

    The current view of the control of spermatogenesis by Fsh and Lh in non-mammalian vertebrates is largely based on studies carried out in teleosts with cystic and cyclic spermatogenesis. Much less is known concerning the specific actions of gonadotropins during semicystic germ cell development, a type of spermatogenesis in which germ cells are released into the tubular lumen where they transform into spermatozoa. In this study, using homologous gonadotropins and a candidate gene approach, for which the genes' testicular cell-type-specific expression was established, we investigated the regulatory effects of Fsh and Lh on gene expression during spermatogenesis in Senegalese sole (Solea senegalensis), a flatfish with asynchronous and semicystic germ cell development. During early spermatogenesis, Fsh and Lh upregulated steroidogenesis-related genes and nuclear steroid receptors, expressed in both somatic and germ cells, through steroid-dependent pathways, although Lh preferentially stimulated the expression of downstream genes involved in androgen and progestin syntheses. In addition, Lh specifically promoted the expression of spermatid-specific genes encoding spermatozoan flagellar proteins through direct interaction with the Lh receptor in these cells. Interestingly, at this spermatogenic stage, Fsh primarily regulated genes encoding Sertoli cell growth factors with potentially antagonistic effects on germ cell proliferation and differentiation through steroid mediation. During late spermatogenesis, fewer genes were regulated by Fsh or Lh, which was associated with a translational and posttranslational downregulation of the Fsh receptor in different testicular compartments. These results reveal that conserved and specialized gonadotropic pathways regulate semicystic spermatogenesis in flatfish, which may spatially adjust cell germ development to maintain a continuous reservoir of spermatids in the testis.

  14. Prolactin affects bovine oocytes through direct and cumulus-mediated pathways.

    PubMed

    Lebedeva, Irina Y; Singina, Galina N; Volkova, Natalia A; Vejlsted, Morten; Zinovieva, Natalia A; Schmidt, Mette

    2014-11-01

    yield both in system 2 (from 20.5%-40.9%, P < 0.01) and in system 3 (from 21.7%-33.9%, P < 0.05). The findings show for the first time the functioning of the direct pathway of PRL signaling into bovine oocytes, as confirmed by the expression of receptors of PRL and its direct meiosis-retarding effect involving activation of tyrosine kinases and NO synthase. Furthermore, this is the first demonstration that the beneficial effect of PRL on the oocyte developmental capacity is achieved via cumulus cells containing PRL receptors.

  15. Anomalies in nanostructure size measurements by AFM

    NASA Astrophysics Data System (ADS)

    Mechler, Ádám; Kopniczky, Judit; Kokavecz, János; Hoel, Anders; Granqvist, Claes-Göran; Heszler, Peter

    2005-09-01

    Anomalies in atomic force microscopy (AFM) based size determination of nanoparticles were studied via comparative analysis of experiments and numerical calculations. Single tungsten oxide nanoparticles with a mean diameter of 3nm were deposited on mica and graphite substrates and were characterised by AFM. The size (height) of the nanoparticles, measured by tapping mode AFM, was found to be sensitive to the free amplitude of the oscillating tip, thus indicating that the images were not purely topographical. By comparing the experimental results to model calculations, we demonstrate that the dependence of the nanoparticle size on the oscillation amplitude of the tip is an inherent characteristic of the tapping mode AFM; it is also a function of physical properties such as elasticity and surface energy of the nanoparticle and the sample surface, and it depends on the radius of curvature of the tip. We show that good approximation of the real size can easily be obtained from plots of particle height vs free amplitude of the oscillating tip, although errors might persist for individual experiments. The results are valid for size (height) determination of any nanometer-sized objects imaged by tapping mode AFM.

  16. Interlaboratory round robin on cantilever calibration for AFM force spectroscopy.

    PubMed

    te Riet, Joost; Katan, Allard J; Rankl, Christian; Stahl, Stefan W; van Buul, Arend M; Phang, In Yee; Gomez-Casado, Alberto; Schön, Peter; Gerritsen, Jan W; Cambi, Alessandra; Rowan, Alan E; Vancso, G Julius; Jonkheijm, Pascal; Huskens, Jurriaan; Oosterkamp, Tjerk H; Gaub, Hermann; Hinterdorfer, Peter; Figdor, Carl G; Speller, Sylvia

    2011-12-01

    Single-molecule force spectroscopy studies performed by Atomic Force Microscopes (AFMs) strongly rely on accurately determined cantilever spring constants. Hence, to calibrate cantilevers, a reliable calibration protocol is essential. Although the thermal noise method and the direct Sader method are frequently used for cantilever calibration, there is no consensus on the optimal calibration of soft and V-shaped cantilevers, especially those used in force spectroscopy. Therefore, in this study we aimed at establishing a commonly accepted approach to accurately calibrate compliant and V-shaped cantilevers. In a round robin experiment involving eight different laboratories we compared the thermal noise and the Sader method on ten commercial and custom-built AFMs. We found that spring constants of both rectangular and V-shaped cantilevers can accurately be determined with both methods, although the Sader method proved to be superior. Furthermore, we observed that simultaneous application of both methods on an AFM proved an accurate consistency check of the instrument and thus provides optimal and highly reproducible calibration. To illustrate the importance of optimal calibration, we show that for biological force spectroscopy studies, an erroneously calibrated cantilever can significantly affect the derived (bio)physical parameters. Taken together, our findings demonstrated that with the pre-established protocol described reliable spring constants can be obtained for different types of cantilevers.

  17. LET Spectrum Measurements In CR-39 PNTD With AFM

    SciTech Connect

    Johnson, C. E.; DeWitt, J. M.; Benton, E. R.; Yasuda, N.; Benton, E. V.

    2011-06-01

    Energetic protons, neutrons, and heavy ions undergoing collisions with target nuclei of varying Z can produce residual heavy recoil fragments via intra-nuclear cascade/evaporation reactions. The particles produced in these non-elastic collisions generally have such extremely short range ({approx}<10 {mu}m) that they cannot be directly observed by conventional detection methods including CR-39 plastic nuclear track detector (PNTD) that has been chemically etched for analysis by standard visible light microscopy. However, high-LET recoil fragments having range on the order of several cell diameters can be produced in tissue during radiotherapy using proton and carbon beams. We have developed a method to analyze short-range, high-LET tracks in CR-39 plastic nuclear track detector (PNTD) using short duration chemical etching ({approx}<1 {mu}m) following by automated atomic force microscope (AFM) scanning. The post-scan data processing used in this work was based on semi-automated matrix analysis opposed to traditional grey-scale image analysis. This method takes advantage of the 3-D data obtained via AFM to achieve robust discrimination of nuclear tracks from other features inherently present in the post-etch detector surface. Through automation of AFM scanning, sufficient AFM scan frames were obtained to attain an LET spectrum spanning the LET range from 200-1500 keV/{mu}m. In addition to our experiments, simulations were carried out with the Monte Carlo transport code, FLUKA. To demonstrate this method, CR-39 PNTD was exposed to the proton therapy beam at Loma Linda University Medical Center (LLUMC) at 60 and 230 MeV. Additionally, detectors were exposed to 1 GeV protons at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). For these exposures CR-39 PNTD, Al and Cu target foils were used between detector layers.

  18. LET Spectrum Measurements In CR-39 PNTD With AFM

    NASA Astrophysics Data System (ADS)

    Johnson, C. E.; DeWitt, J. M.; Benton, E. R.; Yasuda, N.; Benton, E. V.

    2011-06-01

    Energetic protons, neutrons, and heavy ions undergoing collisions with target nuclei of varying Z can produce residual heavy recoil fragments via intra-nuclear cascade/evaporation reactions. The particles produced in these non-elastic collisions generally have such extremely short range (˜<10 μm) that they cannot be directly observed by conventional detection methods including CR-39 plastic nuclear track detector (PNTD) that has been chemically etched for analysis by standard visible light microscopy. However, high-LET recoil fragments having range on the order of several cell diameters can be produced in tissue during radiotherapy using proton and carbon beams. We have developed a method to analyze short-range, high-LET tracks in CR-39 plastic nuclear track detector (PNTD) using short duration chemical etching (˜<1 μm) following by automated atomic force microscope (AFM) scanning. The post-scan data processing used in this work was based on semi-automated matrix analysis opposed to traditional grey-scale image analysis. This method takes advantage of the 3-D data obtained via AFM to achieve robust discrimination of nuclear tracks from other features inherently present in the post-etch detector surface. Through automation of AFM scanning, sufficient AFM scan frames were obtained to attain an LET spectrum spanning the LET range from 200-1500 keV/μm. In addition to our experiments, simulations were carried out with the Monte Carlo transport code, FLUKA. To demonstrate this method, CR-39 PNTD was exposed to the proton therapy beam at Loma Linda University Medical Center (LLUMC) at 60 and 230 MeV. Additionally, detectors were exposed to 1 GeV protons at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). For these exposures CR-39 PNTD, Al and Cu target foils were used between detector layers.

  19. LET spectrum measurements in Cr-39 PNTD with AFM

    SciTech Connect

    Johnson, Carl Edward; De Witt, Joel M; Benton, Eric R; Yasuda, Nakahiro; Benton, Eugene V

    2010-01-01

    Energetic protons, neutrons, and heavy ions undergoing collisions with target nuclei of varying Z can produce residual heavy recoil fragments via intra-nuclear cascade/evaporation reactions. The particles produced in these non-elastic collisions generally have such extremely short range ({approx}< 10 {mu}m) that they cannot be directly observed by conventional detection methods including CR-39 plastic nuclear track detector (PNTD) that has been chemically etched for analysis by standard visible light microscopy. However, high-LET recoil fragments having range on the order of several cell diameters can be produced in tissue during radiotherapy using proton and carbon beams. We have developed a method to analyze short-range, high-LET tracks in CR-39 plastic nuclear track detector (PNTD) using short duration chemical etching ({approx}< 1 {mu}m) followed by automated atomic force microscope (AFM) scanning. The post-scan data processing used in this work was based on semi-automated matrix analysis opposed to traditional grey-scale image analysis. This method takes advantage of the 3-D data obtained via AFM to achieve robust discrimination of nuclear tracks from other features. Through automation of AFM scanning, sufficient AFM scan frames were obtained to attain an LET spectrum spanning the LET range from 200-1500 keV/{mu}m. In addition to our experiments, simulations were carried out with the Monte Carlo transport code, FLUKA. To demonstrate this method, CR-39 PNTD was exposed to the proton therapy beam at Loma Linda University Medical Center (LLUMC) at 60 and 230 MeV. Additionally, detectors were exposed to I GeV protons at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL). For these exposures CR-39 PNTD, Al and Cu target foils were used between detector layers.

  20. Diffusion tensor imaging of dolphin brains reveals direct auditory pathway to temporal lobe

    PubMed Central

    Berns, Gregory S.; Cook, Peter F.; Foxley, Sean; Jbabdi, Saad; Miller, Karla L.; Marino, Lori

    2015-01-01

    The brains of odontocetes (toothed whales) look grossly different from their terrestrial relatives. Because of their adaptation to the aquatic environment and their reliance on echolocation, the odontocetes' auditory system is both unique and crucial to their survival. Yet, scant data exist about the functional organization of the cetacean auditory system. A predominant hypothesis is that the primary auditory cortex lies in the suprasylvian gyrus along the vertex of the hemispheres, with this position induced by expansion of ‘associative′ regions in lateral and caudal directions. However, the precise location of the auditory cortex and its connections are still unknown. Here, we used a novel diffusion tensor imaging (DTI) sequence in archival post-mortem brains of a common dolphin (Delphinus delphis) and a pantropical dolphin (Stenella attenuata) to map their sensory and motor systems. Using thalamic parcellation based on traditionally defined regions for the primary visual (V1) and auditory cortex (A1), we found distinct regions of the thalamus connected to V1 and A1. But in addition to suprasylvian-A1, we report here, for the first time, the auditory cortex also exists in the temporal lobe, in a region near cetacean-A2 and possibly analogous to the primary auditory cortex in related terrestrial mammals (Artiodactyla). Using probabilistic tract tracing, we found a direct pathway from the inferior colliculus to the medial geniculate nucleus to the temporal lobe near the sylvian fissure. Our results demonstrate the feasibility of post-mortem DTI in archival specimens to answer basic questions in comparative neurobiology in a way that has not previously been possible and shows a link between the cetacean auditory system and those of terrestrial mammals. Given that fresh cetacean specimens are relatively rare, the ability to measure connectivity in archival specimens opens up a plethora of possibilities for investigating neuroanatomy in cetaceans and other species

  1. The Interglomerular Circuit Potently Inhibits Olfactory Bulb Output Neurons by Both Direct and Indirect Pathways

    PubMed Central

    Puche, Adam C.; Shipley, Michael T.

    2016-01-01

    Sensory processing shapes our perception. In mammals, odor information is encoded by combinatorial activity patterns of olfactory bulb (OB) glomeruli. Glomeruli are richly interconnected by short axon cells (SACs), which form the interglomerular circuit (IGC). It is unclear how the IGC impacts OB output to downstream neural circuits. We combined in vitro and in vivo electrophysiology with optogenetics in mice and found the following: (1) the IGC potently and monosynaptically inhibits the OB output neurons mitral/tufted cells (MTCs) by GABA release from SACs: (2) gap junction-mediated electrical coupling is strong for the SAC→MTC synapse, but negligible for the SAC→ETC synapse; (3) brief IGC-mediated inhibition is temporally prolonged by the intrinsic properties of MTCs; and (4) sniff frequency IGC activation in vivo generates persistent MTC inhibition. These findings suggest that the temporal sequence of glomerular activation by sensory input determines which stimulus features are transmitted to downstream olfactory networks and those filtered by lateral inhibition. SIGNIFICANCE STATEMENT Odor identity is encoded by combinatorial patterns of activated glomeruli, the initial signal transformation site of the olfactory system. Lateral circuit processing among activated glomeruli modulates olfactory signal transformation before transmission to higher brain centers. Using a combination of in vitro and in vivo optogenetics, this work demonstrates that interglomerular circuitry produces potent inhibition of olfactory bulb output neurons via direct chemical and electrical synapses as well as by indirect pathways. The direct inhibitory synaptic input engages mitral cell intrinsic membrane properties to generate inhibition that outlasts the initial synaptic action. PMID:27629712

  2. Hydration states of AFm cement phases

    SciTech Connect

    Baquerizo, Luis G.; Matschei, Thomas; Scrivener, Karen L.; Saeidpour, Mahsa; Wadsö, Lars

    2015-07-15

    The AFm phase, one of the main products formed during the hydration of Portland and calcium aluminate cement based systems, belongs to the layered double hydrate (LDH) family having positively charged layers and water plus charge-balancing anions in the interlayer. It is known that these phases present different hydration states (i.e. varying water content) depending on the relative humidity (RH), temperature and anion type, which might be linked to volume changes (swelling and shrinkage). Unfortunately the stability conditions of these phases are insufficiently reported. This paper presents novel experimental results on the different hydration states of the most important AFm phases: monocarboaluminate, hemicarboaluminate, strätlingite, hydroxy-AFm and monosulfoaluminate, and the thermodynamic properties associated with changes in their water content during absorption/desorption. This data opens the possibility to model the response of cementitious systems during drying and wetting and to engineer systems more resistant to harsh external conditions.

  3. Anticoagulation beyond direct thrombin and factor Xa inhibitors: indications for targeting the intrinsic pathway?

    PubMed

    van Montfoort, Maurits L; Meijers, Joost C M

    2013-08-01

    Antithrombotic drugs like vitamin K antagonists and heparin have been the gold standard for the treatment and prevention of thromboembolic disease for many years. Unfortunately, there are several disadvantages of these antithrombotic drugs: they are accompanied by serious bleeding problems, it is necessary to monitor the therapeutic window, and there are various interactions with food and other drugs. This has led to the development of new oral anticoagulants, specifically inhibiting either thrombin or factor Xa. In terms of effectiveness, these drugs are comparable to the currently available anticoagulants; however, they are still associated with issues such as bleeding, reversal of the drug and complicated laboratory monitoring. Vitamin K antagonists, heparin, direct thrombin and factor Xa inhibitors have in common that they target key proteins of the haemostatic system. In an attempt to overcome these difficulties we investigated whether the intrinsic coagulation factors (VIII, IX, XI, XII, prekallikrein and high-molecular-weight kininogen) are superior targets for anticoagulation. We analysed epidemiological data concerning thrombosis and bleeding in patients deficient in one of the intrinsic pathway proteins. Furthermore, we discuss several thrombotic models in intrinsic coagulation factor-deficient animals. The combined results suggest that intrinsic coagulation factors could be suitable targets for anticoagulant drugs.

  4. Unraveling the microscopic pathway of homogeneous water crystallization at supercooled conditions from direct simulations

    NASA Astrophysics Data System (ADS)

    Martelli, Fausto; Palmer, Jeremy; Singh, Rakesh; Debenedetti, Pablo; Car, Roberto

    By means of unbiased classical molecular dynamics simulations, we identify the microscopic pathways of spontaneous homogeneous crystallization in supercooled ST2 water. By introducing a new order parameter, we are able to monitor formation/disruption of locally ordered regions characterized by small ice clusters with intermediate range order. When two of these regions are close each other, they percolate and form a larger ordered region. The process is slow enough to allow for polymorphic selection in favor of cubic ice (Ic). The formation of an ice nucleus requires percolation of many small clusters so that the transformations at the interface of the nucleus do not involve its core, thus guaranteeing the stability of the nucleus. The growth of the crystalline nucleus is fast and involves direct transformation of interfacial liquid molecules as well as percolation of small Ic/Ih clusters. The growth is too fast to allow conversion of Ih into Ic sites, originating the formation of a stacking fault in the final crystal. We recognize Euclidean structures in the oxygen configuration of the second shell in Ic and Ih clusters. This new point of view allows us to explain the source of the ordered stacking fault geometry.

  5. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies.

    PubMed

    Doria-Rose, Nicole A; Schramm, Chaim A; Gorman, Jason; Moore, Penny L; Bhiman, Jinal N; DeKosky, Brandon J; Ernandes, Michael J; Georgiev, Ivelin S; Kim, Helen J; Pancera, Marie; Staupe, Ryan P; Altae-Tran, Han R; Bailer, Robert T; Crooks, Ema T; Cupo, Albert; Druz, Aliaksandr; Garrett, Nigel J; Hoi, Kam H; Kong, Rui; Louder, Mark K; Longo, Nancy S; McKee, Krisha; Nonyane, Molati; O'Dell, Sijy; Roark, Ryan S; Rudicell, Rebecca S; Schmidt, Stephen D; Sheward, Daniel J; Soto, Cinque; Wibmer, Constantinos Kurt; Yang, Yongping; Zhang, Zhenhai; Mullikin, James C; Binley, James M; Sanders, Rogier W; Wilson, Ian A; Moore, John P; Ward, Andrew B; Georgiou, George; Williamson, Carolyn; Abdool Karim, Salim S; Morris, Lynn; Kwong, Peter D; Shapiro, Lawrence; Mascola, John R

    2014-05-01

    Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.

  6. Graphene MEMS: AFM probe performance improvement.

    PubMed

    Martin-Olmos, Cristina; Rasool, Haider Imad; Weiller, Bruce H; Gimzewski, James K

    2013-05-28

    We explore the feasibility of growing a continuous layer of graphene in prepatterned substrates, like an engineered silicon wafer, and we apply this as a mold for the fabrication of AFM probes. This fabrication method proves the fabrication of SU-8 devices coated with graphene in a full-wafer parallel technology and with high yield. It also demonstrates that graphene coating enhances the functionality of SU-8 probes, turning them conductive and more resistant to wear. Furthermore, it opens new experimental possibilities such as studying graphene-graphene interaction at the nanoscale with the precision of an AFM or the exploration of properties in nonplanar graphene layers.

  7. Quantitative Measurements of Elastic Properties with Ultrasonic-Based AFM and Conventional Techniques

    NASA Astrophysics Data System (ADS)

    Hurley, D. C.

    A prime motivation for the original development of ultrasonic-based AFM methods was to enable measurements of elastic properties with nanoscale spatial resolution. In this chapter, we discuss the quantitative measurement of elastic modulus with ultrasonic-based AFM methods and compare it to measurement by more conventional or established techniques. First, we present the basic principles of modulus measurement with methods that involve contact resonance spectroscopy, such as atomic force acoustic microscopy (AFAM) and ultrasonic AFM (U-AFM). Fundamental concepts of modulus measurement with more established approaches, especially instrumented (nano-) indentation (NI) and surface acoustic wave spectroscopy (SAWS), are then discussed. We consider the relative strengths and limitations of various approaches, for example measurement accuracy, spatial resolution, and applicability to different materials. Example results for specific material systems are given with an emphasis on studies involving direct intercomparison of different techniques. Finally, current research in this area and opportunities for future work are described.

  8. HbzF catalyzes direct hydrolysis of maleylpyruvate in the gentisate pathway of Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Liu, Kun; Liu, Ting-Ting; Zhou, Ning-Yi

    2013-02-01

    HbzF from Pseudomonas alcaligenes NCIMB 9867 was purified to homogeneity as a His-tagged protein and likely a dimer by SDS-PAGE and gel filtration. This protein was demonstrated to be a novel maleylpyruvate hydrolase, catalyzing direct hydrolysis of maleylpyruvate to maleate and pyruvate, and belongs to the fumarylacetoacetate hydrolase superfamily. This study reveals the genetic determinate for the direct maleylpyruvate hydrolysis in the gentisate pathway, complementary to the well-studied maleylpyruvate isomerization route.

  9. Key modulatory role of presynaptic adenosine A2A receptors in cortical neurotransmission to the striatal direct pathway.

    PubMed

    Quiroz, César; Luján, Rafael; Uchigashima, Motokazu; Simoes, Ana Patrícia; Lerner, Talia N; Borycz, Janusz; Kachroo, Anil; Canas, Paula M; Orru, Marco; Schwarzschild, Michael A; Rosin, Diane L; Kreitzer, Anatol C; Cunha, Rodrigo A; Watanabe, Masahiko; Ferré, Sergi

    2009-11-18

    Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders.

  10. Work Stressors, Social Support, and Burnout in Junior Doctors: Exploring Direct and Indirect Pathways

    ERIC Educational Resources Information Center

    Sochos, Antigonos; Bowers, Alexis; Kinman, Gail

    2012-01-01

    The study tested a pathway model linking different occupational stressors, different sources of social support, and burnout. A sample of 184 junior medical doctors was used. Pathway analysis suggested that doctors who experienced increased time demands, organizational constraints, and a lack of personal confidence perceived their consultants as…

  11. Developmental Pathways to Conduct Disorder: Implications for Future Directions in Research, Assessment, and Treatment

    ERIC Educational Resources Information Center

    Frick, Paul J.

    2012-01-01

    Research has indicated that there are several common pathways through which children and adolescents develop conduct disorder, each with different risk factors and each with different underlying developmental mechanisms leading to the child's aggressive and antisocial behavior. The current article briefly summarizes research on these pathways,…

  12. Education Direct: An Alternative Entry Pathway to Pre-Service Teacher Education

    ERIC Educational Resources Information Center

    Pilkington, Kevin; Lock, Graeme

    2012-01-01

    Universities in Australia are offering alternative entrance pathways to attract students from a range of backgrounds. These alternative pathways will undoubtedly be reviewed due to the recommendation in the Review of Australian Higher Education (Bradley, Noonan, Nugent & Scales, 2008) concerning increasing the diversity of university entrants.…

  13. Conductance of AFM Deformed Carbon Nanotubes

    NASA Technical Reports Server (NTRS)

    Svizhenko, Alexei; Maiti, Amitesh; Anatram, M. P.; Biegel, Bryan (Technical Monitor)

    2002-01-01

    This viewgraph presentation provides information on the electrical conductivity of carbon nanotubes upon deformation by atomic force microscopy (AFM). The density of states and conductance were computed using four orbital tight-binding method with various parameterizations. Different chiralities develop bandgap that varies with chirality.

  14. Contact nanomechanical measurements with the AFM

    NASA Astrophysics Data System (ADS)

    Geisse, Nicholas

    2013-03-01

    The atomic force microscope (AFM) has found broad use in the biological sciences largely due to its ability to make measurements on unfixed and unstained samples under liquid. In addition to imaging at multiple spatial scales ranging from micro- to nanometer, AFMs are commonly used as nanomechanical probes. This is pertinent for cell biology, as it has been demonstrated that the geometrical and mechanical properties of the extracellular microenvironment are important in such processes as cancer, cardiovascular disease, muscular dystrophy, and even the control of cell life and death. Indeed, the ability to control and quantify these external geometrical and mechanical parameters arises as a key issue in the field. Because AFM can quantitatively measure the mechanical properties of various biological samples, novel insights to cell function and to cell-substrate interactions are now possible. As the application of AFM to these types of problems is widened, it is important to understand the performance envelope of the technique and its associated data analyses. This talk will discuss the important issues that must be considered when mechanical models are applied to real-world data. Examples of the effect of different model assumptions on our understanding of the measured material properties will be shown. Furthermore, specific examples of the importance of mechanical stimuli and the micromechanical environment to the structure and function of biological materials will be presented.

  15. Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair.

    PubMed

    Davis, Luther; Maizels, Nancy

    2014-03-11

    DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9(D10A) nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.

  16. Probing ternary solvent effect in high Voc polymer solar cells using advanced AFM techniques

    DOE PAGES

    Li, Chao; Soleman, Mikhael; Lorenzo, Josie; ...

    2016-01-25

    This work describes a simple method to develop a high Voc low band gap PSCs. In addition, two new atomic force microscopy (AFM)-based nanoscale characterization techniques to study the surface morphology and physical properties of the structured active layer are introduced. With the help of ternary solvent processing of the active layer and C60 buffer layer, a bulk heterojunction PSC with Voc more than 0.9 V and conversion efficiency 7.5% is developed. In order to understand the fundamental properties of the materials ruling the performance of the PSCs tested, AFM-based nanoscale characterization techniques including Pulsed-Force-Mode AFM (PFM-AFM) and Mode-Synthesizing AFMmore » (MSAFM) are introduced. Interestingly, MSAFM exhibits high sensitivity for direct visualization of the donor–acceptor phases in the active layer of the PSCs. Lastly, conductive-AFM (cAFM) studies reveal local variations in conductivity in the donor and acceptor phases as well as a significant increase in photocurrent in the PTB7:ICBA sample obtained with the ternary solvent processing.« less

  17. Redesign, reconstruction, and directed extension of the Brevibacterium linens C40 carotenoid pathway in Escherichia coli.

    PubMed

    Kim, Se Hyeuk; Park, Yun Hee; Schmidt-Dannert, Claudia; Lee, Pyung Cheon

    2010-08-01

    In this study, the carotenoid biosynthetic pathways of Brevibacterium linens DSMZ 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host Escherichia coli. The modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. Extension of the novel 3,4-didehydrolycopene pathway with the mutant Pantoea lycopene cyclase CrtY(2) and the Rhodobacter spheroidene monooxygenase CrtA generated monocyclic torulene and acyclic oxocarotenoids, respectively. The reconstructed beta-carotene pathway synthesized an unexpected 7,8-dihydro-beta-carotene in addition to beta-carotene. Extension of the beta-carotene pathway with the B. linens beta-ring desaturase CrtU and Pantoea beta-carotene hydroxylase CrtZ generated asymmetric carotenoid agelaxanthin A, which had one aromatic ring at the one end of carotene backbone and one hydroxyl group at the other end, as well as aromatic carotenoid isorenieratene and dihydroxy carotenoid zeaxanthin. These results demonstrate that reconstruction of the biosynthetic pathways and extension with promiscuous enzymes in a heterologous host holds promise as a rational strategy for generating structurally diverse compounds that are hardly accessible in nature.

  18. Detection of Pathogens Using AFM and SPR

    NASA Astrophysics Data System (ADS)

    Vaseashta, Ashok

    2005-03-01

    A priori detection of pathogens in food and water has become a subject of paramount importance. Several recent incidents have resulted in the government passing stringent regulations for tolerable amounts of contamination of food products. Identification and/or monitoring of bacterial contamination in food are critical. The conventional methods of pathogen detection require time-consuming steps to arrive disembark at meaningful measurement in a timely manner as the detection time exceeds the time in which perishable food recycles through the food chain distribution. The aim of this presentation is to outline surface plasmon resonance (SPR) and atomic force microscopy (AFM) as two methods for fast detect6ion of pathogens. Theoretical basis of SPR and experimental results of SPR and AFM on E. coli O157:H7 and prion are presented.

  19. A Multidisciplinary Self-Directed Learning Module Improves Knowledge of a Quality Improvement Instrument: The HEART Pathway.

    PubMed

    Hartman, Nicholas D; Harper, Erin N; Leppert, Lauren M; Browning, Brittany M; Askew, Kim; Manthey, David E; Mahler, Simon A

    2016-07-20

    We created and tested an educational intervention to support implementation of an institution wide QI project (the HEART Pathway) designed to improve care for patients with acute chest pain. Although online learning modules have been shown effective in imparting knowledge regarding QI projects, it is unknown whether these modules are effective across specialties and healthcare professions. Participants, including nurses, advanced practice clinicians, house staff and attending physicians (N = 486), were enrolled into an online, self-directed learning course exploring the key concepts of the HEART Pathway. The module was completed by 97% of enrollees (469/486) and 90% passed on the first attempt (422/469). Out of 469 learners, 323 completed the pretest, learning module and posttest in the correct order. Mean test scores across learners improved significantly from 74% to 89% from the pretest to the posttest. Following the intervention, the HEART Pathway was used for 88% of patients presenting to our institution with acute chest pain. Our data demonstrate that this online, self-directed learning module can improve knowledge of the HEART Pathway across specialties-paving the way for more efficient and informed care for acute chest pain patients.

  20. KynR, a Lrp/AsnC-type transcriptional regulator, directly controls the kynurenine pathway in Pseudomonas aeruginosa.

    PubMed

    Knoten, Claire A; Hudson, L Lynn; Coleman, James P; Farrow, John M; Pesci, Everett C

    2011-12-01

    The opportunistic pathogen Pseudomonas aeruginosa can utilize a variety of carbon sources and produces many secondary metabolites to help survive harsh environments. P. aeruginosa is part of a small group of bacteria that use the kynurenine pathway to catabolize tryptophan. Through the kynurenine pathway, tryptophan is broken down into anthranilate, which is further degraded into tricarboxylic acid cycle intermediates or utilized to make numerous aromatic compounds, including the Pseudomonas quinolone signal (PQS). We have previously shown that the kynurenine pathway is a critical source of anthranilate for PQS synthesis and that the kynurenine pathway genes (kynA and kynBU) are upregulated in the presence of kynurenine. A putative Lrp/AsnC-type transcriptional regulator (gene PA2082, here called kynR), is divergently transcribed from the kynBU operon and is highly conserved in gram-negative bacteria that harbor the kynurenine pathway. We show that a mutation in kynR renders P. aeruginosa unable to utilize L-tryptophan as a sole carbon source and decreases PQS production. In addition, we found that the increase of kynA and kynB transcriptional activity in response to kynurenine was completely abolished in a kynR mutant, further indicating that KynR mediates the kynurenine-dependent expression of the kynurenine pathway genes. Finally, we found that purified KynR specifically bound the kynA promoter in the presence of kynurenine and bound the kynB promoter in the absence or presence of kynurenine. Taken together, our data show that KynR directly regulates the kynurenine pathway genes.

  1. RDM16 and STA1 regulate differential usage of exon/intron in RNA directed DNA methylation pathway.

    PubMed

    Sharma, Ravi Datta; Bogaerts, Bert; Goyal, Neha

    2017-04-20

    The splicing factors RDM16 and STA1 have been reported to play a role in the RNA directed DNA Methylation (RdDM) pathway. In this pathway, small interfering RNAs guide de-novo methylation of homologous DNA sequences. DNA methylation is epigenetic marks, which can suppress transposable elements, repeat sequences and genes. It also affects the chromatin structure. Upon deletion of RDM16 and STA1, previous studies based on gene level analysis were unable to find differentially spliced events implicated in RdDM pathway. In this study, RNA-seq data from RDM16 &STA1 mutants were analyzed. Differential expression analysis was performed at exon and intron level. This analysis revealed 3474 genes with potential differential expression events in the RDM16 mutant and 3058 in the STA1 mutant. We found 17 genes that have been reported to be involved in the RdDM pathway. These results suggest involvement of RDM16 and STA1 by influencing the expression of key gene components such as MORC6 in the RdDM pathway. Therefore, this study increases the understanding of the role of RDM16 and STA1 splicing factors in DNA methylation.

  2. Directed random walks and constraint programming reveal active pathways in hepatocyte growth factor signaling.

    PubMed

    Kittas, Aristotelis; Delobelle, Aurélien; Schmitt, Sabrina; Breuhahn, Kai; Guziolowski, Carito; Grabe, Niels

    2016-01-01

    An effective means to analyze mRNA expression data is to take advantage of established knowledge from pathway databases, using methods such as pathway-enrichment analyses. However, pathway databases are not case-specific and expression data could be used to infer gene-regulation patterns in the context of specific pathways. In addition, canonical pathways may not always describe the signaling mechanisms properly, because interactions can frequently occur between genes in different pathways. Relatively few methods have been proposed to date for generating and analyzing such networks, preserving the causality between gene interactions and reasoning over the qualitative logic of regulatory effects. We present an algorithm (MCWalk) integrated with a logic programming approach, to discover subgraphs in large-scale signaling networks by random walks in a fully automated pipeline. As an exemplary application, we uncover the signal transduction mechanisms in a gene interaction network describing hepatocyte growth factor-stimulated cell migration and proliferation from gene-expression measured with microarray and RT-qPCR using in-house perturbation experiments in a keratinocyte-fibroblast co-culture. The resulting subgraphs illustrate possible associations of hepatocyte growth factor receptor c-Met nodes, differentially expressed genes and cellular states. Using perturbation experiments and Answer Set programming, we are able to select those which are more consistent with the experimental data. We discover key regulator nodes by measuring the frequency with which they are traversed when connecting signaling between receptors and significantly regulated genes and predict their expression-shift consistently with the measured data. The Java implementation of MCWalk is publicly available under the MIT license at: https://bitbucket.org/akittas/biosubg.

  3. Characterizing Cell Mechanics with AFM and Microfluidics

    NASA Astrophysics Data System (ADS)

    Walter, N.; Micoulet, A.; Suresh, S.; Spatz, J. P.

    2007-03-01

    Cell mechanical properties and functionality are mainly determined by the cytoskeleton, besides the cell membrane, the nucleus and the cytosol, and depend on various parameters e.g. surface chemistry and rigidity, surface area and time available for cell spreading, nutrients and drugs provided in the culture medium. Human epithelial pancreatic and mammary cancer cells and their keratin intermediate filaments are the main focus of our work. We use Atomic Force Microscopy (AFM) to study cells adhering to substrates and Microfluidic Channels to probe cells in suspension, respectively. Local and global properties are extracted by varying AFM probe tip size and the available adhesion area for cells. Depth-sensing, instrumented indentation tests with AFM show a clear difference in contact stiffness for cells that are spread of controlled substrates and those that are loosely attached. Microfluidic Channels are utilized in parallel to evaluate cell deformation and ``flow resistance'', which are dependent on channel cross section, flow rate, cell nucleus size and the mechanical properties of cytoskeleton and membrane. The results from the study are used to provide some broad and quantitative assessments of the connections between cellular/subcellular mechanics and biochemical origins of disease states.

  4. Vibration signature analysis of AFM images

    SciTech Connect

    Joshi, G.A.; Fu, J.; Pandit, S.M.

    1995-12-31

    Vibration signature analysis has been commonly used for the machine condition monitoring and the control of errors. However, it has been rarely employed for the analysis of the precision instruments such as an atomic force microscope (AFM). In this work, an AFM was used to collect vibration data from a sample positioning stage under different suspension and support conditions. Certain structural characteristics of the sample positioning stage show up as a result of the vibration signature analysis of the surface height images measured using an AFM. It is important to understand these vibration characteristics in order to reduce vibrational uncertainty, improve the damping and structural design, and to eliminate the imaging imperfections. The choice of method applied for vibration analysis may affect the results. Two methods, the data dependent systems (DDS) analysis and the Welch`s periodogram averaging method were investigated for application to this problem. Both techniques provide smooth spectrum plots from the data. Welch`s periodogram provides a coarse resolution as limited by the number of samples and requires a choice of window to be decided subjectively by the user. The DDS analysis provides sharper spectral peaks at a much higher resolution and a much lower noise floor. A decomposition of the signal variance in terms of the frequencies is provided as well. The technique is based on an objective model adequacy criterion.

  5. Nanomechanics of Yeast Surfaces Revealed by AFM

    NASA Astrophysics Data System (ADS)

    Dague, Etienne; Beaussart, Audrey; Alsteens, David

    Despite the large and well-documented characterization of the microbial cell wall in terms of chemical composition, the determination of the mechanical properties of surface molecules in relation to their function remains a key challenge in cell biology.The emergence of powerful tools allowing molecular manipulations has already revolutionized our understanding of the surface properties of fungal cells. At the frontier between nanophysics and molecular biology, atomic force microscopy (AFM), and more specifically single-molecule force spectroscopy (SMFS), has strongly contributed to our current knowledge of the cell wall organization and nanomechanical properties. However, due to the complexity of the technique, measurements on live cells are still at their infancy.In this chapter, we describe the cell wall composition and recapitulate the principles of AFM as well as the main current methodologies used to perform AFM measurements on live cells, including sample immobilization and tip functionalization.The current status of the progress in probing nanomechanics of the yeast surface is illustrated through three recent breakthrough studies. Determination of the cell wall nanostructure and elasticity is presented through two examples: the mechanical response of mannoproteins from brewing yeasts and elasticity measurements on lacking polysaccharide mutant strains. Additionally, an elegant study on force-induced unfolding and clustering of adhesion proteins located at the cell surface is also presented.

  6. Nanoscale thermal AFM of polymers: transient heat flow effects.

    PubMed

    Duvigneau, Joost; Schönherr, Holger; Vancso, G Julius

    2010-11-23

    Thermal transport around the nanoscale contact area between the heated atomic force microscopy (AFM) probe tip and the specimen under investigation is a central issue in scanning thermal microscopy (SThM). Polarized light microscopy and AFM imaging of the temperature-induced crystallization of poly(ethylene terephthalate) (PET) films in the region near the tip were used in this study to unveil the lateral heat transport. The radius of the observed lateral surface isotherm at 133 °C ranged from 2.2 ± 0.5 to 18.7 ± 0.5 μm for tip-polymer interface temperatures between 200 and 300 °C with contact times varying from 20 to 120 s, respectively. In addition, the heat transport into polymer films was assessed by measurements of the thermal expansion of poly(dimethyl siloxane) (PDMS) films with variable thickness on silicon supports. Our data showed that heat transport in the specimen normal (z) direction occurred to depths exceeding 1000 μm using representative non-steady-state SThM conditions (i.e., heating from 40 to 180 °C at a rate of 10 °C s(-1)). On the basis of the experimental results, a 1D steady-state model for heat transport was developed, which shows the temperature profile close to the tip-polymer contact. The model also indicates that ≤1% of the total power generated in the heater area, which is embedded in the cantilever end, is transported into the polymer through the tip-polymer contact interface. Our results complement recent efforts in the evaluation and improvement of existing theoretical models for thermal AFM, as well as advance further developments of SThM for nanoscale thermal materials characterization and/or manipulation via scanning thermal lithography (SThL).

  7. Starter unit specificity directs genome mining of polyketide synthase pathways in fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Search of the protein database with the aflatoxin pathway polyketide synthase (PKS) revealed putative PKSs in the pathogenic fungi Coccidioides immitis and Coccidioides posadasii that could require partnerships with a pair of fatty acid synthase (FAS) subunits for the biosynthesis of fatty acid-poly...

  8. Diversity in Pathways to Parenthood: Patterns, Implications, and Emerging Research Directions

    ERIC Educational Resources Information Center

    Smock, Pamela J.; Greenland, Fiona Rose

    2010-01-01

    This review examines and synthesizes recent research on pathways to parenthood. We begin by providing basic information about patterns, differentials, and trends and discussing adoption and new reproductive technologies. We next turn to several areas of inquiry that became particularly prominent in the last decade: the continued "decoupling" of…

  9. A direct pathway for the conversion of propionate into pyruvate in Moraxella lwoffi

    PubMed Central

    Hodgson, B.; McGarry, J. D.

    1968-01-01

    ]propionate was chemically degraded. The C(1) of propionate was found only in C(1) of pyruvate. At least 86% of C(2) of pyruvate was derived from C(2) of propionate and at least 92% of C(3) of pyruvate from C(3) of propionate. 8. These results are incompatible with the operation of any of the previously described pathways for propionate metabolism except the direct one, perhaps via an activated acrylate. ImagesPLATE 2PLATE 1 PMID:4296055

  10. Directed evolution of a second xylitol catabolic pathway in Klebsiella pneumoniae.

    PubMed Central

    Doten, R C; Mortlock, R P

    1984-01-01

    Klebsiella pneumoniae PRL-R3 has inducible catabolic pathways for the degradation of ribitol and D-arabitol but cannot utilize xylitol as a growth substrate. A mutation in the rbtB regulatory gene of the ribitol operon permits the constitutive synthesis of the ribitol catabolic enzymes and allows growth on xylitol. The evolved xylitol catabolic pathway consists of an induced D-arabitol permease system that also transports xylitol, a constitutively synthesized ribitol dehydrogenase that oxidizes xylitol at the C-2 position to produce D-xylulose, and an induced D-xylulokinase from either the D-arabitol or D-xylose catabolic pathway. To investigate the potential of K. pneumoniae to evolve a different xylitol catabolic pathway, strains were constructed which were unable to synthesize ribitol dehydrogenase or either type of D-xylulokinase but constitutively synthesized the D-arabitol permease system. These strains had an inducible L-xylulokinase; therefore, the evolution of an enzyme which oxidized xylitol at the C-4 position to L-xylulose would establish a new xylitol catabolic pathway. Four independent xylitol-utilizing mutants were isolated, each of which had evolved a xylitol-4-dehydrogenase activity. The four dehydrogenases appeared to be identical because they comigrated during nondenaturing polyacrylamide gel electrophoresis. This novel xylitol dehydrogenase was constitutively synthesized, whereas L-xylulokinase remained inducible. Transductional analysis showed that the evolved dehydrogenase was not an altered ribitol or D-arabitol dehydrogenase and that the evolved dehydrogenase structural gene was not linked to the pentitol gene cluster. This evolved dehydrogenase had the highest activity with xylitol as a substrate, a Km for xylitol of 1.4 M, and a molecular weight of 43,000. Images PMID:6378891

  11. Direct demonstration of 25- and 50-microm arteriovenous pathways in healthy human and baboon lungs.

    PubMed

    Lovering, Andrew T; Stickland, Michael K; Kelso, Amy J; Eldridge, Marlowe W

    2007-04-01

    Postmortem microsphere studies in adult human lungs have demonstrated the existence of intrapulmonary arteriovenous pathways using nonphysiological conditions. The aim of the current study was to determine whether large diameter (>25 and 50 microm) intrapulmonary arteriovenous pathways are functional in human and baboon lungs under physiological perfusion and ventilation pressures. We used fresh healthy human donor lungs obtained for transplantation and fresh lungs from baboons (Papio c. anubis). Lungs were ventilated with room air by using a peak inflation pressure of 15 cm H(2)O and a positive end-expiratory pressure of 5 cm H(2)O. Lungs were perfused between 10 and 20 cm H(2)O by using a phosphate-buffered saline solution with 5% albumin. We infused a mixture of 25- and 50-microm microspheres (0.5 and 1 million total for baboons and human studies, respectively) into the pulmonary artery and collected the entire pulmonary venous outflow. Under these conditions, evidence of intrapulmonary arteriovenous anastomoses was found in baboon (n = 3/4) and human (n = 4/6) lungs. In those lungs showing evidence of arteriovenous pathways, 50-microm microspheres were always able to traverse the pulmonary circulation, and the fraction of transpulmonary passage ranged from 0.0003 to 0.42%. These data show that intrapulmonary arteriovenous pathways >50 microm in diameter are functional under physiological ventilation and perfusion pressures in the isolated lung. These pathways provide an alternative conduit for pulmonary blood flow that likely bypasses the areas of gas exchange at the capillary-alveolar interface that could compromise both gas exchange and the ability of the lung to filter out microemboli.

  12. Role of Direct vs. Indirect Pathways from the Motor Cortex to Spinal Motoneurons in the Control of Hand Dexterity

    PubMed Central

    Isa, Tadashi; Kinoshita, Masaharu; Nishimura, Yukio

    2013-01-01

    Evolutionally, development of the direct connection from the motor cortex to spinal motoneurons [corticomotoneuronal (CM) pathway] parallels the ability of hand dexterity. Damage to the corticofugal fibers in higher primates resulted in deficit of fractionated digit movements. Based on such observations, it was generally believed that the CM pathway plays a critical role in the control of hand dexterity. On the other hand, a number of “phylogenetically older” indirect pathways from the motor cortex to motoneurons still exist in primates. The indirect pathways are mediated by intercalated neurons such as segmental interneurons (sINs), propriospinal neurons (PNs) reticulospinal neurons (RSNs), or rubrospinal neurons (RuSNs). However, their contribution to hand dexterity remains elusive. Lesion of the brainstem pyramid sparing the transmission through the RuSNs and RSNs, resulted in permanent deficit of fractionated digit movements in macaque monkeys. On the other hand, in our recent study, after lesion of the dorsolateral funiculus (DLF) at the C5 segment, which removed the lateral corticospinal tract (l-CST) including the CM pathway and the transmission through sINs and RuSNs but spared the processing through the PNs and RSNs, fractionated digit movements recovered within several weeks. These results suggest that the PNs can be involved in the recovery of fractionated digit movements, but the RSNs and RuSNs have less capacity in this regard. However, on closer inspection, it was found that the activation pattern of hand and arm muscles considerably changed after the C5 lesion, suggesting limitation of PNs for the compensation of hand dexterity. Altogether, it is suggested that PNs, RSNs RuSNs, and the CM pathway (plus sINs) make a different contribution to the hand dexterity and appearance of motor deficit of the hand dexterity caused by damage to the corticofugal fibers and potential of recovery varies depending on the rostrocaudal level of the lesion. PMID

  13. A novel pathway of direct methane production and emission by eukaryotes including plants, animals and fungi: An overview

    NASA Astrophysics Data System (ADS)

    Liu, Jiangong; Chen, Huai; Zhu, Qiuan; Shen, Yan; Wang, Xue; Wang, Meng; Peng, Changhui

    2015-08-01

    Methane (CH4) is a powerful greenhouse gas with a global warming potential 28 times that of carbon dioxide (CO2). CH4 is responsible for approximately 20% of the Earth's warming since pre-industrial times. Knowledge of the sources of CH4 is crucial due to the recent substantial interannual variability of growth rates and uncertainties regarding individual sources. The prevailing paradigm is that methanogenesis carried out by methanogenic archaea occurs primarily under strictly anaerobic conditions. However, in the past decade, studies have confirmed direct CH4 release from three important kingdoms of eukaryotes-Plantae, Animalia and Fungi-even in the presence of oxygen. This novel CH4 production pathway has been aptly termed "aerobic CH4 production" to distinguish it from the well-known anaerobic CH4 production pathway, which involves catalytic activity by methanogenic archaeal enzymes. In this review, we collated recent experimental evidence from the published literature and documented this novel pathway of direct CH4 production and emission by eukaryotes. The mechanisms involved in this pathway may be related to protective strategies of eukaryotes in response to changing environmental stresses, with CH4 a by-product or end-product during or at the end of the process(es) that originates from organic methyl-type compounds. Based on the existing, albeit uncertain estimates, plants seem to contribute less to the global CH4 budget (3-24%) compared to previous estimates (10-37%). We still lack estimates of CH4 emissions by animals and fungi. Overall, there is an urgent need to identify the precursors for this novel CH4 source and improve our understanding of the mechanisms of direct CH4 production and the impacts of environmental stresses. An estimate of this new CH4 source, which was not considered as a CH4 source by the Intergovernmental Panel on Climate Change (IPCC) (2013), could be useful for better quantitation of the global CH4 budget.

  14. Identification of pathways directly regulated by SHORT VEGETATIVE PHASE during vegetative and reproductive development in Arabidopsis

    PubMed Central

    2013-01-01

    Background MADS-domain transcription factors play important roles during plant development. The Arabidopsis MADS-box gene SHORT VEGETATIVE PHASE (SVP) is a key regulator of two developmental phases. It functions as a repressor of the floral transition during the vegetative phase and later it contributes to the specification of floral meristems. How these distinct activities are conferred by a single transcription factor is unclear, but interactions with other MADS domain proteins which specify binding to different genomic regions is likely one mechanism. Results To compare the genome-wide DNA binding profile of SVP during vegetative and reproductive development we performed ChIP-seq analyses. These ChIP-seq data were combined with tiling array expression analysis, induction experiments and qRT-PCR to identify biologically relevant binding sites. In addition, we compared genome-wide target genes of SVP with those published for the MADS domain transcription factors FLC and AP1, which interact with SVP during the vegetative and reproductive phases, respectively. Conclusions Our analyses resulted in the identification of pathways that are regulated by SVP including those controlling meristem development during vegetative growth and flower development whereas floral transition pathways and hormonal signaling were regulated predominantly during the vegetative phase. Thus, SVP regulates many developmental pathways, some of which are common to both of its developmental roles whereas others are specific to only one of them. PMID:23759218

  15. Advances in the development of cancer therapeutics directed against the RAS-mitogen-activated protein kinase pathway.

    PubMed

    Sebolt-Leopold, Judith S

    2008-06-15

    Among mammalian mitogen-activated protein kinase (MAPK) signaling cascades, the extracellular signal-related kinase (ERK) pathway has received the most attention in the oncology drug discovery arena. By virtue of its central role in promoting proliferation, survival, and metastasis, this pathway directly affects both the formation and progression of human tumors. The identification of non-ATP-competitive inhibitors of the MAPK kinase MAPK/ERK kinase (MEK) resulted in the first demonstration that the ERK pathway could be effectively shut down in a highly selective fashion. Subsequent discovery of the oncogenic nature of B-raf kinase led to the escalation of drug discovery efforts revolving around MEK and RAF. The emergence of multiple drug candidates targeting these downstream kinases provides us with the means for validating the importance of the RAS-RAF-MEK-ERK signaling cascade in human tumors. This article highlights the lessons learned in the clinical evaluation of MAPK pathway inhibitors as anticancer agents and the complexities surrounding optimization of their therapeutic potential in light of the challenges posed by genetic heterogeneity within patient populations.

  16. Direct AFM observations of impurity effects on a lysozyme crystal

    NASA Astrophysics Data System (ADS)

    Nakada, Toshitaka; Sazaki, Gen; Miyashita, Satoru; Durbin, Stephen D.; Komatsu, Hiroshi

    1999-01-01

    Impurity effects on the growth of tetragonal lysozyme crystals have been studied using in situ atomic force microscopy. Commercially available hen egg white lysozyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining, and purified by re-crystallization and successive high pressure liquid chromatography. On the (1 1 0) crystal surface, there was no significant difference in morphology between crystals grown in commercial and in purified solutions. On the (1 0 1) surface, however, a large number of small particles were found when the crystal was grown in the commercial solution, while the surface grown in the purified solution was quite smooth. Among the typical residual impurities contained in commercial lysozyme, only covalently bound lysozyme dimer yielded such particles. From measurements of particle separation and an estimate of the critical nucleation size, we infer that the particles reduced the step velocity according to the mechanism described by Cabrera et al. [N. Cabrera, D.A. Vermilyea, in: R.H. Doremus et al. (Eds.), Growth and Perfection of Crystals, 1958, P. 393].

  17. Direct accumulation pathway of radioactive cesium to fruit-bodies of edible mushroom from contaminated wood logs

    PubMed Central

    Ohnuki, Toshihiko; Aiba, Yukitoshi; Sakamoto, Fuminori; Kozai, Naofumi; Niizato, Tadafumi; Sasaki, Yoshito

    2016-01-01

    This paper presents the accumulation process of radioactive Cs in edible mushrooms. We here first report the direct accumulation pathway of radioactive Cs from contaminated wood logs to the fruit-bodies of shiitake mushrooms through the basal portion of the stipe. In this pathway, radioactive Cs is not transported through the hyphae. This pathway results in a high accumulation of radioactive Cs in the fruit-body, more by the excess accumulation of radioactive Cs from the wood logs than that through the hyphae. We grew the fruit-bodies of Shiitake mushroom from radioactive-Cs-contaminated wood logs. The spatial distributions of radioactive Cs and Prussian blue as a tracer of interstitial water in the cross section of the wood log measured after the harvest of the fruit-body from the inoculated sawdust spawn area indicated that some fraction of the radioactive Cs and Prussian blue were transported directly to the basal portion of the stipe during the growth of the fruit-bodies. PMID:27430163

  18. Direct accumulation pathway of radioactive cesium to fruit-bodies of edible mushroom from contaminated wood logs

    NASA Astrophysics Data System (ADS)

    Ohnuki, Toshihiko; Aiba, Yukitoshi; Sakamoto, Fuminori; Kozai, Naofumi; Niizato, Tadafumi; Sasaki, Yoshito

    2016-07-01

    This paper presents the accumulation process of radioactive Cs in edible mushrooms. We here first report the direct accumulation pathway of radioactive Cs from contaminated wood logs to the fruit-bodies of shiitake mushrooms through the basal portion of the stipe. In this pathway, radioactive Cs is not transported through the hyphae. This pathway results in a high accumulation of radioactive Cs in the fruit-body, more by the excess accumulation of radioactive Cs from the wood logs than that through the hyphae. We grew the fruit-bodies of Shiitake mushroom from radioactive-Cs-contaminated wood logs. The spatial distributions of radioactive Cs and Prussian blue as a tracer of interstitial water in the cross section of the wood log measured after the harvest of the fruit-body from the inoculated sawdust spawn area indicated that some fraction of the radioactive Cs and Prussian blue were transported directly to the basal portion of the stipe during the growth of the fruit-bodies.

  19. Drug uptake pathways of multidrug transporter AcrB studied by molecular simulations and site-directed mutagenesis experiments.

    PubMed

    Yao, Xin-Qiu; Kimura, Nobuhiro; Murakami, Satoshi; Takada, Shoji

    2013-05-22

    Multidrug resistance has been a critical issue in current chemotherapy. In Escherichia coli , a major efflux pump responsible for the multidrug resistance contains a transporter AcrB. Crystallographic studies and mutational assays of AcrB provided much of structural and overall functional insights, which led to the functionally rotating mechanism. However, the drug uptake pathways are somewhat controversial because at least two possible pathways, the vestibule and the cleft paths, were suggested. Here, combining molecular simulations and site-directed mutagenesis experiments, we addressed the uptake mechanism finding that the drug uptake pathways can be significantly different depending on the properties of drugs. First, in the computational free energy analysis of drug movements along AcrB tunnels, we found a ligand-dependent drug uptake mechanism. With the same molecular sizes, drugs that are both strongly hydrophobic and lipophilic were preferentially taken in via the vestibule path, while other drugs favored the cleft path. Second, direct simulations realized totally about 3500 events of drug uptake by AcrB for a broad range of drug property. These simulations confirmed the ligand-dependent drug uptake and further suggested that a smaller drug favors the vestibule path, while a larger one is taken in via the cleft path. Moreover, the direct simulations identified an alternative uptake path which is not visible in the crystal structure. Third, site-directed mutagenesis of AcrB in E. coli verified that mutations of residues located along the newly identified path significantly reduced the efflux efficiency, supporting its relevance in in vivo function.

  20. Resveratrol directly targets DDX5 resulting in suppression of the mTORC1 pathway in prostate cancer

    PubMed Central

    Taniguchi, T; Iizumi, Y; Watanabe, M; Masuda, M; Morita, M; Aono, Y; Toriyama, S; Oishi, M; Goi, W; Sakai, T

    2016-01-01

    Resveratrol has various attractive bioactivities, such as prevention of cancer, neurodegenerative disorders, and obesity-related diseases. Therefore, identifying its direct binding proteins is expected to discover druggable targets. Sirtuin 1 and phosphodiesterases have so far been found as the direct molecular targets of resveratrol. We herein identified 11 novel resveratrol-binding proteins, including the DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5, also known as p68), using resveratrol-immobilized beads. Treatment with resveratrol induced degradation of DDX5 in prostate cancer cells. Depletion of DDX5 caused apoptosis by inhibiting mammalian target of rapamycin complex 1 (mTORC1) signaling. Moreover, knockdown of DDX5 attenuated the inhibitory activities of resveratrol against mTORC1 signaling and cancer cell growth. These data show that resveratrol directly targets DDX5 and induces cancer cell death by inhibiting the mTORC1 pathway. PMID:27148684

  1. BOREAS AFM-6 Surface Meteorological Data

    NASA Technical Reports Server (NTRS)

    Wilczak, James; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-6 team from the National Oceanic and Atmospheric Adminsitration/Environment Technology Laboratory (NOAA/ETL) collected surface meteorological data from 21 May to 20 Sep 1994 near the Southern Study Area-Old Jack Pine (SSA-OJP) tower site. The data are in tabular ASCII files. The surface meteorological data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  2. Direct, two-step synthetic pathway to novel dibenzo[a,c]phenanthridines.

    PubMed

    Churruca, Fátima; SanMartin, Raul; Carril, Mónica; Urtiaga, Miren Karmele; Solans, Xavier; Tellitu, Imanol; Domínguez, Esther

    2005-04-15

    Novel dibenzo[a,c]phenanthridines are prepared regioselectively by the application of a straightforward synthetic pathway, starting from new 3,4-diaryl- and 3,4-dihydro-3,4-diarylisoquinolines prepared via Ritter-type heterocyclization and the more classical two-step reductive amination/Bischler-Napieralski cyclization of triarylethanones, respectively. A comparative study of nonphenolic oxidative coupling methodologies provides a highly efficient procedure, based on the hypervalent iodine reagent phenyliodine(III) bis(trifluoroacetate) (PIFA), to accomplish the final coupling step.

  3. Eukaryotic Chemotaxis: A Network of Signaling Pathways Controls Motility, Directional Sensing, and Polarity

    PubMed Central

    Swaney, Kristen F.; Huang, Chuan-Hsiang; Devreotes, Peter N.

    2015-01-01

    Chemotaxis, the directed migration of cells in chemical gradients, is a vital process in normal physiology and in the pathogenesis of many diseases. Chemotactic cells display motility, directional sensing, and polarity. Motility refers to the random extension of pseudopodia, which may be driven by spontaneous actin waves that propagate through the cytoskeleton. Directional sensing is mediated by a system that detects temporal and spatial stimuli and biases motility toward the gradient. Polarity gives cells morphologically and functionally distinct leading and lagging edges by relocating proteins or their activities selectively to the poles. By exploiting the genetic advantages of Dictyostelium, investigators are working out the complex network of interactions between the proteins that have been implicated in the chemotactic processes of motility, directional sensing, and polarity. PMID:20192768

  4. Eukaryotic chemotaxis: a network of signaling pathways controls motility, directional sensing, and polarity.

    PubMed

    Swaney, Kristen F; Huang, Chuan-Hsiang; Devreotes, Peter N

    2010-01-01

    Chemotaxis, the directed migration of cells in chemical gradients, is a vital process in normal physiology and in the pathogenesis of many diseases. Chemotactic cells display motility, directional sensing, and polarity. Motility refers to the random extension of pseudopodia, which may be driven by spontaneous actin waves that propagate through the cytoskeleton. Directional sensing is mediated by a system that detects temporal and spatial stimuli and biases motility toward the gradient. Polarity gives cells morphologically and functionally distinct leading and lagging edges by relocating proteins or their activities selectively to the poles. By exploiting the genetic advantages of Dictyostelium, investigators are working out the complex network of interactions between the proteins that have been implicated in the chemotactic processes of motility, directional sensing, and polarity.

  5. AFM1 in Milk: Physical, Biological, and Prophylactic Methods to Mitigate Contamination

    PubMed Central

    Giovati, Laura; Magliani, Walter; Ciociola, Tecla; Santinoli, Claudia; Conti, Stefania; Polonelli, Luciano

    2015-01-01

    Aflatoxins (AFs) are toxic, carcinogenic, immunosuppressive secondary metabolites produced by some Aspergillus species which colonize crops, including many dietary staple foods and feed components. AFB1 is the prevalent and most toxic among AFs. In the liver, it is biotransformed into AFM1, which is then excreted into the milk of lactating mammals, including dairy animals. AFM1 has been shown to be cause of both acute and chronic toxicoses. The presence of AFM1 in milk and dairy products represents a worldwide concern since even small amounts of this metabolite may be of importance as long-term exposure is concerned. Contamination of milk may be mitigated either directly, decreasing the AFM1 content in contaminated milk, or indirectly, decreasing AFB1 contamination in the feed of dairy animals. Current strategies for AFM1 mitigation include good agricultural practices in pre-harvest and post-harvest management of feed crops (including storage) and physical or chemical decontamination of feed and milk. However, no single strategy offers a complete solution to the issue. PMID:26512694

  6. In situ nanomanipulators as a tool to separate individual tobermorite crystals for AFM studies.

    PubMed

    Yang, Tianhe; Holzer, Lorenz; Kägi, Ralf; Winnefeld, Frank; Keller, Bruno

    2007-10-01

    Atomic force microscopy (AFM) studies of cementitious materials are limited, mainly due to the lack of appropriate sample preparation techniques. In porous autoclaved aerated concrete (AAC), calcium silicate hydrate (C-S-H) is produced in its crystalline form, tobermorite. The crystals are lath-like with a length of several micrometers. In this work, we demonstrate the application of nanomanipulators to separate an individual tobermorite crystal from the bulk AAC for subsequent AFM investigations. The nanomanipulators are operated directly in an environmental scanning electron microscope (ESEM). We studied the interaction between moisture and the tobermorite surface under controlled relative humidity (RH). The results of topography and adhesion force measurements with AFM suggest that the surface of tobermorite is hydrophobic, which contrasts the macroscopic material properties (e.g. moisture transport in capillary pores).

  7. New Direction in Hydrogeochemical Transport Modeling: Incorporating Multiple Kinetic and Equilibrium Reaction Pathways

    SciTech Connect

    Steefel, C.I.

    2000-02-02

    At least two distinct kinds of hydrogeochemical models have evolved historically for use in analyzing contaminant transport, but each has important limitations. One kind, focusing on organic contaminants, treats biodegradation reactions as parts of relatively simple kinetic reaction networks with no or limited coupling to aqueous and surface complexation and mineral dissolution/precipitation reactions. A second kind, evolving out of the speciation and reaction path codes, is capable of handling a comprehensive suite of multicomponent complexation (aqueous and surface) and mineral precipitation and dissolution reactions, but has not been able to treat reaction networks characterized by partial redox disequilibrium and multiple kinetic pathways. More recently, various investigators have begun to consider biodegradation reactions in the context of comprehensive equilibrium and kinetic reaction networks (e.g. Hunter et al. 1998, Mayer 1999). Here we explore two examples of multiple equilibrium and kinetic reaction pathways using the reactive transport code GIMRT98 (Steefel, in prep.): (1) a computational example involving the generation of acid mine drainage due to oxidation of pyrite, and (2) a computational/field example where the rates of chlorinated VOC degradation are linked to the rates of major redox processes occurring in organic-rich wetland sediments overlying a contaminated aerobic aquifer.

  8. Role of the direct and indirect pathways for glycogen synthesis in rat liver in the postprandial state

    SciTech Connect

    Huang, M.T.; Veech, R.L.

    1988-03-01

    The pathway for hepatic glycogen synthesis in the postprandial state was studied in meal-fed rats chronically cannulated in the portal vein. Plasma glucose concentration in the portal vein was found to be 4.50 +/- 1.01 mM (mean +/- SE; n = 3) before a meal and 11.54 +/- 0.70 mM (mean +/- SE; n = 4) after a meal in rats meal-fed a diet consisting of 100% commercial rat chow for 7 d. The hepatic-portal difference of plasma glucose concentration showed that liver released glucose in the fasted state and either extracted or released glucose after feeding depending on plasma glucose concentration in the portal vein. The concentration of portal vein glucose at which liver changes from glucose releasing to glucose uptake was 8 mM, the Km of glucokinase. The rate of glycogen synthesis in liver during meal-feeding was found to be approximately 1 mumol glucosyl U/g wet wt/min in rats meal-fed a 50% glucose supplemented chow diet. The relative importance of the direct vs. indirect pathway for the replenishment of hepatic glycogen was determined by the incorporation of (3-/sup 3/H,U-/sup 14/C)glucose into liver glycogen. Labeled glucose was injected into the portal vein at the end of meal-feeding. The ratio of /sup 3/H//sup 14/C in the glucosyl units of glycogen was found to be 83-92% of the ratio in liver free glucose six minutes after the injection, indicating that the majority of exogenous glucose incorporated into glycogen did not go through glycolysis. The percent contribution of the direct versus indirect pathway was quantitated from the difference in the relative specific activity (RSA) of (/sup 3/H) and (/sup 14/C)-glycogen in rats infused with (3-/sup 3/H,U-/sup 14/C)glucose. No significant difference was found between the RSA of (/sup 3/H)glycogen and (/sup 14/C)glycogen, indicating further that the pathway for glycogen synthesis in liver from exogenous glucose is from the direct pathway.

  9. Direct contact between dust and HBCD-treated fabrics is an important pathway of source-to-dust transfer.

    PubMed

    Rauert, Cassandra; Kuribara, Isamu; Kataoka, Toshiyuki; Wada, Takeharu; Kajiwara, Natsuko; Suzuki, Go; Takigami, Hidetaka; Harrad, Stuart

    2016-03-01

    Hexabromocyclododecanes (HBCDs) are a class of brominated flame retardant that have found extensive application in consumer products used widely in indoor environments. Although uncertainty remains about the human health impacts of HBCDs, ingestion of HBCD-contaminated indoor dust has been shown to be a particularly significant exposure pathway for young children. Despite this, understanding of the mechanisms via which HBCD transfer from products to indoor dust remains incomplete. In this study, an in-house test chamber was used to investigate transfer of HBCDs from a treated textile sample to indoor dust via direct textile:dust contact. Results were compared with previous data using the same test chamber to examine other pathways via which HBCDs transfer from products to dust, and highlighted HBCD transfer via direct source:dust contact as being particularly important. This novel finding was corroborated by complementary experiments that examined HBCD transfer via direct contact, from other treated textiles to three major components of indoor dust: artificial indoor dust, soil particles, and cotton linters.

  10. The effects of cervical transcutaneous spinal direct current stimulation on motor pathways supplying the upper limb in humans

    PubMed Central

    D’Amico, Jessica M.; Butler, Jane E.; Taylor, Janet L.

    2017-01-01

    Non-invasive, weak direct current stimulation can induce changes in excitability of underlying neural tissue. Many studies have used transcranial direct current stimulation to induce changes in the brain, however more recently a number of studies have used transcutaneous spinal direct current stimulation to induce changes in the spinal cord. This study further characterises the effects following cervical transcutaneous spinal direct current stimulation on motor pathways supplying the upper limb. In Study 1, on two separate days, participants (n = 12, 5 F) received 20 minutes of either real or sham direct current stimulation at 3 mA through electrodes placed in an anterior-posterior configuration over the neck (anode anterior). Biceps brachii, flexor carpi radialis and first dorsal interosseous responses to transcranial magnetic stimulation (motor evoked potentials) and cervicomedullary stimulation (cervicomedullary motor evoked potentials) were measured before and after real or sham stimulation. In Study 2, on two separate days, participants (n = 12, 7 F) received either real or sham direct current stimulation in the same way as for Study 1. Before and after real or sham stimulation, median nerve stimulation elicited M waves and H reflexes in the flexor carpi radialis. H-reflex recruitment curves and homosynaptic depression of the H reflex were assessed. Results show that the effects of real and sham direct current stimulation did not differ for motor evoked potentials or cervicomedullary motor evoked potentials for any muscle, nor for H-reflex recruitment curve parameters or homosynaptic depression. Cervical transcutaneous spinal direct current stimulation with the parameters described here does not modify motor responses to corticospinal stimulation nor does it modify H reflexes of the upper limb. These results are important for the emerging field of transcutaneous spinal direct current stimulation. PMID:28225813

  11. The effects of cervical transcutaneous spinal direct current stimulation on motor pathways supplying the upper limb in humans.

    PubMed

    Dongés, Siobhan C; D'Amico, Jessica M; Butler, Jane E; Taylor, Janet L

    2017-01-01

    Non-invasive, weak direct current stimulation can induce changes in excitability of underlying neural tissue. Many studies have used transcranial direct current stimulation to induce changes in the brain, however more recently a number of studies have used transcutaneous spinal direct current stimulation to induce changes in the spinal cord. This study further characterises the effects following cervical transcutaneous spinal direct current stimulation on motor pathways supplying the upper limb. In Study 1, on two separate days, participants (n = 12, 5 F) received 20 minutes of either real or sham direct current stimulation at 3 mA through electrodes placed in an anterior-posterior configuration over the neck (anode anterior). Biceps brachii, flexor carpi radialis and first dorsal interosseous responses to transcranial magnetic stimulation (motor evoked potentials) and cervicomedullary stimulation (cervicomedullary motor evoked potentials) were measured before and after real or sham stimulation. In Study 2, on two separate days, participants (n = 12, 7 F) received either real or sham direct current stimulation in the same way as for Study 1. Before and after real or sham stimulation, median nerve stimulation elicited M waves and H reflexes in the flexor carpi radialis. H-reflex recruitment curves and homosynaptic depression of the H reflex were assessed. Results show that the effects of real and sham direct current stimulation did not differ for motor evoked potentials or cervicomedullary motor evoked potentials for any muscle, nor for H-reflex recruitment curve parameters or homosynaptic depression. Cervical transcutaneous spinal direct current stimulation with the parameters described here does not modify motor responses to corticospinal stimulation nor does it modify H reflexes of the upper limb. These results are important for the emerging field of transcutaneous spinal direct current stimulation.

  12. Blood vessel endothelium-directed tumor cell streaming in breast tumors requires the HGF/C-Met signaling pathway.

    PubMed

    Leung, E; Xue, A; Wang, Y; Rougerie, P; Sharma, V P; Eddy, R; Cox, D; Condeelis, J

    2016-11-28

    During metastasis to distant sites, tumor cells migrate to blood vessels. In vivo, breast tumor cells utilize a specialized mode of migration known as streaming, where a linear assembly of tumor cells migrate directionally towards blood vessels on fibronectin-collagen I-containing extracellular matrix (ECM) fibers in response to chemotactic signals. We have successfully reconstructed tumor cell streaming in vitro by co-plating tumors cells, macrophages and endothelial cells on 2.5 μm thick ECM-coated micro-patterned substrates. We found that tumor cells and macrophages, when plated together on the micro-patterned substrates, do not demonstrate sustained directional migration in only one direction (sustained directionality) but show random bi-directional walking. Sustained directionality of tumor cells as seen in vivo was established in vitro when beads coated with human umbilical vein endothelial cells were placed at one end of the micro-patterned 'ECM fibers' within the assay. We demonstrated that these endothelial cells supply the hepatocyte growth factor (HGF) required for the chemotactic gradient responsible for sustained directionality. Using this in vitro reconstituted streaming system, we found that directional streaming is dependent on, and most effectively blocked, by inhibiting the HGF/C-Met signaling pathway between endothelial cells and tumor cells. Key observations made with the in vitro reconstituted system implicating C-Met signaling were confirmed in vivo in mammary tumors using the in vivo invasion assay and intravital multiphoton imaging of tumor cell streaming. These results establish HGF/C-Met as a central organizing signal in blood vessel-directed tumor cell migration in vivo and highlight a promising role for C-Met inhibitors in blocking tumor cell streaming and metastasis in vivo, and for use in human trials.Oncogene advance online publication, 28 November 2016; doi:10.1038/onc.2016.421.

  13. Probing ternary solvent effect in high Voc polymer solar cells using advanced AFM techniques

    SciTech Connect

    Li, Chao; Soleman, Mikhael; Lorenzo, Josie; Dhasmana, Nitesh; Chantharasupawong, Panit; Ievlev, Anton; Gesquiere, Andre; Tetard, Laurene; Thomas, Jayan

    2016-01-25

    This work describes a simple method to develop a high Voc low band gap PSCs. In addition, two new atomic force microscopy (AFM)-based nanoscale characterization techniques to study the surface morphology and physical properties of the structured active layer are introduced. With the help of ternary solvent processing of the active layer and C60 buffer layer, a bulk heterojunction PSC with Voc more than 0.9 V and conversion efficiency 7.5% is developed. In order to understand the fundamental properties of the materials ruling the performance of the PSCs tested, AFM-based nanoscale characterization techniques including Pulsed-Force-Mode AFM (PFM-AFM) and Mode-Synthesizing AFM (MSAFM) are introduced. Interestingly, MSAFM exhibits high sensitivity for direct visualization of the donor–acceptor phases in the active layer of the PSCs. Lastly, conductive-AFM (cAFM) studies reveal local variations in conductivity in the donor and acceptor phases as well as a significant increase in photocurrent in the PTB7:ICBA sample obtained with the ternary solvent processing.

  14. Bypassing primary sensory cortices--a direct thalamocortical pathway for transmitting salient sensory information.

    PubMed

    Liang, M; Mouraux, A; Iannetti, G D

    2013-01-01

    Detection and appropriate reaction to sudden and intense events happening in the sensory environment is crucial for survival. By combining Bayesian model selection with dynamic causal modeling of functional magnetic resonance imaging data, a novel analysis approach that allows inferring the causality between neural activities in different brain areas, we demonstrate that salient sensory information reaches the multimodal cortical areas responsible for its detection directly from the thalamus, without being first processed in primary and secondary sensory-specific areas. This direct thalamocortical transmission of multimodal salient information is parallel to the processing of finer stimulus attributes, which are transmitted in a modality-specific fashion from the thalamus to the relevant primary sensory areas. Such direct thalamocortical connections bypassing primary sensory cortices provide a fast and efficient way for transmitting information from subcortical structures to multimodal cortical areas, to allow the early detection of salient events and, thereby, trigger immediate and appropriate behavior.

  15. Lack of a primary physicochemical determinant in the direct transport of drugs to the brain after nasal administration in rats: potential involvement of transporters in the pathway.

    PubMed

    Lee, Kyeong-Ryoon; Maeng, Han-Joo; Chae, Jung-Byung; Chong, Saeho; Kim, Dae-Duk; Shim, Chang-Koo; Chung, Suk-Jae

    2010-01-01

    The objectives of this study were to evaluate the relative contribution of the direct pathway in overall brain transport for 17 model drugs with different physicochemical properties after nasal administrations and to identify factors that govern the fraction of the dose transported to the brain via the direct pathway (F(a, direct)). When the model drugs were nasally administered to rats, 5 of the 17 model drugs were delivered to a significant extent to the brain via the direct pathway. Multiple linear regression analyses showed that the correlation between various physicochemical properties and F(a, direct) was not statistically significant, indicative of a lack of primary physicochemical determinants in the direct transport pathway. Transporters such as rOAT3 and rOCT2 were expressed at significant levels in rat olfactory epithelia, and uptakes of standard substrates were significantly decreased in HEK293 cells expressing rOAT3 and rOCT2 in the presence of the five model drugs that were delivered to appreciable extents to the brain via the direct pathway. Therefore, these observations indicate that carrier-mediated transport may play a role in the brain delivery of drugs from the nose via the direct transport pathway.

  16. Device level 3D characterization using PeakForce AFM

    NASA Astrophysics Data System (ADS)

    Timoney, Padraig; Zhang, Xiaoxiao; Vaid, Alok; Hand, Sean; Osborne, Jason; Milligan, Eric; Feinstein, Adam

    2016-03-01

    Traditional metrology solutions face a range of challenges at the 1X node such as three dimensional (3D) measurement capabilities, shrinking overlay and critical dimension (CD) error budgets driven by multi-patterning and via in trench CD measurements. With advent of advanced technology nodes and 3D processing, an increasing need is emerging for in-die metrology including across-structure and structure-to-structure characterization. A myriad of work has emerged in the past few years intending to address these challenges from various aspects; in-die OCD with reduced spot size and tilt beam on traditional critical dimension scanning electron microscopy (CDSEM) for height measurements. This paper explores the latest capability offered by PeakForceTM Tapping Atomic Force Microscopy (PFT-AFM). The use of traditional harmonic tapping mode for scanning high aspect ratio, and complex "3D" wafer structures, results in limited depth probing capability as well as excessive tip wear. These limitations arise due to the large tip-sample interaction volume in such confined spaces. PeakForce Tapping eliminates these limitations through direct real time control of the tip-sample interaction contact force. The ability of PeakForce to measure, and respond directly to tip- sample interaction forces results in more detailed feature resolution, reduced tip wear, and improved depth capability. In this work, the PFT-AFM tool was applied for multiple applications, including the 14nm fin and replacement metal gate (RMG) applications outlined below. Results from DOE wafers, detailed measurement precision studies and correlation to reference metrology are presented for validation of this methodology. With the fin application, precision of 0.3nm is demonstrated by measuring 5 dies with 10 consecutive runs. Capability to resolve within-die and localized within-macro height variation is also demonstrated. Results obtained from the fin measurements support the increasing trend that measurements

  17. BOREAS AFM-04 Twin Otter Aircraft Sounding Data

    NASA Technical Reports Server (NTRS)

    MacPherson, J. Ian; Desjardins, Raymond L.; Hall, Forrest G. (Editor); Knapp, David E. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-4 team used the National Research Council, Canada (NRC) Twin Otter aircraft to make sounding measurements through the boundary layer. These measurements included concentrations of carbon dioxide and ozone, atmospheric pressure, dry bulb temperature, potential temperature, dewpoint temperature, calculated mixing ratio, and wind speed and direction. Aircraft position, heading, and altitude were also recorded. Data were collected at both the Northern Study Area (NSA) and the Southern Study Area (SSA) in 1994 and 1996. These data are stored in tabular ASCII files. The Twin Otter aircraft sounding data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files also are available on a CD-ROM (see document number 20010000884).

  18. Pathways to Adult Sexual Revictimization: Direct and Indirect Behavioral Risk Factors across the Lifespan

    ERIC Educational Resources Information Center

    Fargo, Jamison D.

    2009-01-01

    The purpose of this study is to investigate direct and indirect social and behavioral risk factors for adult sexual revictimization. Participants include 147 adult, predominantly African American (88%) women, 59% of whom had a documented history of child sexual abuse. Participants are interviewed in adulthood about adolescent and adult sexual…

  19. Constrained spherical deconvolution analysis of the limbic network in human, with emphasis on a direct cerebello-limbic pathway

    PubMed Central

    Arrigo, Alessandro; Mormina, Enricomaria; Anastasi, Giuseppe Pio; Gaeta, Michele; Calamuneri, Alessandro; Quartarone, Angelo; De Salvo, Simona; Bruschetta, Daniele; Rizzo, Giuseppina; Trimarchi, Fabio; Milardi, Demetrio

    2014-01-01

    The limbic system is part of an intricate network which is involved in several functions like memory and emotion. Traditionally the role of the cerebellum was considered mainly associated to motion control; however several evidences are raising about a role of the cerebellum in learning skills, emotions control, mnemonic and behavioral processes involving also connections with limbic system. In 15 normal subjects we studied limbic connections by probabilistic Constrained Spherical Deconvolution (CSD) tractography. The main result of our work was to prove for the first time in human brain the existence of a direct cerebello-limbic pathway which was previously hypothesized but never demonstrated. We also extended our analysis to the other limbic connections including cingulate fasciculus, inferior longitudinal fasciculus, uncinated fasciculus, anterior thalamic connections and fornix. Although these pathways have been already described in the tractographic literature we provided reconstruction, quantitative analysis and Fractional Anisotropy (FA) right-left symmetry comparison using probabilistic CSD tractography that is known to provide a potential improvement compared to previously used Diffusion Tensor Imaging (DTI) techniques. The demonstration of the existence of cerebello-limbic pathway could constitute an important step in the knowledge of the anatomic substrate of non-motor cerebellar functions. Finally the CSD statistical data about limbic connections in healthy subjects could be potentially useful in the diagnosis of pathological disorders damaging this system. PMID:25538606

  20. Electrocatalytic oxygen reduction reaction on perovskite oxides: series versus direct pathway.

    PubMed

    Poux, Tiphaine; Bonnefont, Antoine; Kéranguéven, Gwénaëlle; Tsirlina, Galina A; Savinova, Elena R

    2014-07-21

    The mechanism of the oxygen reduction reaction (ORR) on LaCoO(3) and La(0.8)Sr(0.2)MnO(3) perovskite oxides is studied in 1 M NaOH by using the rotating ring disc electrode (RRDE) method. By combining experimental studies with kinetic modeling, it was demonstrated that on perovskite, as well as on perovskite/carbon electrodes, the ORR follows a series pathway through the intermediate formation of hydrogen peroxide. The escape of this intermediate from the electrode strongly depends on: 1) The loading of perovskite; high loadings lead to an overall 4 e(-) oxygen reduction due to efficient hydrogen peroxide re-adsorption on the active sites and its further reduction. 2) The addition of carbon to the catalytic layer, which affects both the utilization of the perovskite surface and the production of hydrogen peroxide. 3) The type of oxide; La(0.8)Sr(0.2)MnO(3) displays higher (compared to LaCoO(3)) activity in the reduction of oxygen to hydrogen peroxide and in the reduction/oxidation of the latter.

  1. Mechanisms of direct inhibition of the respiratory sulfate-reduction pathway by (per)chlorate and nitrate

    PubMed Central

    Carlson, Hans K; Kuehl, Jennifer V; Hazra, Amrita B; Justice, Nicholas B; Stoeva, Magdalena K; Sczesnak, Andrew; Mullan, Mark R; Iavarone, Anthony T; Engelbrektson, Anna; Price, Morgan N; Deutschbauer, Adam M; Arkin, Adam P; Coates, John D

    2015-01-01

    We investigated perchlorate (ClO4−) and chlorate (ClO3−) (collectively (per)chlorate) in comparison with nitrate as potential inhibitors of sulfide (H2S) production by mesophilic sulfate-reducing microorganisms (SRMs). We demonstrate the specificity and potency of (per)chlorate as direct SRM inhibitors in both pure cultures and undefined sulfidogenic communities. We demonstrate that (per)chlorate and nitrate are antagonistic inhibitors and resistance is cross-inducible implying that these compounds share at least one common mechanism of resistance. Using tagged-transposon pools we identified genes responsible for sensitivity and resistance in Desulfovibrio alaskensis G20. We found that mutants in Dde_2702 (Rex), a repressor of the central sulfate-reduction pathway were resistant to both (per)chlorate and nitrate. In general, Rex derepresses its regulon in response to increasing intracellular NADH:NAD+ ratios. In cells in which respiratory sulfate reduction is inhibited, NADH:NAD+ ratios should increase leading to derepression of the sulfate-reduction pathway. In support of this, in (per)chlorate or nitrate-stressed wild-type G20 we observed higher NADH:NAD+ ratios, increased transcripts and increased peptide counts for genes in the core Rex regulon. We conclude that one mode of (per)chlorate and nitrate toxicity is as direct inhibitors of the central sulfate-reduction pathway. Our results demonstrate that (per)chlorate are more potent inhibitors than nitrate in both pure cultures and communities, implying that they represent an attractive alternative for controlling sulfidogenesis in industrial ecosystems. Of these, perchlorate offers better application logistics because of its inhibitory potency, solubility, relative chemical stability, low affinity for mineral cations and high mobility in environmental systems. PMID:25405978

  2. Mechanisms of direct inhibition of the respiratory sulfate-reduction pathway by (per)chlorate and nitrate.

    PubMed

    Carlson, Hans K; Kuehl, Jennifer V; Hazra, Amrita B; Justice, Nicholas B; Stoeva, Magdalena K; Sczesnak, Andrew; Mullan, Mark R; Iavarone, Anthony T; Engelbrektson, Anna; Price, Morgan N; Deutschbauer, Adam M; Arkin, Adam P; Coates, John D

    2015-06-01

    We investigated perchlorate (ClO(4)(-)) and chlorate (ClO(3)(-)) (collectively (per)chlorate) in comparison with nitrate as potential inhibitors of sulfide (H(2)S) production by mesophilic sulfate-reducing microorganisms (SRMs). We demonstrate the specificity and potency of (per)chlorate as direct SRM inhibitors in both pure cultures and undefined sulfidogenic communities. We demonstrate that (per)chlorate and nitrate are antagonistic inhibitors and resistance is cross-inducible implying that these compounds share at least one common mechanism of resistance. Using tagged-transposon pools we identified genes responsible for sensitivity and resistance in Desulfovibrio alaskensis G20. We found that mutants in Dde_2702 (Rex), a repressor of the central sulfate-reduction pathway were resistant to both (per)chlorate and nitrate. In general, Rex derepresses its regulon in response to increasing intracellular NADH:NAD(+) ratios. In cells in which respiratory sulfate reduction is inhibited, NADH:NAD(+) ratios should increase leading to derepression of the sulfate-reduction pathway. In support of this, in (per)chlorate or nitrate-stressed wild-type G20 we observed higher NADH:NAD(+) ratios, increased transcripts and increased peptide counts for genes in the core Rex regulon. We conclude that one mode of (per)chlorate and nitrate toxicity is as direct inhibitors of the central sulfate-reduction pathway. Our results demonstrate that (per)chlorate are more potent inhibitors than nitrate in both pure cultures and communities, implying that they represent an attractive alternative for controlling sulfidogenesis in industrial ecosystems. Of these, perchlorate offers better application logistics because of its inhibitory potency, solubility, relative chemical stability, low affinity for mineral cations and high mobility in environmental systems.

  3. BOREAS AFM-6 Boundary Layer Height Data

    NASA Technical Reports Server (NTRS)

    Wilczak, James; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-6 team from National Oceanic and Atmospheric Adminsitration/Environment Technology Laboratory (NOAA/ETL) operated a 915-MHz wind/Radio Acoustic Sounding System (RASS) profiler system in the Southern Study Area (SSA) near the Old Jack Pine (OJP) site. This data set provides boundary layer height information over the site. The data were collected from 21 May 1994 to 20 Sep 1994 and are stored in tabular ASCII files. The boundary layer height data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  4. BOREAS AFM-06 Mean Temperature Profile Data

    NASA Technical Reports Server (NTRS)

    Wilczak, James; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-6 team from the National Oceanic and Atmospheric Adminsitration/Environment Technology Laboratory (NOAA/ETL) operated a 915-MHz wind/Radio Acoustic Sounding System (RASS) profiler system in the Southern Study Area (SSA) near the Old Jack Pine (OJP) tower from 21 May 1994 to 20 Sep 1994. The data set provides temperature profiles at 15 heights, containing the variables of virtual temperature, vertical velocity, the speed of sound, and w-bar. The data are stored in tabular ASCII files. The mean temperature profile data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  5. Cryogenic AFM-STM for mesoscopic physics

    NASA Astrophysics Data System (ADS)

    Le Sueur, H.

    Electronic spectroscopy based on electron tunneling gives access to the electronic density of states (DOS) in conductive materials, and thus provides detailed information about their electronic properties. During this thesis work, we have developed a microscope in order to perform spatially resolved (10 nm) tunneling spectroscopy, with an unprecedented energy resolution (10 μeV), on individual nanocircuits. This machine combines an Atomic Force Microscope (AFM mode) together with a Scanning Tunneling Spectroscope (STS mode) and functions at very low temperatures (30 mK). In the AFM mode, the sample topography is recorded using a piezoelectric quartz tuning fork, which allows us to locate and image nanocircuits. Tunneling can then be performed on conductive areas of the circuit. With this microscope, we have measured the local DOS in a hybrid Superconductor-Normal metal-Superconductor (S-N-S) structure. In such circuit, the electronic properties of N and S are modified by the superconducting proximity effect. In particular, for short N wires, we have observed a minigap independent of position in the DOS of the N wire, as was previously predicted. Moreover, when varying the superconducting phase difference between the S electrodes, we have measured the modification of the minigap and its disappearance when the phase difference equals π. Our experimental results for the DOS, and its dependences (on phase, position, N length), are quantitatively accounted for by the quasiclassical theory of superconductivity. Some predictions of this theory are observed for the first time. La spectroscopie électronique basée sur l'effet tunnel donne accès à la densité d'états des électrons (DOS) dans les matériaux conducteurs, et renseigne ainsi en détail sur leurs propriétés électroniques. Au cours de cette thèse, nous avons développé un microscope permettant d'effectuer la spectroscopie tunnel résolue spatialement (10 nm) de nanocircuits individuels, avec une r

  6. Different Corticostriatal Integration in Spiny Projection Neurons from Direct and Indirect Pathways

    PubMed Central

    Flores-Barrera, Edén; Vizcarra-Chacón, Bianca J.; Tapia, Dagoberto; Bargas, José; Galarraga, Elvira

    2010-01-01

    The striatum is the principal input structure of the basal ganglia. Major glutamatergic afferents to the striatum come from the cerebral cortex and make monosynaptic contacts with medium spiny projection neurons (MSNs) and interneurons. Also: glutamatergic afferents to the striatum come from the thalamus. Despite differences in axonal projections, dopamine (DA) receptors expression and differences in excitability between MSNs from “direct” and “indirect” basal ganglia pathways, these neuronal classes have been thought as electrophysiologically very similar. Based on work with bacterial artificial chromosome (BAC) transgenic mice, here it is shown that corticostriatal responses in D1- and D2-receptor expressing MSNs (D1- and D2-MSNs) are radically different so as to establish an electrophysiological footprint that readily differentiates between them. Experiments in BAC mice allowed us to predict, with high probability (P > 0.9), in rats or non-BAC mice, whether a recorded neuron, from rat or mouse, was going to be substance P or enkephalin (ENK) immunoreactive. Responses are more prolonged and evoke more action potentials in D1-MSNs, while they are briefer and exhibit intrinsic autoregenerative responses in D2-MSNs. A main cause for these differences was the interaction of intrinsic properties with the inhibitory contribution in each response. Inhibition always depressed corticostriatal depolarization in D2-MSNs, while it helped in sustaining prolonged depolarizations in D1-MSNs, in spite of depressing early discharge. Corticostriatal responses changed dramatically after striatal DA depletion in 6-hydroxy-dopamine (6-OHDA) lesioned animals: a response reduction was seen in substance P (SP)+ MSNs whereas an enhanced response was seen in ENK+ MSNs. The end result was that differences in the responses were greatly diminished after DA depletion. PMID:20589098

  7. Tissue-Specific Whole Transcriptome Sequencing in Castor, Directed at Understanding Triacylglycerol Lipid Biosynthetic Pathways

    PubMed Central

    Swarbreck, David; Febrer, Melanie; Larson, Tony R.; Graham, Ian A.; Caccamo, Mario; Slabas, Antoni R.

    2012-01-01

    Background Storage triacylglycerols in castor bean seeds are enriched in the hydroxylated fatty acid ricinoleate. Extensive tissue-specific RNA-Seq transcriptome and lipid analysis will help identify components important for its biosynthesis. Methodology/Findings Storage triacylglycerols (TAGs) in the endosperm of developing castor (Ricinus communis) seeds are highly enriched in ricinoleic acid (18:1-OH). We have analysed neutral lipid fractions from other castor tissues using TLC, GLC and mass spectrometry. Cotyledons, like the endosperm, contain high levels of 18:1-OH in TAG. Pollen and male developing flowers accumulate TAG but do not contain 18:1-OH and leaves do not contain TAG or 18:1-OH. Analysis of acyl-CoAs in developing endosperm shows that ricinoleoyl-CoA is not the dominant acyl-CoA, indicating that either metabolic channelling or enzyme substrate selectivity are important in the synthesis of tri-ricinolein in this tissue. RNA-Seq transcriptomic analysis, using Illumina sequencing by synthesis technology, has been performed on mRNA isolated from two stages of developing seeds, germinating seeds, leaf and pollen-producing male flowers in order to identify differences in lipid-metabolic pathways and enzyme isoforms which could be important in the biosynthesis of TAG enriched in 18:1-OH. This study gives comprehensive coverage of gene expression in a variety of different castor tissues. The potential role of differentially expressed genes is discussed against a background of proteins identified in the endoplasmic reticulum, which is the site of TAG biosynthesis, and transgenic studies aimed at increasing the ricinoleic acid content of TAG. Conclusions/Significance Several of the genes identified in this tissue-specific whole transcriptome study have been used in transgenic plant research aimed at increasing the level of ricinoleic acid in TAG. New candidate genes have been identified which might further improve the level of ricinoleic acid in transgenic

  8. Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway

    PubMed Central

    1986-01-01

    Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20- fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane. PMID:3007532

  9. Intranasal administration of carbamazepine to mice: a direct delivery pathway for brain targeting.

    PubMed

    Serralheiro, Ana; Alves, Gilberto; Fortuna, Ana; Falcão, Amílcar

    2014-08-18

    The currently available antiepileptic drugs are typically administered via oral or intravenous (IV) routes which commonly exhibit high systemic distribution into non-targeted tissues, leading to peripheral adverse effects and limited brain uptake. In order to improve the efficacy and tolerability of the antiepileptic drug therapy, alternative administration strategies have been investigated. The purpose of the present study was to assess the pharmacokinetics of carbamazepine administered via intranasal (IN) and IV routes to mice, and to investigate whether a direct transport of the drug from nose to brain could be involved. The similar pharmacokinetic profiles obtained in all matrices following both administration routes indicate that, after IN delivery, carbamazepine reaches quickly and extensively the bloodstream, achieving the brain predominantly via systemic circulation. However, the uneven biodistribution of carbamazepine through the brain regions with higher concentrations in the olfactory bulb and frontal cortex following IN instillation, in comparison with the homogenous brain distribution pattern after IV injection, strongly suggests the involvement of a direct transport of carbamazepine from nose to brain. Therefore, it seems that IN delivery represents a suitable and promising alternative route to administer carbamazepine not only for the chronically use of the drug but also in emergency conditions.

  10. Synthesis of Bulk BC8 Silicon Allotrope by Direct Transformation and Reduced-Pressure Chemical Pathways.

    PubMed

    Kurakevych, Oleksandr O; Le Godec, Yann; Crichton, Wilson A; Guignard, Jérémy; Strobel, Timothy A; Zhang, Haidong; Liu, Hanyu; Coelho Diogo, Cristina; Polian, Alain; Menguy, Nicolas; Juhl, Stephen J; Gervais, Christel

    2016-09-06

    Phase-pure samples of a metastable allotrope of silicon, Si-III or BC8, were synthesized by direct elemental transformation at 14 GPa and ∼900 K and also at significantly reduced pressure in the Na-Si system at 9.5 GPa by quenching from high temperatures ∼1000 K. Pure sintered polycrystalline ingots with dimensions ranging from 0.5 to 2 mm can be easily recovered at ambient conditions. The chemical route also allowed us to decrease the synthetic pressures to as low as 7 GPa, while pressures required for direct phase transition in elemental silicon are significantly higher. In situ control of the synthetic protocol, using synchrotron radiation, allowed us to observe the underlying mechanism of chemical interactions and phase transformations in the Na-Si system. Detailed characterization of Si-III using X-ray diffraction, Raman spectroscopy, (29)Si NMR spectroscopy, and transmission electron microscopy are discussed. These large-volume syntheses at significantly reduced pressures extend the range of possible future bulk characterization methods and applications.

  11. Direct neural pathways convey distinct visual information to Drosophila mushroom bodies

    PubMed Central

    Vogt, Katrin; Aso, Yoshinori; Hige, Toshihide; Knapek, Stephan; Ichinose, Toshiharu; Friedrich, Anja B; Turner, Glenn C; Rubin, Gerald M; Tanimoto, Hiromu

    2016-01-01

    Previously, we demonstrated that visual and olfactory associative memories of Drosophila share mushroom body (MB) circuits (Vogt et al., 2014). Unlike for odor representation, the MB circuit for visual information has not been characterized. Here, we show that a small subset of MB Kenyon cells (KCs) selectively responds to visual but not olfactory stimulation. The dendrites of these atypical KCs form a ventral accessory calyx (vAC), distinct from the main calyx that receives olfactory input. We identified two types of visual projection neurons (VPNs) directly connecting the optic lobes and the vAC. Strikingly, these VPNs are differentially required for visual memories of color and brightness. The segregation of visual and olfactory domains in the MB allows independent processing of distinct sensory memories and may be a conserved form of sensory representations among insects. DOI: http://dx.doi.org/10.7554/eLife.14009.001 PMID:27083044

  12. Pathways to adult sexual revictimization: direct and indirect behavioral risk factors across the lifespan.

    PubMed

    Fargo, Jamison D

    2009-11-01

    The purpose of this study is to investigate direct and indirect social and behavioral risk factors for adult sexual revictimization. Participants include 147 adult, predominantly African American (88%) women, 59% of whom had a documented history of child sexual abuse. Participants are interviewed in adulthood about adolescent and adult sexual victimization as well as other background and lifestyle characteristics. Structural equation modeling indicates that the relationship between child and adolescent sexual victimization is indirect, mediated by adolescent risk-taking behavior. The relationship between adolescent and adult sexual victimization is also indirect, mediated by risky sexual behavior. The residual effects of early childhood family environment and childhood physical abuse also indirectly predict sexual revictimization. Results provide empirical support for the general supposition that the relationship between child and adult sexual victimization is complex and that many intermediary factors differentially affect risk for a heightened vulnerability to sexual revictimization.

  13. Adiposity Has No Direct Effect on Carotid Intima-Media Thickness in Adolescents and Young Adults: Use of Structural Equation Modeling to Elucidate Indirect & Direct Pathways

    PubMed Central

    Gao, Zhiqian; Khoury, Philip R.; McCoy, Connie E.; Shah, Amy S.; Kimball, Thomas R.; Dolan, Lawrence M.; Urbina, Elaine M.

    2016-01-01

    Background Carotid intima-media thickness (cIMT) is associated with CV events in adults. Thicker cIMT is found in youth with CV risk factors including obesity. Which risk factors have the most effect upon cIMT in youth and whether obesity has direct or indirect effects is not known. We used structural equation modeling to elucidate direct and indirect pathways through which obesity and other risk factors were associated with cIMT. Methods We collected demographics, anthropometrics and laboratory data on 784 subjects age 10–24 years (mean 18.0 ± 3.3 years). Common, bulb and internal carotid cIMT were measured by ultrasound. Multivariable regression analysis was performed to assess independent determinants of cIMT. Analyses were repeated with structural equation modeling to determine direct and indirect effects. Results Multivariable regression models explained 11%–22% of variation of cIMT. Age, sex and systolic blood pressure (BP) z-score were significant determinants of all cIMT segments. Body mass index (BMI) z-score, race, presence of type 2 diabetes mellitus (T2DM), hemoglobin A1c (HbA1c) and non-HDL were significant for some segments (all p=0.05). The largest direct effect on cIMT was age (0.312) followed by BP (0.228), Blood glucose control (0.108) and non-HDL (0.134). BMI only had a significant indirect effect through blood glucose control, BP & non-HDL. High sensitivity C-reactive protein (CRP) had a small indirect effect through blood glucose control (all p=0.05). Conclusions Age and BP are the major factors with direct effect on cIMT. Glucose and non-HDL were also important in this cohort with a high prevalence of T2DM. BMI only has indirect effects, through other risk factors. Traditional CV risk factors have important direct effects on cIMT in the young, but adiposity exerts its influence only through other CV risk factors. PMID:26752690

  14. In-plane information from tapping mode AFM images

    NASA Astrophysics Data System (ADS)

    Marcus, Matthew

    2003-03-01

    Phase contrast in intermittent-contact atomic force microscopy is shown to reveal in-plane structural and mechanical properties of poly(diacetylene) monolayer films. This is surprising because measurements of in-plane properties typically require a contact mode of microscopy. Such measurements are possible because the tilt in the oscillating cantilever provides components of motion not just perpendicular to the surface, but also parallel to the sample surface. Lateral tip displacement is virtually universal in AFM, implying that any oscillating tip-AFM technique is sensitive to in-plane material properties. Although the tilt in the cantilever is small ( 10^o) it produces a component of motion that is 20% of the total tip displacement, and this motion accounts for 5-10% of dissipated energy through the tip-sample interaction[1]. The data is used in conjunction with a numerical model to extract in-plane material parameters. The effect of the cantilever tilt on phase measurements is directly verified through measurements on silicon samples tilted at a variety of angles with respect to the cantilever. The lateral tip displacement we make use of allows measurements of in-plane properties of soft samples such as polymer and biological samples. This work was done in collaboration with M. D'Amato, R.W. Carpick, and M.A. Eriksson, and was supported by the NSF CAREER and MRSEC programs and the Research Corporation. 1. M.S. Marcus, R.W. Carpick, D.Y. Sasaki, M.A. Eriksson, Phys. Rev. Lett. 88, 226103 (2002)

  15. BRCA1 Directs the Repair Pathway to Homologous Recombination by Promoting 53BP1 Dephosphorylation.

    PubMed

    Isono, Mayu; Niimi, Atsuko; Oike, Takahiro; Hagiwara, Yoshihiko; Sato, Hiro; Sekine, Ryota; Yoshida, Yukari; Isobe, Shin-Ya; Obuse, Chikashi; Nishi, Ryotaro; Petricci, Elena; Nakada, Shinichiro; Nakano, Takashi; Shibata, Atsushi

    2017-01-10

    BRCA1 promotes homologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G2 phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR.

  16. Multiple Smaller Missions as a Direct Pathway to Mars Sample Return

    NASA Technical Reports Server (NTRS)

    Niles, P. B.; Draper, D. S.; Evans, C. A.; Gibson, E. K.; Graham, L. D.; Jones, J. H.; Lederer, S. M.; Ming, D.; Seaman, C. H.; Archer, P. D.; Andrews-Hanna, J.; Baldridge, A. M.; Bourke, M. C.; Crown, D. A.; Fries, M.; Knudson, A. T.; Michalski, J.; Dobrea, E. Noe; Vaniman, D.; Weitz, C. M.; Williams, R. M. E.; Bell, J. F., III; Knauth, L. P.

    2012-01-01

    Recent discoveries by the Mars Exploration Rovers, Mars Express, Mars Odyssey, and Mars Reconnaissance Orbiter spacecraft include multiple, tantalizing astrobiological targets representing both past and present environments on Mars. The most desirable path to Mars Sample Return (MSR) would be to collect and return samples from that site which provides the clearest examples of the variety of rock types considered a high priority for sample return (pristine igneous, sedimentary, and hydrothermal). Here we propose an MSR architecture in which the next steps (potentially launched in 2018) would entail a series of smaller missions, including caching, to multiple landing sites to verify the presence of high priority sample return targets through in situ analyses. This alternative architecture to one flagship-class sample caching mission to a single site would preserve a direct path to MSR as stipulated by the Planetary Decadal Survey, while permitting investigation of diverse deposit types and providing comparison of the site of returned samples to other aqueous environments on early Mars

  17. Direct Pathway to Molecular Photodissociation on Metal Surfaces Using Visible Light.

    PubMed

    Kazuma, Emiko; Jung, Jaehoon; Ueba, Hiromu; Trenary, Michael; Kim, Yousoo

    2017-03-01

    We demonstrate molecular photodissociation on single-crystalline metal substrates, driven by visible-light irradiation. The visible-light-induced photodissociation on metal substrates has long been thought to never occur, either because visible-light energy is much smaller than the optical energy gap between the frontier electronic states of the molecule or because the molecular excited states have short lifetimes due to the strong hybridization between the adsorbate molecular orbitals (MOs) and metal substrate. The S-S bond in dimethyl disulfide adsorbed on both Cu(111) and Ag(111) surfaces was dissociated through direct electronic excitation from the HOMO-derived MO (the nonbonding lone-pair type orbitals on the S atoms (nS)) to the LUMO-derived MO (the antibonding orbital localized on the S-S bond (σ*SS)) by irradiation with visible light. A combination of scanning tunneling microscopy and density functional theory calculations revealed that visible-light-induced photodissociation becomes possible due to the interfacial electronic structures constructed by the hybridization between molecular orbitals and the metal substrate states. The molecule-metal hybridization decreases the gap between the HOMO- and LUMO-derived MOs into the visible-light energy region and forms LUMO-derived MOs that have less overlap with the metal substrate, which results in longer excited-state lifetimes.

  18. Spatiotemporal trajectories of reactivation of somatosensory cortex by direct and secondary pathways after dorsal column lesions in squirrel monkeys.

    PubMed

    Qi, Hui-Xin; Wang, Feng; Liao, Chia-Chi; Friedman, Robert M; Tang, Chaohui; Kaas, Jon H; Avison, Malcolm J

    2016-11-15

    After lesions of the somatosensory dorsal column (DC) pathway, the cortical hand representation can become unresponsive to tactile stimuli, but considerable responsiveness returns over weeks of post-lesion recovery. The reactivation suggests that preserved subthreshold sensory inputs become potentiated and axon sprouting occurs over time to mediate recovery. Here, we studied the recovery process in 3 squirrel monkeys, using high-resolution cerebral blood volume-based functional magnetic resonance imaging (CBV-fMRI) mapping of contralateral somatosensory cortex responsiveness to stimulation of distal finger pads with low and high level electrocutaneous stimulation (ES) before and 2, 4, and 6weeks after a mid-cervical level contralateral DC lesion. Both low and high intensity ES of digits revealed the expected somatotopy of the area 3b hand representation in pre-lesion monkeys, while in areas 1 and 3a, high intensity stimulation was more effective in activating somatotopic patterns. Six weeks post-lesion, and irrespective of the severity of loss of direct DC inputs (98%, 79%, 40%), somatosensory cortical area 3b of all three animals showed near complete recovery in terms of somatotopy and responsiveness to low and high intensity ES. However there was significant variability in the patterns and amplitudes of reactivation of individual digit territories within and between animals, reflecting differences in the degree of permanent and/or transient silencing of primary DC and secondary inputs 2weeks post-lesion, and their spatio-temporal trajectories of recovery between 2 and 6weeks. Similar variations in the silencing and recovery of somatotopy and responsiveness to high intensity ES in areas 3a and 1 are consistent with individual differences in damage to and recovery of DC and spinocuneate pathways, and possibly the potentiation of spinothalamic pathways. Thus, cortical deactivation and subsequent reactivation depends not only on the degree of DC lesion, but also on

  19. Interleukin-18 directly protects cortical neurons by activating PI3K/AKT/NF-κB/CREB pathways.

    PubMed

    Zhou, Jia; Ping, Feng-feng; Lv, Wen-ting; Feng, Jun-yi; Shang, Jing

    2014-09-01

    Interleukin-18 (IL-18), a member of the IL-1 family of cytokines, was initially identified as an interferon (IFN)-γ-inducing factor. IL-18 is expressed in both immune and non-immune cells and participates in the adjustment of multitude cellular functions. Nonetheless, the effects of IL-18 on cortical neurons have not been explored. The present study was conducted to investigate the influence of IL-18 on rat primary cortical neurons and elucidate the underlying mechanisms. We proved that rrIL-18 increased the brain-derived neurotrophic factor (BDNF) expression in a time-dependent manner. Treatment with rrIL-18 (50 ng/ml) deactivated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by facilitating its phosphorylation, enhanced the expression of Phosphoinositide 3-OH kinase (PI3K) and p-Akt, standing for the activation of the PI3K/Akt pathway. As its pivotal downstream pathways, nuclear factor-kappa B (NF-κB), cAMP-responsive element binding protein (CREB)/Bcl-2 and glycogen synthase kinase-3β (GSK-3β) were examined in further steps. Our data revealed that rrIL-18 stimulated NF-κB activation, improved p-CREB and anti-apoptotic Bcl-2 expression levels. But rrIL-18 had little or no effect on GSK-3β pathway. Besides, rrIL-18 increased levels of BDNF and Bcl-2/Bax ratio and decreased cleaved caspase-3 expression to protect cortical neurons from damage induced by oxygen-glucose deprivation (OGD). These results in vitro showed the protection of IL-18 on cortical neurons. And this direct neuroprotective effect of IL-18 is crippled by PI3K inhibitor wortmannin.

  20. Understanding the direct radiative effect of dust aerosols on transport pathways using the NASA GEOS-5 AGCM

    NASA Astrophysics Data System (ADS)

    Nowottnick, E. P.; Colarco, P. R.; Lau, W. K.; Kim, K.

    2012-12-01

    African dust aerosols are transported across the Atlantic Ocean to the Caribbean by the easterly trade winds.While in transport, dust aerosols interact with the Earth system in various ways, ranging from influencing the local radiation balance to serving as a nutrient for tropical ecosystems.However, our current understanding of these processes is incomplete and serves as a source of uncertainty in Earth system modeling.Here, we focus on understanding the direct radiative impacts of African dust aerosols on the atmosphere using the NASA GEOS-5 atmospheric general circulation model that simulates aerosols with an online version of the GOCART model. For this study, we compare a high resolution GEOS-5 climate simulation where aerosols have been radiatively coupled to the atmosphere to one where aerosols are treated as passive tracers for June - September, 2009. Utilizing streamfunction and velocity potentials of the simulated dust mass flux, we isolate differences in dust transport pathways caused by the direct radiative effect of dust by comparing the rotational and divergent components of the dust flow in the horizontal and vertical on various timescales.Additionally, we pay special attention to the influence of dust aerosols on African Easterly Jet (AEJ) position and strength, as well as temperature profiles, cloudiness, and precipitation to gain further insight into the direct radiative effect of dust aerosols on the atmosphere

  1. Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli.

    PubMed

    Clarke, Justin E; Kime, Louise; Romero A, David; McDowall, Kenneth J

    2014-10-01

    Escherichia coli endoribonuclease E has a major influence on gene expression. It is essential for the maturation of ribosomal and transfer RNA as well as the rapid degradation of messenger RNA. The latter ensures that translation closely follows programming at the level of transcription. Recently, one of the hallmarks of RNase E, i.e. its ability to bind via a 5'-monophosphorylated end, was shown to be unnecessary for the initial cleavage of some polycistronic tRNA precursors. Here we show using RNA-seq analyses of ribonuclease-deficient strains in vivo and a 5'-sensor mutant of RNase E in vitro that, contrary to current models, 5'-monophosphate-independent, 'direct entry' cleavage is a major pathway for degrading and processing RNA. Moreover, we present further evidence that direct entry is facilitated by RNase E binding simultaneously to multiple unpaired regions. These simple requirements may maximize the rate of degradation and processing by permitting multiple sites to be surveyed directly without being constrained by 5'-end tethering. Cleavage was detected at a multitude of sites previously undescribed for RNase E, including ones that regulate the activity and specificity of ribosomes. A potentially broad role for RNase G, an RNase E paralogue, in the trimming of 5'-monophosphorylated ends was also revealed.

  2. Transcranial direct current stimulation modulates human color discrimination in a pathway-specific manner.

    PubMed

    Costa, Thiago L; Nagy, Balázs V; Barboni, Mirella T S; Boggio, Paulo S; Ventura, Dora F

    2012-01-01

    Previous research showed that transcranial direct current stimulation (tDCS) can modulate visual cortex excitability. However, there is no experiment on the effects of tDCS on color perception to date. The present study aimed to investigate the effects of tDCS on color discrimination tasks. Fifteen healthy subjects (mean age of 25.6 ± 4.4 years) were tested with Cambridge Color Test 2.0 (Trivector and ellipses protocols) and a Forced-choice Spatial Color Contrast Sensitivity task (vertical red-green sinusoidal grating) while receiving tDCS. Anodal, cathodal, and sham tDCS were delivered at Oz for 22 min using two square electrodes (25 cm(2) with a current of 1.5 mA) in sessions separated by 7 days. Anodal tDCS significantly increased tritan sensitivity (p < 0.01) and had no significant effect on protan, deutan, or red-green grating discrimination. The effects on the tritan discrimination returned to baseline after 15 min (p < 0.01). Cathodal tDCS reduced the sensitivity in the deutan axis and increased sensitivity in the tritan axis (p < 0.05). The lack of anodal tDCS effects in the protan, deutan, and red-green grating sensitivities could be explained by a "ceiling effect" since adults in this age range tend to have optimal color discrimination performance for these hues. The differential effects of cathodal tDCS on tritan and deutan sensitivities and the absence of the proposed ceiling effects for the tritan axes might be explained by Parvocellular (P) and Koniocellular (K) systems with regard to their functional, physiological, and anatomical differences. The results also support the existence of a systematic segregation of P and K color-coding cells in V1. Future research and possible clinical implications are discussed.

  3. Control of torque direction by spinal pathways at the cat ankle joint.

    PubMed

    Nichols, T R; Lawrence, J H; Bonasera, S J

    1993-01-01

    To study the biomechanics of the calcaneal tendon's complex insertion onto the calcaneus, we measured torque-time trajectories exerted by the triceps surae and tibialis anterior muscles in eight unanesthetized decerebrate cats using a multi-axis force-moment sensor placed at the ankle joint. The ankle was constrained to an angle of 110 degrees plantarflexion. Muscles were activated using crossed-extension (XER), flexion (FWR), and caudal cutaneous sural nerve (SNR) reflexes. Torque contributions of other muscles activated by these reflexes were eliminated by denervation or tenotomy. In two animals, miniature pressure transducers were implanted among tendon fibers from the lateral gastrocnemius (LG) muscle that insert straight into the calcaneus or among tendon fibers from the medial gastrocnemius (MG) that cross over and insert on the lateral aspect of calcaneus. Reflexively evoked torques had the following directions: FWR, dorsiflexion and adduction; SNR, plantarflexion and abduction; and XER, plantarflexion and modest abduction or adduction. The proportion of abduction torque to plantarflexion torque was always greater for SNR than XER; this difference was about 50% of the magnitude of abduction torque generated by tetanic stimulation of the peronei. During SNR, pressures were higher in regions of the calcaneal tendon originating from MG than regions originating from LG. Similarly, pressures within the MG portion of the calcaneal tendon were higher during SNR than during XER, although these two reflexes produced matched ankle plantarflexion forces. Selective tenotomies and electromyographic recordings further demonstrated that MG generated most of the torque in response to SNR, while soleus, LG, and MG all generated torques in response to XER.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. The Notch Signaling Pathway Controls the Size of the Ocular Lens by Directly Suppressing p57Kip2 Expression▿ †

    PubMed Central

    Jia, Junling; Lin, Min; Zhang, Lingna; York, J. Philippe; Zhang, Pumin

    2007-01-01

    The size of an organ must be tightly controlled so that it fits within an organism. The mammalian lens is a relatively simple organ composed of terminally differentiated, amitotic lens fiber cells capped on the anterior surface by a layer of immature, mitotic epithelial cells. The proliferation of lens epithelial cells fuels the growth of the lens, thus controling the size of the lens. We report that the Notch signaling pathway defines the boundary between proliferation and differentiation in the developing lens. The loss of Notch signaling results in the loss of epithelial cells to differentiation and a much smaller lens. We found that the Notch effector Herp2 is expressed in lens epithelium and directly suppresses p57Kip2 expression, providing a molecular link between Notch signaling and the cell cycle control machinery during lens development. PMID:17709399

  5. Molecular mechanism of direct proflavine-DNA intercalation: evidence for drug-induced minimum base-stacking penalty pathway.

    PubMed

    Sasikala, Wilbee D; Mukherjee, Arnab

    2012-10-11

    DNA intercalation, a biophysical process of enormous clinical significance, has surprisingly eluded molecular understanding for several decades. With appropriate configurational restraint (to prevent dissociation) in all-atom metadynamics simulations, we capture the free energy surface of direct intercalation from minor groove-bound state for the first time using an anticancer agent proflavine. Mechanism along the minimum free energy path reveals that intercalation happens through a minimum base stacking penalty pathway where nonstacking parameters (Twist→Slide/Shift) change first, followed by base stacking parameters (Buckle/Roll→Rise). This mechanism defies the natural fluctuation hypothesis and provides molecular evidence for the drug-induced cavity formation hypothesis. The thermodynamic origin of the barrier is found to be a combination of entropy and desolvation energy.

  6. A direct amygdala-motor pathway for emotional displays to influence action: A diffusion tensor imaging study.

    PubMed

    Grèzes, Julie; Valabrègue, Romain; Gholipour, Bahar; Chevallier, Coralie

    2014-12-01

    An important evolutionary function of emotions is to prime individuals for action. Although functional neuroimaging has provided evidence for such a relationship, little is known about the anatomical substrates allowing the limbic system to influence cortical motor-related areas. Using diffusion-weighted magnetic resonance imaging and probabilistic tractography on a cohort of 40 participants, we provide evidence of a structural connection between the amygdala and motor-related areas (lateral and medial precentral, motor cingulate and primary motor cortices, and postcentral gyrus) in humans. We then compare this connection with the connections of the amygdala with emotion-related brain areas (superior temporal sulcus, fusiform gyrus, orbitofrontal cortex, and lateral inferior frontal gyrus) and determine which amygdala nuclei are at the origin of these projections. Beyond the well-known subcortical influences over automatic and stereotypical emotional behaviors, a direct amygdala-motor pathway might provide a mechanism by which the amygdala can influence more complex motor behaviors.

  7. Preparation of DNA and nucleoprotein samples for AFM imaging

    PubMed Central

    Lyubchenko, Yuri L.

    2010-01-01

    Sample preparation techniques allowing reliable and reproducible imaging of DNA with various structures, topologies and complexes with proteins are reviewed. The major emphasis is given to methods utilizing chemical functionalization of mica, enabling preparation of the surfaces with required characteristics. The methods are illustrated by examples of imaging of different DNA structures. Special attention is given to the possibility of AFM to image the dynamics of DNA at the nanoscale. The capabilities of time-lapse AFM in aqueous solutions are illustrated by imaging of dynamic processes as transitions of local alternative structures (transition of DNA between H and B forms). The application of AFM to studies of protein-DNA complexes is illustrated by a few examples of imaging site-specific complexes, as well as such systems as chromatin. The time-lapse AFM studies of protein-DNA complexes including very recent advances with the use of high-speed AFM are reviewed. PMID:20864349

  8. AFM studies of the crystallization and habit modification of an excipient material, adipic acid.

    PubMed

    Keel, T R; Thompson, C; Davies, M C; Tendler, S J B; Roberts, C J

    2004-08-06

    Atomic force microscopy (AFM) has been used to investigate the (1 0 0) face of crystalline adipic acid, both in air and liquid environments. In air, surface reorganization occurred during scanning of the AFM probe, which has been investigated using single point force-distance analysis under a controlled relative humidity (RH) environment. We suggest such reorganization can be attributed to the influence of a network of water molecules bound to the hydrophilic (1 0 0) surface permitting local AFM tip-enhanced dissolution and reorganization of the solute. In situ imaging was also carried out on the crystals, revealing etch-pit formation during dissolution, and rapid growth at higher levels of supersaturation (sigma), both of which are direct consequences of the hydrophilic nature of the (1 0 0) face. Also presented here are nanoscale observations of the effect of octanoic acid, a structurally-related habit modifier, on crystalline adipic acid. Using AFM, we have been able to show that the presence of octanoic acid at low concentration has little observable affect on the development of the (1 0 0) face; however, as this concentration is increased, there are clear changes in step morphology and growth mode on the (1 0 0) face of the crystal. At a concentration of 1.26 mmol dm(-3) (a concentration corresponding to a molar ratio of approximately 1:175 octanoic acid:adipic acid), growth on the (1 0 0) face is inhibited, with in situ AFM imaging indicating this is a direct consequence of octanoic acid binding to the surface, and pinning the monomolecular growth steps.

  9. An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles

    SciTech Connect

    Zhang, Xiaojun; Chen, Yuan; Chen, Yong

    2014-03-28

    Highlights: • Air drying induced the transformation of cell-surface membrane vesicles into pits. • An AFM-based pit-measuring method was developed to measure cell-surface vesicles. • Our method detected at least two populations of cell-surface membrane vesicles. - Abstract: Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM) has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release.

  10. Manufacturing process of nanofluidics using afm probe

    NASA Astrophysics Data System (ADS)

    Karingula, Varun Kumar

    A new process for fabricating a nano fluidic device that can be used in medical application is developed and demonstrated. Nano channels are fabricated using a nano tip in indentation mode on AFM (Atomic Force Microscopy). The nano channels are integrated between the micro channels and act as a filter to separate biomolecules. Nano channels of 4 to7 m in length, 80nm in width, and at varying depths from 100nm to 850 nm allow the resulting device to separate selected groups of lysosomes and other viruses. Sharply developed vertical micro channels are produced from a deep reaction ion etching followed by deposition of different materials, such as gold and polymers, on the top surface, allowing the study of alternative ways of manufacturing a nanofluidic device. PDMS (Polydimethylsiloxane) bonding is performed to close the top surface of the device. An experimental setup is used to test and validate the device by pouring fluid through the channels. A detailed cost evaluation is conducted to compare the economical merits of the proposed process. It is shown that there is a 47:7% manufacturing time savings and a 60:6% manufacturing cost savings.

  11. Microrheology using a custom-made AFM

    NASA Astrophysics Data System (ADS)

    Kosgodagan Acharige, Sebastien; Benzaquen, Michael; Steinberger, Audrey

    In the past few years, a new method was developed to measure local properties of liquids (X. Xiong et al., Phys. Rev. E 80, 2009). This method consists of gluing a micron-sized glass fiber at the tip of an AFM cantilever and probing the liquid with it. In ENS Lyon, this method was perfected (C. Devailly et al., EPL, 106 5, 2014) with the help of an interferometer developped in the same laboratory (L. Bellon et al., Opt. Commun. 207 49, 2002 and P. Paolino et al., Rev. Sci. Instrum. 84, 2013), which background noise can reach 10-14 m /√{ Hz } . This method allows us to measure a wide range of viscosities (1 mPa . s to 500 mPa . s) of transparent and opaque fluids using a small sample volume ( 5 mL). In this presentation, I will briefly describe the interferometer developped in ENS Lyon, then explain precisely the microrheology measurements and then compare the experimental results to a model developped by M. Benzaquen. This work is supported financially by the ANR project NANOFLUIDYN (Grant Number ANR-13-BS10-0009).

  12. BOREAS AFM-07 SRC Surface Meteorological Data

    NASA Technical Reports Server (NTRS)

    Osborne, Heather; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Young, Kim; Wittrock, Virginia; Shewchuck, Stan; Smith, David E. (Technical Monitor)

    2000-01-01

    The Saskatchewan Research Council (SRC) collected surface meteorological and radiation data from December 1993 until December 1996. The data set comprises Suite A (meteorological and energy balance measurements) and Suite B (diffuse solar and longwave measurements) components. Suite A measurements were taken at each of ten sites, and Suite B measurements were made at five of the Suite A sites. The data cover an approximate area of 500 km (North-South) by 1000 km (East-West) (a large portion of northern Manitoba and northern Saskatchewan). The measurement network was designed to provide researchers with a sufficient record of near-surface meteorological and radiation measurements. The data are provided in tabular ASCII files, and were collected by Aircraft Flux and Meteorology (AFM)-7. The surface meteorological and radiation data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  13. Non-collinear magnetism and exchange bias at the FM NiFe/AFM NiMn interface: local spin density FLAPW study

    NASA Astrophysics Data System (ADS)

    Nakamura, K.; Freeman, A. J.; Wang, D.-S.; Zhong, L.; Fernandez-de-Castro, J.

    2001-03-01

    Magnetism at interfaces, such as the exchange bias between ferromagnetic (FM) and antiferromagnetic (AFM) materials, has attracted great attention because of technological applications. In order to investigate magnetic structures at the FM/AFM interface, we have implemented the FLAPW (E. Wimmer, H. Krakauer, M. Weinert and A.J. Freeman, PRB 24, 864(1981)) methodologies including non-collinear magnetism, in which the magnetic moment direction as well as the magnitude can vary continuously all over space. We first demonstrate this approach to determine the structure of a magnetic structure at an interface between FM NiFe and AFM NiMn. Although both bulk systems each show collinear FM and AFM structures, we found that a perpendicular magnetic orientation at their interface is energetically favorable, where the magnetic moments of the FM NiFe tend to lie perpendicular to those of AFM NiMn.

  14. AFM imaging of fenestrated liver sinusoidal endothelial cells.

    PubMed

    Braet, F; Wisse, E

    2012-12-01

    Each microscope with its dedicated sample preparation technique provides the investigator with a specific set of data giving an instrument-determined (or restricted) insight into the structure and function of a tissue, a cell or parts thereof. Stepwise improvements in existing techniques, both instrumental and preparative, can sometimes cross barriers in resolution and image quality. Of course, investigators get really excited when completely new principles of microscopy and imaging are offered in promising new instruments, such as the AFM. The present paper summarizes a first phase of studies on the thin endothelial cells of the liver. It describes the preparation-dependent differences in AFM imaging of these cells after isolation. Special point of interest concerned the dynamics of the fenestrae, thought to filter lipid-carrying particles during their transport from the blood to the liver cells. It also describes the attempts to image the details of these cells when alive in cell cultures. It explains what physical conditions, mainly contributed to the scanning stylus, are thought to play a part in the limitations in imaging these cells. The AFM also offers promising specifications to those interested in cell surface details, such as membrane-associated structures, receptors, coated pits, cellular junctions and molecular aggregations or domains. The AFM also offers nano-manipulation possibilities, strengths and elasticity measurements, force interactions, affinity measurements, stiffness and other physical aspects of membranes and cytoskeleton. The potential for molecular approaches is there. New developments in cantilever construction and computer software promise to bring real time video imaging to the AFM. Home made accessories for the first generation of AFM are now commodities in commercial instruments and make the life of the AFM microscopist easier. Also, the combination of different microscopies, such as AFM and TEM, or AFM and SEM find their way to the

  15. [Application of atomic force microscopy (AFM) in ophthalmology].

    PubMed

    Milka, Michał; Mróz, Iwona; Jastrzebska, Maria; Wrzalik, Roman; Dobrowolski, Dariusz; Roszkowska, Anna M; Moćko, Lucyna; Wylegała, Edward

    2012-01-01

    Atomic force microscopy (AFM) allows to examine surface of different biological objects in the nearly physiological conditions at the nanoscale. The purpose of this work is to present the history of introduction and the potential applications of the AFM in ophthalmology research and clinical practice. In 1986 Binnig built the AFM as a next generation of the scanning tunnelling microscope (STM). The functional principle of AFM is based on the measurement of the forces between atoms on the sample surface and the probe. As a result, the three-dimensional image of the surface with the resolution on the order of nanometres can be obtained. Yamamoto used as the first the AFM on a wide scale in ophthalmology. The first investigations used the AFM method to study structure of collagen fibres of the cornea and of the sclera. Our research involves the analysis of artificial intraocular lenses (IOLs). According to earlier investigations, e.g. Lombardo et al., the AFM was used to study only native IOLs. Contrary to the earlier investigations, we focused our measurements on lenses explanted from human eyes. The surface of such lenses is exposed to the influence of the intraocular aqueous environment, and to the related impacts of biochemical processes. We hereby present the preliminary results of our work in the form of AFM images depicting IOL surface at the nanoscale. The images allowed us to observe early stages of the dye deposit formation as well as local calcinosis. We believe that AFM is a very promising tool for studying the structure of IOL surface and that further observations will make it possible to explain the pathomechanism of artificial intraocular lens opacity formation.

  16. The cAMP Signaling Pathway and Direct Protein Kinase A Phosphorylation Regulate Polycystin-2 (TRPP2) Channel Function*

    PubMed Central

    Cantero, María del Rocío; Velázquez, Irina F.; Streets, Andrew J.; Ong, Albert C. M.; Cantiello, Horacio F.

    2015-01-01

    Polycystin-2 (PC2) is a TRP-type, Ca2+-permeable non-selective cation channel that plays an important role in Ca2+ signaling in renal and non-renal cells. The effect(s) of the cAMP pathway and kinase mediated phosphorylation of PC2 seem to be relevant to PC2 trafficking and its interaction with polycystin-1. However, the role of PC2 phosphorylation in channel function is still poorly defined. Here we reconstituted apical membranes of term human syncytiotrophoblast (hST), containing endogenous PC2 (PC2hst), and in vitro translated channel protein (PC2iv). Addition of the catalytic subunit of PKA increased by 566% the spontaneous PC2hst channel activity in the presence of ATP. Interestingly, 8-Br-cAMP also stimulated spontaneous PC2hst channel activity in the absence of the exogenous kinase. Either stimulation was inhibited by addition of alkaline phosphatase, which in turn, was reversed by the phosphatase inhibitor vanadate. Neither maneuver modified the single channel conductance but instead increased channel mean open time. PKA directly phosphorylated PC2, which increased the mean open time but not the single channel conductance of the channel. PKA phosphorylation did not modify either R742X truncated or S829A-mutant PC2iv channel function. The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. The relevant PKA site for PC2 channel regulation centers on a single residue serine 829, in the carboxyl terminus. PMID:26269590

  17. High-speed AFM of human chromosomes in liquid

    NASA Astrophysics Data System (ADS)

    Picco, L. M.; Dunton, P. G.; Ulcinas, A.; Engledew, D. J.; Hoshi, O.; Ushiki, T.; Miles, M. J.

    2008-09-01

    Further developments of the previously reported high-speed contact-mode AFM are described. The technique is applied to the imaging of human chromosomes at video rate both in air and in water. These are the largest structures to have been imaged with high-speed AFM and the first imaging in liquid to be reported. A possible mechanism that allows such high-speed contact-mode imaging without significant damage to the sample is discussed in the context of the velocity dependence of the measured lateral force on the AFM tip.

  18. Raman and AFM study of gamma irradiated plastic bottle sheets

    NASA Astrophysics Data System (ADS)

    Ali, Yasir; Kumar, Vijay; Sonkawade, R. G.; Dhaliwal, A. S.

    2013-02-01

    In this investigation, the effects of gamma irradiation on the structural properties of plastic bottle sheet are studied. The Plastic sheets were exposed with 1.25MeV 60Co gamma rays source at various dose levels within the range from 0-670 kGy. The induced modifications were followed by micro-Raman and atomic force microscopy (AFM). The Raman spectrum shows the decrease in Raman intensity and formation of unsaturated bonds with an increase in the gamma dose. AFM image displays rough surface morphology after irradiation. The detailed Raman analysis of plastic bottle sheets is presented here, and the results are correlated with the AFM observations.

  19. AFM-based quantification of conformational changes in DNA caused by reactive oxygen species.

    PubMed

    Berg, Florian; Wilken, Janine; Helm, Christiane A; Block, Stephan

    2015-01-08

    Radical induced modification of DNA plays an important role in many pathological pathways like cancer development, aging, etc. In this work, we quantify radical-induced DNA damage that causes transitions from double to single stranded DNA using atomic force microscopy (AFM). The plasmid pBR322 is attacked by free hydroxyl radicals that are produced by Fenton's reaction; the strength of the radical attack is controlled via the ratio of hydroxyl radical molecules to DNA base pairs. The extent of DNA modification is assessed by AFM tapping mode (TM) imaging of the plasmids (after adsorption onto PAH-functionalized mica) in air. As single stranded DNA chains (height ∼2 Å) are much smaller than intact DNA strands (∼5 Å), their fraction can be quantified based on the height distribution, which allows a simplified data analysis in comparison to similar AFM-based approaches. It is found that the amount of damaged DNA strands increases with increasing strength of radical attack, and decreases if ROS scavengers like sodium acetate are added. Competition curves are calculated for the interaction of hydroxyl radicals with DNA and sodium acetate, which finally allows calculation of relative rate constants for the respective reactions.

  20. Genome-wide analysis of N1ICD/RBPJ targets in vivo reveals direct transcriptional regulation of Wnt, SHH, and hippo pathway effectors by Notch1.

    PubMed

    Li, Yaochen; Hibbs, Matthew Aaron; Gard, Ashley Lauren; Shylo, Natalia Aliakseeuna; Yun, Kyuson

    2012-04-01

    The Notch pathway plays a pivotal role in regulating cell fate decisions in many stem cell systems. However, the full repertoire of Notch target genes in vivo and the mechanisms through which this pathway activity is integrated with other signaling pathways are largely unknown. Here, we report a transgenic mouse in which the activation of the Notch pathway massively expands the neural stem cell (NSC) pool in a cell context-dependent manner. Using this in vivo system, we identify direct targets of RBPJ/N1ICD in cortical NSCs at a genome-wide level through combined ChIP-Seq and transcriptome analyses. Through a highly conservative analysis of these datasets, we identified 98 genes that are directly regulated by N1ICD/RPBJ in vivo. These include many transcription factors that are known to be critical for NSC self-renewal (Sox2, Pax6, Tlx, and Id4) and the transcriptional effectors of the Wnt, SHH, and Hippo pathways, TCF4, Gli2, Gli3, Yap1, and Tead2. Since little is known about the function of the Hippo-Yap pathway in NSCs, we analyzed Yap1 expression and function in NSCs. We show that Yap1 expression is restricted to the stem cell compartment in the developing forebrain and that its expression is sufficient to rescue Notch pathway inhibition in NSC self-renewal assays. Together, results of this study reveal a previously underappreciated complexity and breadth of Notch1 targets in vivo and show direct interaction between Notch and Hippo-Yap pathways in NSCs.

  1. Introduction to atomic force microscopy (AFM) in biology.

    PubMed

    Goldsbury, Claire S; Scheuring, Simon; Kreplak, Laurent

    2009-11-01

    The atomic force microscope (AFM) has the unique capability of imaging biological samples with molecular resolution in buffer solution. In addition to providing topographical images of surfaces with nanometer- to angstrom-scale resolution, forces between single molecules and mechanical properties of biological samples can be investigated from the nanoscale to the microscale. Importantly, the measurements are made in buffer solutions, allowing biological samples to "stay alive" within a physiological-like environment while temporal changes in structure are measured-e.g., before and after addition of chemical reagents. These qualities distinguish AFM from conventional imaging techniques of comparable resolution, e.g., electron microscopy (EM). This unit provides an introduction to AFM on biological systems and describes specific examples of AFM on proteins, cells, and tissues. The physical principles of the technique and methodological aspects of its practical use and applications are also described.

  2. Msi2 Maintains Quiescent State of Hair Follicle Stem Cells by Directly Repressing the Hh Signaling Pathway.

    PubMed

    Ma, Xianghui; Tian, Yuhua; Song, Yongli; Shi, Jianyun; Xu, Jiuzhi; Xiong, Kai; Li, Jia; Xu, Wenjie; Zhao, Yiqiang; Shuai, Jianwei; Chen, Lei; Plikus, Maksim V; Lengner, Christopher J; Ren, Fazheng; Xue, Lixiang; Yu, Zhengquan

    2017-01-29

    Hair follicles (HFs) undergo precisely regulated cycles of active regeneration (anagen), involution (catagen), and relative quiescence (telogen). Hair follicle stem cells (HFSCs) play important roles in regenerative cycling. Elucidating mechanisms that govern HFSC behavior can help uncover the underlying principles of hair development, hair growth disorders, and skin cancers. RNA-binding proteins of the Musashi (Msi) have been implicated in the biology of different stem cell types, yet they have not been studied in HFSCs. Here we utilized gain- and loss-of-function mouse models to demonstrate that forced MSI2 expression retards anagen entry and consequently delays hair growth, whereas loss of Msi2 enhances hair regrowth. Furthermore, our findings show that Msi2 maintains quiescent state of HFSCs in the process of the telogen-to-anagen transition. At the molecular level, our unbiased transcriptome profiling shows that Msi2 represses Hedgehog signaling activity and that Shh is its direct target in the hair follicle. Taken together, our findings reveal the importance of Msi2 in suppressing hair regeneration and maintaining HFSC quiescence. The previously unreported Msi2-Shh-Gli1 pathway adds to the growing understanding of the complex network governing cyclic hair growth.

  3. USP39, a direct target of microRNA-133a, promotes progression of pancreatic cancer via the AKT pathway.

    PubMed

    Cai, Jing; Liu, Tiande; Huang, Peng; Yan, Wei; Guo, Changkuo; Xiong, Le; Liu, Anwen

    2017-04-22

    Ubiquitin specific protease 39 (USP39) is one of the deubiquitinating enzymes without ubiquitin protease activity, which has been implicated in the progression of several cancers. However, the role of USP39 in pancreatic cancer (PC) is largely unknown. In present study, we found that USP39 expression was elevated in PC tissues than adjacent non-tumor tissues. Importantly, we demonstrated that overexpression of USP39 is closely correlated with tumor progression and poor survival in PC patients. Furthermore, high USP39 expression was observed in PC cell lines and ectopic expression of USP39 significantly enhanced in vitro cell proliferation and promoted in vivo tumor growth, whereas silencing USP39 suppressed growth of PC cells. Besides, our experimental data revealed that knockdown of USP39 induced cell apoptosis through inhibition of AKT signaling pathway in PC cells. Moreover, USP39 was a direct target of miR-133a, a microRNA that has been reported to be involved in progression of PC. Taken together, our data provide a novel PC regulatory axis that is miR-133a/USP39, the dysfunction of which drives diverse aspects of the progression of PC.

  4. Direct observation of kinetic traps associated with structural transformations leading to multiple pathways of S-layer assembly.

    PubMed

    Shin, Seong-Ho; Chung, Sungwook; Sanii, Babak; Comolli, Luis R; Bertozzi, Carolyn R; De Yoreo, James J

    2012-08-07

    The concept of a folding funnel with kinetic traps describes folding of individual proteins. Using in situ Atomic Force Microscopy to investigate S-layer assembly on mica, we show this concept is equally valid during self-assembly of proteins into extended matrices. We find the S-layer-on-mica system possesses a kinetic trap associated with conformational differences between a long-lived transient state and the final stable state. Both ordered tetrameric states emerge from clusters of the monomer phase, however, they then track along two different pathways. One leads directly to the final low-energy state and the other to the kinetic trap. Over time, the trapped state transforms into the stable state. By analyzing the time and temperature dependencies of formation and transformation we find that the energy barriers to formation of the two states differ by only 0.7 kT, but once the high-energy state forms, the barrier to transformation to the low-energy state is 25 kT. Thus the transient state exhibits the characteristics of a kinetic trap in a folding funnel.

  5. BOREAS AFM-03-NCAR Electra 1994 Aircraft Sounding Data

    NASA Technical Reports Server (NTRS)

    Lenschow, Donald H.; Oncley, Steven P.; Hall, Forrest G. (Editor); Knapp, David E. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-3 team used the National Center for Atmospheric Research's (NCAR) Electra aircraft to make sounding measurements to study the planetary boundary layer using in situ and remote-sensing measurements. Measurements were made of wind speed and direction, air pressure and temperature, potential temperature, dewpoint, mixing ratio of H, O, CO, concentration, and ozone concentration. Twenty-five research missions were flown over the Northern Study Area (NSA), Southern Study Area (SSA), and the transect during BOREAS Intensive Field Campaigns (IFCs) 1, 2, and 3 during 1994. All missions had from four to ten soundings through the top of the planetary boundary layer. This sounding data set contains all of the in situ vertical profiles through the boundary layer top that were made (with the exception of 'porpoise' maneuvers). Data were recorded in one-second time intervals. These data are stored in tabular ASCII files. The NCAR Electra 1994 aircraft sounding data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  6. BOREAS AFM-2 Wyoming King Air 1994 Aircraft Sounding Data

    NASA Technical Reports Server (NTRS)

    Kelly, Robert D.; Hall, Forrest G. (Editor); Knapp, David E. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The BOREAS AFM-2 team used the University of Wyoming King Air aircraft during IFCs 1, 2, and 3 in 1994 to collected pass-by-pass fluxes (and many other statistics) for the large number of level (constant altitude), straight-line passes used in a variety of flight patterns over the SSA and NSA and areas along the transect between these study areas. The data described here form a second set, namely soundings that were incorporated into nearly every research flight by the King Air in 1994. These soundings generally went from near the surface to above the inversion layer. Most were flown immediately after takeoff or immediately after finishing the last flux pattern of that particular day's flights. The parameters that were measured include wind direction, wind speed, west wind component (u), south wind component (v), static pressure, air dry bulb temperature, potential temperature, dewpoint, temperature, water vapor mixing ratio, and CO2 concentration. Data on the aircraft's location, attitude, and altitude during data collection are also provided. These data are stored in tabular ASCH files. The data files are available on a CD-ROM (see document number 20010000884) or from the Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC).

  7. Nanoscale structural features determined by AFM for single virus particles

    NASA Astrophysics Data System (ADS)

    Chen, Shu-Wen W.; Odorico, Michael; Meillan, Matthieu; Vellutini, Luc; Teulon, Jean-Marie; Parot, Pierre; Bennetau, Bernard; Pellequer, Jean-Luc

    2013-10-01

    In this work, we propose ``single-image analysis'', as opposed to multi-image averaging, for extracting valuable information from AFM images of single bio-particles. This approach allows us to study molecular systems imaged by AFM under general circumstances without restrictions on their structural forms. As feature exhibition is a resolution correlation, we have performed AFM imaging on surfaces of tobacco mosaic virus (TMV) to demonstrate variations of structural patterns with probing resolution. Two AFM images were acquired with the same tip at different probing resolutions in terms of pixel width, i.e., 1.95 and 0.49 nm per pixel. For assessment, we have constructed an in silico topograph based on the three-dimensional crystal structure of TMV as a reference. The prominent artifacts observed in the AFM-determined shape of TMV were attributed to tip convolutions. The width of TMV rod was systematically overestimated by ~10 nm at both probing resolutions of AFM. Nevertheless, the effects of tip convolution were less severe in vertical orientation so that the estimated height of TMV by AFM imaging was in close agreement with the in silico X-ray topograph. Using dedicated image processing algorithms, we found that at low resolution (i.e., 1.95 nm per pixel), the extracted surface features of TMV can be interpreted as a partial or full helical repeat (three complete turns with ~7.0 nm in length), while individual protein subunits (~2.5 nm) were perceivable only at high resolution. The present study shows that the scales of revealed structural features in AFM images are subject to both probing resolution and processing algorithms for image analysis.

  8. Robust strategies for automated AFM force curve analysis--I. Non-adhesive indentation of soft, inhomogeneous materials.

    PubMed

    Lin, David C; Dimitriadis, Emilios K; Horkay, Ferenc

    2007-06-01

    The atomic force microscope (AFM) has found wide applicability as a nanoindentation tool to measure local elastic properties of soft materials. An automated approach to the processing of AFM indentation data, namely, the extraction of Young's modulus, is essential to realizing the high-throughput potential of the instrument as an elasticity probe for typical soft materials that exhibit inhomogeneity at microscopic scales. This paper focuses on Hertzian analysis techniques, which are applicable to linear elastic indentation. We compiled a series of synergistic strategies into an algorithm that overcomes many of the complications that have previously impeded efforts to automate the fitting of contact mechanics models to indentation data. AFM raster data sets containing up to 1024 individual force-displacement curves and macroscopic compression data were obtained from testing polyvinyl alcohol gels of known composition. Local elastic properties of tissue-engineered cartilage were also measured by the AFM. All AFM data sets were processed using customized software based on the algorithm, and the extracted values of Young's modulus were compared to those obtained by macroscopic testing. Accuracy of the technique was verified by the good agreement between values of Young's modulus obtained by AFM and by direct compression of the synthetic gels. Validation of robustness was achieved by successfully fitting the vastly different types of force curves generated from the indentation of tissue-engineered cartilage. For AFM indentation data that are amenable to Hertzian analysis, the method presented here minimizes subjectivity in preprocessing and allows for improved consistency and minimized user intervention. Automated, large-scale analysis of indentation data holds tremendous potential in bioengineering applications, such as high-resolution elasticity mapping of natural and artificial tissues.

  9. Information seeking and anxiety among colonoscopy-naïve adults: Direct-to-colonoscopy vs traditional consult-first pathways

    PubMed Central

    Silvester, Jocelyn A; Kalkat, Harmandeep; Graff, Lesley A; Walker, John R; Singh, Harminder; Duerksen, Donald R

    2016-01-01

    AIM To investigate the effects of direct to colonoscopy pathways on information seeking behaviors and anxiety among colonoscopy-naïve patients. METHODS Colonoscopy-naïve patients at two tertiary care hospitals completed a survey immediately prior to their scheduled outpatient procedure and before receiving sedation. Survey items included clinical pathway (direct or consult), procedure indication (cancer screening or symptom investigation), telephone and written contact from the physician endoscopist office, information sources, and pre-procedure anxiety. Participants reported pre-procedure anxiety using a 10 point scale anchored by “very relaxed” (1) and “very nervous” (10). At least three months following the procedure, patient medical records were reviewed to determine sedative dose, procedure indications and any adverse events. The primary comparison was between the direct and consult pathways. Given the very different implications, a secondary analysis considering the patient-reported indication for the procedure (symptoms or screening). Effects of pathway (direct vs consult) were compared both within and between the screening and symptom subgroups. RESULTS Of 409 patients who completed the survey, 34% followed a direct pathway. Indications for colonoscopy were similar in each group. The majority of the participants were women (58%), married (61%), and internet users (81%). The most important information source was family physicians (Direct) and specialist physicians (Consult). Use of other information sources, including the internet (20% vs 18%) and Direct family and friends (64% vs 53%), was similar in the Direct and Consult groups, respectively. Only 31% of the 81% who were internet users accessed internet health information. Most sought fundamental information such as what a colonoscopy is or why it is done. Pre-procedure anxiety did not differ between care pathways. Those undergoing colonoscopy for symptoms reported greater anxiety [mean 5.3, 95

  10. AFM investigation and optical band gap study of chemically deposited PbS thin films

    NASA Astrophysics Data System (ADS)

    Zaman, S.; Mansoor, M.; Abubakar; Asim, M. M.

    2016-08-01

    The interest into deposition of nanocrystalline PbS thin films, the potential of designing and tailoring both the topographical features and the band gap energy (Eg) by controlling growth parameters, has significant technological importance. Nanocrystalline thin films of lead sulfide were grown onto glass substrates by chemical bath deposition (CBD) method. The experiments were carried out by varying deposition temperature. We report on the modification of structural and optical properties as a function of deposition temperature. The morphological changes of the films were analyzed by using SEM and AFM. AFM was also used to calculate average roughness of the films. XRD spectra indicated preferred growth of cubic phase of PbS films in (200) direction with increasing deposition time. Optical properties have been studied by UV-Spectrophotometer. From the diffused reflectance spectra we have calculated the optical Eg shift from 0.649-0.636 eV with increasing deposition time.

  11. New insights into the mucoadhesion of pectins by AFM roughness parameters in combination with SPR.

    PubMed

    Joergensen, Lars; Klösgen, Beate; Simonsen, Adam Cohen; Borch, Jonas; Hagesaether, Ellen

    2011-06-15

    The object of this study was to assess the mucoadhesion of the three main commercially available types of pectin by atomic force microscopy (AFM) and surface Plasmon resonance (SPR). Polyacrylic acid and polyvinyl pyrrolidone were used as positive and negative control, respectively. Image analysis of the AFM scans revealed a significant change of roughness parameters when low-ester pectin was introduced to mica supported bovine submaxillarymucin, indicating a high mucoadhesion for this type of pectin. Only minor changes were observed with high-ester and amidated pectin. The same ranking order of adhesion affinity was confirmed by SPR. In conclusion, a high specific mucin interaction of pectin with a high charge density was demonstrated directly on a molecular scale without interference from the viscoelastic properties or the intra-molecular interactions between the polymer chains themselves, using two independent methods.

  12. Influence of Fluid Cell Design on the Frequency Response of AFM Microcantilevers in Liquid Media

    PubMed Central

    Motamedi, Ramin; Wood-Adams, Paula M.

    2008-01-01

    A study of the frequency response of AFM microcantilevers in liquid media contained in a commercial fluid cell is presented. Such systems exhibit complicated dynamics which are often not well described by available theories. Their dynamic behavior has a direct effect on the use of the AFM in dynamic mode while imaging in liquid or while extracting the rheological properties of the fluid. We explore the issues related to the design of the cantilever holder/fluid cell and propose an approach for evaluating, minimizing and recognizing the ultimate limitations of commercial cantilever holders. A technique for estimating the frequency response spectrum of the fluid cell itself from experimental data is presented. This spectrum can then be used to evaluate whether or not the fluid cell is suited for the desired purpose. PMID:27873849

  13. Production of resident fish benefits from experimental salmon subsidies via direct and indirect pathways across stream-riparian boundaries

    USGS Publications Warehouse

    Collins, Scott F.; Baxter, Colden V.; Marcarelli, Amy M.; Wipfli, Mark S.

    2016-01-01

    Artificial additions of nutrients of differing forms such as salmon carcasses and analog pellets (i.e. pasteurized fishmeal) have been proposed as a means of stimulating aquatic productivity and enhancing populations of anadromous and resident fishes. Nutrient mitigation to enhance fish production in stream ecosystems assumes that the central pathway by which effects occur is bottom-up, through aquatic primary and secondary production, with little consideration of reciprocal aquatic-terrestrial pathways. The net outcome (i.e. bottom-up vs. top-down) of adding salmon-derived materials to streams depend on whether or not these subsidies indirectly intensify predation on in situ prey via increases in a shared predator or alleviate such predation pressure. We conducted a 3-year experiment across nine tributaries of the N. Fork Boise River, Idaho, USA, consisting of 500-m stream reaches treated with salmon carcasses (n = 3), salmon carcass analog (n = 3), and untreated control reaches (n = 3). We observed 2–8 fold increases in streambed biofilms in the 2–6 weeks following additions of both salmon subsidy treatments in years 1 and 2 and a 1.5-fold increase in standing crop biomass of aquatic invertebrates to carcass additions in the second year of our experiment. The consumption of benthic invertebrates by stream fishes increased 110–140% and 44–66% in carcass and analog streams in the same time frame, which may have masked invertebrate standing crop responses in years 3 and 4. Resident trout directly consumed 10.0–24.0 g·m−2·yr−1 of salmon carcass and <1–11.0 g·m−2·yr−1 of analog material, which resulted in 1.2–2.9 g·m−2·yr−1 and 0.03–1.4 g·m−2·yr−1 of tissue produced. In addition, a feedback flux of terrestrial maggots to streams contributed 0.0–2.0 g·m−2·yr−1 to trout production. Overall, treatments increased annual trout production by 2–3 fold, though density and biomass were unaffected. Our results

  14. In situ AFM Study of Amelogenin Assembly and Disassembly Dynamics on Charged Surfaces Provides Insights on Matrix Protein Self-Assembly

    PubMed Central

    Chen, Chun-Long; Bromley, Keith M.

    2012-01-01

    Because self-assembly of matrix proteins is a key step in hard tissue mineralization, developing an understanding of the assembly pathways and underlying mechanisms is likely to be important for successful hard tissue engineering. While many studies of matrix protein assembly have been performed on bulk solutions, in vivo these proteins are likely to be in contact with charged biological surfaces composed of lipids, proteins, or minerals. Here we report the results of an in situ AFM study of self-assembly by amelogenin - the principal protein of the extracellular matrix in developing enamel - in contact with two different charged substrates: hydrophilic negatively charged bare mica and positively charged 3-aminopropyl triethoxysilane (APS) silanized mica. First we demonstrate an AFM-based protocol for determining the size of both amelogenin monomers and oligomers. Using this protocol, we find that, although amelogenin exists primarily as ∼26 nm in diameter nanospheres in bulk solution at pH8.0 studied by DLS, it behaves dramatically differently upon interacting with charged substrates at the same pH, and exhibits complex substrate-dependent assembly pathways and dynamics. On positively charged APS-treated mica surfaces, amelogenin forms a relatively uniform population of decameric oligomers which then transforms into two main populations: higher-order assemblies of oligomers and amelogenin monomers, while on negatively charged bare mica surfaces, it forms a film of monomers that exhibits tip-induced desorption and patterning. The present study represents a successful attempt to identify the size of amelogenin oligomers and to directly monitor assembly and disassembly dynamics on surfaces. The findings have implications for amelogenin-controlled calcium phosphate mineralization in vitro and may offer new insights into in vivo self-assembly of matrix proteins, as well as their control over hard tissue formation. PMID:21916473

  15. Asymmetric direct α-alkylation of 2-oxindoles with Michler's hydrol catalyzed by bis-cinchona alkaloid-Brønsted acid via an SN1-type pathway.

    PubMed

    Zhang, Tao; Qiao, Zhen; Wang, Yan; Zhong, Nengjun; Liu, Li; Wang, Dong; Chen, Yong-Jun

    2013-02-25

    An enantioselective direct α-alkylation of 2-oxindoles with Michler's hydrol via an S(N)1-type pathway in the non-covalent activation mode using the bis-cinchona alkaloid and Brønsted acid as a co-catalyst was developed and good to high yields and enantioselectivities were obtained.

  16. Dopamine D1 Receptor-Mediated Transmission Maintains Information Flow Through the Cortico-Striato-Entopeduncular Direct Pathway to Release Movements.

    PubMed

    Chiken, Satomi; Sato, Asako; Ohta, Chikara; Kurokawa, Makoto; Arai, Satoshi; Maeshima, Jun; Sunayama-Morita, Tomoko; Sasaoka, Toshikuni; Nambu, Atsushi

    2015-12-01

    In the basal ganglia (BG), dopamine plays a pivotal role in motor control, and dopamine deficiency results in severe motor dysfunctions as seen in Parkinson's disease. According to the well-accepted model of the BG, dopamine activates striatal direct pathway neurons that directly project to the output nuclei of the BG through D1 receptors (D1Rs), whereas dopamine inhibits striatal indirect pathway neurons that project to the external pallidum (GPe) through D2 receptors. To clarify the exact role of dopaminergic transmission via D1Rs in vivo, we developed novel D1R knockdown mice in which D1Rs can be conditionally and reversibly regulated. Suppression of D1R expression by doxycycline treatment decreased spontaneous motor activity and impaired motor ability in the mice. Neuronal activity in the entopeduncular nucleus (EPN), one of the output nuclei of the rodent BG, was recorded in awake conditions to examine the mechanism of motor deficits. Cortically evoked inhibition in the EPN mediated by the cortico-striato-EPN direct pathway was mostly lost during suppression of D1R expression, whereas spontaneous firing rates and patterns remained unchanged. On the other hand, GPe activity changed little. These results suggest that D1R-mediated dopaminergic transmission maintains the information flow through the direct pathway to appropriately release motor actions.

  17. A theory about a role of the hyper direct pathway in pattern expression by the basal ganglia.

    PubMed

    Jourdan, Ivan; Barttfeld, Pablo; Zanutto, B Silvano

    2010-01-01

    The Basal Ganglia (BG) are a group of nuclei, in the brain of mammalians and other vertebrates, strongly connected with the cerebral cortex, thalamus and other brain areas. The BG are associated with several brain functions including learning and motor control. When there is cortical activation, there is a strong synchronization between BG and cortex, i.e. when a given task is being executed or in the case of Parkinson disease[1], [2]. If we consider the internal segment of the Globus Pallidus (GPi) there is synchronism between GPi-cortex at frequencies as low as 3Hz to as high as 85Hz [1], [3]. In the other hand, in a delta sleep or in an anesthetized case, a very low frequency correlation is observed (1-10 Hz), but no high frequency correlation between GPi-cortex [1], [2], [3]. It is unknown why this decorrelation happens. But It is agreement that when there is no pattern to select, like in delta sleep or with an anesthetized model, the BG network would maintain the GPi and cortex decorrelated at high frequencies. Many thalamus-BG and thalamus-BG-cortex loops are modulators of the BG activity. Particularly there exists an anatomic thalamus-BG loop, formed by GPi, intralaminar thalamic nuclei (IL) and Subthalamic Nucleus (STN) [4]. Using a computational model, based on an "Integrate and Fire" neural network, we analyzed the IL nucleus as a modulator of the so-called hyper direct pathway. Our results show that, in an anesthetic case, this thalamic path could be relevant to allow a high frequency decorrelated state between the GPi and cortex.

  18. Molecular dynamics study on the mechanism of AFM-based nanoscratching process with water-layer lubrication

    NASA Astrophysics Data System (ADS)

    Ren, Jiaqi; Zhao, Jinsheng; Dong, Zeguang; Liu, Pinkuan

    2015-08-01

    The atomic force microscopy (AFM) based direct nanoscratching has been thoroughly studied but the mechanism of nanoscratching with water-layer lubrication is yet to be well understood. In current study, three-dimensional molecular dynamics (MD) simulations are conducted to evaluate the effects of the water-layer lubrication on the AFM-based nanoscratching process on monocrystalline copper. Comparisons of workpiece deformation, scratching forces, and friction coefficients are made between the water-lubricated and dry scratching under various thickness of water layer, scratching depth and scratching velocity. Simulation results reveal that the water layer has positive impact on the surface quality and significant influence on the scratching forces (normal forces and tangential forces). The friction coefficients of the tip in water-lubricated nanoscratching are significantly bigger than those in the dry process. Our simulation results shed lights on a promising AFM-based nanofabrication method, which can assist to get nanoscale surface morphologies with higher quality than traditional approaches.

  19. New AFM Techniques for Investigating Molecular Growth Mechanisms of Protein Crystals

    NASA Technical Reports Server (NTRS)

    Li, Huayu; Nadarajah, Arunan; Konnert, John H.; Pusey, Marc L.

    1998-01-01

    . Measuring these units was not attempted before and most studies have assumed that the growth unit consisted of individual protein molecules. The linescan mode of AFM instruments allows the crystal surface to be scanned along a single line. By scanning across a growth step an image showing the motion of the step is obtained. Normally such an image shows a straight line for continuous and constant step velocity. In this study by increasing the scan rate and by decreasing the step velocity (by decreasing the supersaturation), we were able to capture images of individual growth events, shown by jump discontinuities in the step line. By suitable integration of the image the growth unit dimension in the scanned direction can be obtained. Since multiple units can be involved in the growth process it is necessary to collect a statistically relevant sample before drawing conclusions about the growth mechanism. This technique was successfully employed to obtain the dimensions of growth units for the (110) face, showing that they consisted of various aggregates corresponding to the 43 helices in the crystal structure.

  20. Granzyme H induces cell death primarily via a Bcl-2-sensitive mitochondrial cell death pathway that does not require direct Bid activation.

    PubMed

    Ewen, Catherine L; Kane, Kevin P; Bleackley, R Chris

    2013-07-01

    Natural killer and T cell-mediated cytotoxicity is important for the elimination of viruses and transformed cells. The granule lytic pathway utilizes perforin and granzymes to induce cell death, while receptor-mediated lytic pathways rely on molecules such as FasL. Pro-apoptotic activities of Granzyme B (GrB) and Fas are well-established, and many of their cellular targets have been identified. However, humans express additional related granzymes - GrA, GrM, GrK, and GrH. Neither the cytotoxic potential of GrH, nor the mechanism by which GrH may induce target cell death is currently understood. We proposed that GrH would have pro-apoptotic activity that would be distinct from that of GrB and FasL, which could be relevant when Fas/FasL or GrB activity or death pathways were impaired. Our results, using a purified recombinant form of GrH, revealed that GrH induced cell death via a Bcl-2-sensitive mitochondrial pathway without direct processing of Bid. Additionally, neither the apoptosome nor caspase-3 was essential to the induction of GrH-mediated cell death. However, GrH did directly process DFF45, potentially leading to DNA damage. Our findings support the idea that multiple, non-redundant death pathways may be initiated by cytotoxic cells to counteract various immune evasion strategies.

  1. Low tip damage AFM technique development for nano structures characterization

    NASA Astrophysics Data System (ADS)

    Liu, Biao; Wang, Charles C.; Huang, Po-Fu; Uritsky, Yuri

    2010-06-01

    Ambient dynamic mode (tapping mode or intermittent-contact mode) AFM imaging has been used extensively for the characterization of the topography of nano structures. However, the results are beset with artifacts, because hard tapping of the AFM tip on sample surface usually causes premature tip damage. Through careful study of the cantilever amplitude and phase signals as functions of tip-to-sample distance, principle of non-contact AFM operation was discovered to enable high resolution and low tip damage AFM image acquisition [1, 2]. However, current study discovers that the conventional way of acquiring amplitude and phase versus distance curves gives erroneous non-contact operating range, because the tip gets damaged during the data acquisition process. A new technique is developed to reliably map the operating parameters of an intact tip that ensures the AFM be operated with the correct non-contact settings. Two examples are given to illustrate the successful applications of this new technique. The first example involves the size characterization of polystyrene latex (PSL) nano particles used for light scattering tool calibration. The second example is the development of robust recipes for the measurement of the depth of phase-shift mask trenches.

  2. A low-cost AFM setup with an interferometer for undergraduates and secondary-school students

    NASA Astrophysics Data System (ADS)

    Bergmann, Antje; Feigl, Daniela; Kuhn, David; Schaupp, Manuel; Quast, Günter; Busch, Kurt; Eichner, Ludwig; Schumacher, Jens

    2013-07-01

    Atomic force microscopy (AFM) is an important tool in nanotechnology. This method makes it possible to observe nanoscopic surfaces beyond the resolution of light microscopy. In order to provide undergraduate and secondary-school students with insights into this world, we have developed a very robust low-cost AFM setup with a Fabry-Perot interferometer as a detecting device. This setup is designed to be operated almost completely manually and its simplicity gives access to a profound understanding of the working principle. Our AFM is operated in a constant height mode, i.e. the topography of the sample surface is represented directly by the deflection of the cantilever. Thus, the measuring procedure can be understood even by secondary-school students; furthermore, it is the method with the lowest cost, totalling not more than 10-15 k Euros. Nevertheless, we are able to examine a large variety of sample topographies such as CD and DVD surfaces, IC structures, blood cells, butterfly wings or moth eyes. Furthermore, force-distance curves can be recorded and the tensile moduli of some materials can be evaluated. We present our setup in detail and describe its working principles. In addition, we show various experiments which have already been performed by students.

  3. Graphene Nanopore Support System for Simultaneous High-Resolution AFM Imaging and Conductance Measurements

    PubMed Central

    2015-01-01

    Accurately defining the nanoporous structure and sensing the ionic flow across nanoscale pores in thin films and membranes has a wide range of applications, including characterization of biological ion channels and receptors, DNA sequencing, molecule separation by nanoparticle films, sensing by block co-polymers films, and catalysis through metal–organic frameworks. Ionic conductance through nanopores is often regulated by their 3D structures, a relationship that can be accurately determined only by their simultaneous measurements. However, defining their structure–function relationships directly by any existing techniques is still not possible. Atomic force microscopy (AFM) can image the structures of these pores at high resolution in an aqueous environment, and electrophysiological techniques can measure ion flow through individual nanoscale pores. Combining these techniques is limited by the lack of nanoscale interfaces. We have designed a graphene-based single-nanopore support (∼5 nm thick with ∼20 nm pore diameter) and have integrated AFM imaging and ionic conductance recording using our newly designed double-chamber recording system to study an overlaid thin film. The functionality of this integrated system is demonstrated by electrical recording (<10 pS conductance) of suspended lipid bilayers spanning a nanopore and simultaneous AFM imaging of the bilayer. PMID:24581087

  4. Fractal properties of macrophage membrane studied by AFM.

    PubMed

    Bitler, A; Dover, R; Shai, Y

    2012-12-01

    Complexity of cell membrane poses difficulties to quantify corresponding morphology changes during cell proliferation and damage. We suggest using fractal dimension of the cell membrane to quantify its complexity and track changes produced by various treatments. Glutaraldehyde fixed mouse RAW 264.7 macrophage membranes were chosen as model system and imaged in PeakForce QNM (quantitative nanomechanics) mode of AFM (atomic force microscope). The morphology of the membranes was characterized by fractal dimension. The parameter was calculated for set of AFM images by three different methods. The same calculations were done for the AFM images of macrophages treated with colchicine, an inhibitor of the microtubule polymerization, and microtubule stabilizing agent taxol. We conclude that fractal dimension can be additional and useful parameter to characterize the cell membrane complexity and track the morphology changes produced by different treatments.

  5. AFM of biological complexes: what can we learn?

    PubMed Central

    Gaczynska, Maria; Osmulski, Pawel A.

    2009-01-01

    The term “biological complexes” broadly encompasses particles as diverse as multisubunit enzymes, viral capsids, transport cages, molecular nets, ribosomes, nucleosomes, biological membrane components and amyloids. The complexes represent a broad range of stability and composition. Atomic force microscopy offers a wealth of structural and functional data about such assemblies. For this review, we choose to comment on the significance of AFM to study various aspects of biology of selected nonmembrane protein assemblies. Such particles are large enough to reveal many structural details under the AFM probe. Importantly, the specific advantages of the method allow for gathering dynamic information about their formation, stability or allosteric structural changes critical for their function. Some of them have already found their way to nanomedical or nanotechnological applications. Here we present examples of studies where the AFM provided pioneering information about the biology of complexes, and examples of studies where the simplicity of the method is used toward the development of potential diagnostic applications. PMID:19802337

  6. Sub-diffraction nano manipulation using STED AFM.

    PubMed

    Chacko, Jenu Varghese; Canale, Claudio; Harke, Benjamin; Diaspro, Alberto

    2013-01-01

    In the last two decades, nano manipulation has been recognized as a potential tool of scientific interest especially in nanotechnology and nano-robotics. Contemporary optical microscopy (super resolution) techniques have also reached the nanometer scale resolution to visualize this and hence a combination of super resolution aided nano manipulation ineluctably gives a new perspective to the scenario. Here we demonstrate how specificity and rapid determination of structures provided by stimulated emission depletion (STED) microscope can aid another microscopic tool with capability of mechanical manoeuvring, like an atomic force microscope (AFM) to get topological information or to target nano scaled materials. We also give proof of principle on how high-resolution real time visualization can improve nano manipulation capability within a dense sample, and how STED-AFM is an optimal combination for this job. With these evidences, this article points to future precise nano dissections and maybe even to a nano-snooker game with an AFM tip and fluorospheres.

  7. Optimization of phase contrast in bimodal amplitude modulation AFM

    PubMed Central

    Damircheli, Mehrnoosh; Payam, Amir F

    2015-01-01

    Summary Bimodal force microscopy has expanded the capabilities of atomic force microscopy (AFM) by providing high spatial resolution images, compositional contrast and quantitative mapping of material properties without compromising the data acquisition speed. In the first bimodal AFM configuration, an amplitude feedback loop keeps constant the amplitude of the first mode while the observables of the second mode have not feedback restrictions (bimodal AM). Here we study the conditions to enhance the compositional contrast in bimodal AM while imaging heterogeneous materials. The contrast has a maximum by decreasing the amplitude of the second mode. We demonstrate that the roles of the excited modes are asymmetric. The operational range of bimodal AM is maximized when the second mode is free to follow changes in the force. We also study the contrast in trimodal AFM by analyzing the kinetic energy ratios. The phase contrast improves by decreasing the energy of second mode relative to those of the first and third modes. PMID:26114079

  8. Mounting of Escherichia coli spheroplasts for AFM imaging.

    SciTech Connect

    Sullivan, Claretta J; Morrell-Falvey, Jennifer L; Allison, David P; Doktycz, Mitchel John

    2005-11-01

    The cytoplasmic membrane of Escherichia coli (E. coli) is the location of numerous, chemically specific transporters and recognition elements. Investigation of this membrane in vivo by atomic force microscopy (AFM) requires removal of the cell wall and stable immobilization of the spheroplast. AFM images demonstrate that spheroplasts can be secured with warm gelatin applied to the mica substrate just before the addition of a spheroplast suspension. The resulting preparation can be repeatedly imaged by AFM over the course of several hours. Confocal fluorescence imaging confirms the association of the spheroplasts with the gelatin layer. Gelatin molecules are known to reorder into a network after heating. Entrapment within this gelatin network is believed to be responsible for the immobilization of spheroplasts on mica.

  9. Evaluating legacy contaminants and emerging chemicals in marine environments using adverse outcome pathways and biological effects-directed analysis.

    PubMed

    Hutchinson, Thomas H; Lyons, Brett P; Thain, John E; Law, Robin J

    2013-09-30

    important scientific, economic and health challenges. In order to meet these challenges and pursue cost-effective scientific approaches that can provide evidence necessary to support policy needs (e.g. the European Marine Strategy Framework Directive), it is widely recognised that there is a need to (i) provide marine exposure assessments for priority contaminants using a range of validated models, passive samplers and biomarkers; (ii) integrate chemical monitoring data with biological effects data across spatial and temporal scales (including quality controls); and (iii) strengthen the evidence base to understand the relationship between exposure to complex chemical mixtures, biological and ecological impacts through integrated approaches and molecular data (e.g. genomics, proteomics and metabolomics). Additionally, we support the widely held view that (iv) that rather than increasing the analytical chemistry monitoring of large number of emerging contaminants, it will be important to target analytical chemistry towards key groups of chemicals of concern using effects-directed analysis. It is also important to evaluate to what extent existing biomarkers and bioassays can address various classes of emerging chemicals using the adverse outcome pathway (AOP) approach now being developed by the Organization for Economic Cooperation and Development (OECD) with respect to human toxicology and ecotoxicology.

  10. BOREAS AFM-04 Twin Otter Aircraft Flux Data

    NASA Technical Reports Server (NTRS)

    MacPherson, J. Ian; Hall, Forrest G. (Editor); Knapp, David E. (Editor); Desjardins, Raymond L.; Smith, David E. (Technical Monitor)

    2000-01-01

    The BOREAS AFM-5 team collected and processed data from the numerous radiosonde flights during the project. The goals of the AFM-05 team were to provide large-scale definition of the atmosphere by supplementing the existing AES aerological network, both temporally and spatially. This data set includes basic upper-air parameters collected from the network of upper-air stations during the 1993, 1994, and 1996 field campaigns over the entire study region. The data are contained in tabular ASCII files. The data files are available on a CD-ROM (see document number 20010000884) or from the Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC).

  11. Determining surface properties with bimodal and multimodal AFM.

    PubMed

    Forchheimer, D; Borysov, Stanislav S; Platz, D; Haviland, David B

    2014-12-05

    Conventional dynamic atomic force microscopy (AFM) can be extended to bimodal and multimodal AFM in which the cantilever is simultaneously excited at two or more resonance frequencies. Such excitation schemes result in one additional amplitude and phase images for each driven resonance, and potentially convey more information about the surface under investigation. Here we present a theoretical basis for using this information to approximate the parameters of a tip-surface interaction model. The theory is verified by simulations with added noise corresponding to room-temperature measurements.

  12. Direct comparison of 3-centre and 4-centre HBr elimination pathways in methyl-substituted vinyl bromides.

    PubMed

    Pandit, Shubhrangshu; Hornung, Balázs; Orr-Ewing, Andrew J

    2016-10-12

    Elimination of HBr from UV-photoexcited vinyl bromides can occur through both 3-centre and 4-centre transition states (TSs). The competition between these pathways is examined using velocity map imaging of HBr (v = 0-2, J) photofragments. The three vinyl bromides chosen for study have methyl substituents that block either the 3-centre or the 4-centre TS, or leave both pathways open. The kinetic energy distributions extracted from velocity map images of HBr from 193 nm photolysis of the three vinyl bromide compounds are approximately described by a statistical model of energy disposal among the degrees of freedom of the photoproducts, and are attributed to dissociation on the lowest electronic state of the molecule after internal conversion. Dissociation via the 4-centre TS gives greater average kinetic energy release than for the 3-centre TS pathway. The resonance enhanced multi-photon ionization (REMPI) schemes used to detect HBr restrict measurements to J ≤ 7 for v = 2 and J ≤ 15 for v = 0. Within this spectroscopic range, the HBr rotational temperature is colder for the 4-centre than for the 3-centre elimination pathway. Calculations of the intrinsic reaction coordinates and RRKM calculations of HBr elimination rate coefficients provide mechanistic insights into the competition between the pathways.

  13. Measurement of Cationic and Intracellular Modulation of Integrin Binding Affinity by AFM-Based Nanorobot

    PubMed Central

    Patterson, Kevin C.; Yang, Ruiguo; Zeng, Bixi; Song, Bo; Wang, Shouye; Xi, Ning; Basson, Marc D.

    2013-01-01

    Integrins are dynamic transmembrane cation-dependent heterodimers that both anchor cells in position and transduce signals into and out of cells. We used an atomic force microscope (AFM)-based nanorobotic system to measure integrin-binding forces in intact human intestinal epithelial Caco-2 cells. The AFM-based nanorobot enables human-directed, high-accuracy probe positioning and site-specific investigations. Functionalizing the AFM probe with an arginine-glycine-aspartate (RGD)-containing sequence (consensus binding sequence for integrins) allowed us to detect a series of peptide-cell membrane interactions with a median binding force of 115.1 ± 4.9 pN that were not detected in control interactions. Chelating divalent cations from the culture medium abolished these interactions, as did inhibiting intracellular focal adhesion kinase (FAK) using Y15. Adding 1 mM Mg2+ to the medium caused a rightward shift in the force-binding curve. Adding 1 mM Ca2+ virtually abolished the RGD-membrane specific interactions and blocked the Mg2+ effects. Cell adhesion assays demonstrated parallel effects of divalent cations and the FAK inhibitor on cell adhesion. These results demonstrate direct modulation of integrin-binding affinity by both divalent cations and intracellular signal inhibition. Additionally, three binding states (nonspecific, specific inactivated, and specific activated) were delineated from affinity measurements. Although other research has assumed that this process of integrin conformational change causes altered ligand binding, in this work we directly measured these three states in individual integrins in a physiologically based study. PMID:23823222

  14. Measurement of cationic and intracellular modulation of integrin binding affinity by AFM-based nanorobot.

    PubMed

    Patterson, Kevin C; Yang, Ruiguo; Zeng, Bixi; Song, Bo; Wang, Shouye; Xi, Ning; Basson, Marc D

    2013-07-02

    Integrins are dynamic transmembrane cation-dependent heterodimers that both anchor cells in position and transduce signals into and out of cells. We used an atomic force microscope (AFM)-based nanorobotic system to measure integrin-binding forces in intact human intestinal epithelial Caco-2 cells. The AFM-based nanorobot enables human-directed, high-accuracy probe positioning and site-specific investigations. Functionalizing the AFM probe with an arginine-glycine-aspartate (RGD)-containing sequence (consensus binding sequence for integrins) allowed us to detect a series of peptide-cell membrane interactions with a median binding force of 115.1 ± 4.9 pN that were not detected in control interactions. Chelating divalent cations from the culture medium abolished these interactions, as did inhibiting intracellular focal adhesion kinase (FAK) using Y15. Adding 1 mM Mg(2+) to the medium caused a rightward shift in the force-binding curve. Adding 1 mM Ca(2+) virtually abolished the RGD-membrane specific interactions and blocked the Mg(2+) effects. Cell adhesion assays demonstrated parallel effects of divalent cations and the FAK inhibitor on cell adhesion. These results demonstrate direct modulation of integrin-binding affinity by both divalent cations and intracellular signal inhibition. Additionally, three binding states (nonspecific, specific inactivated, and specific activated) were delineated from affinity measurements. Although other research has assumed that this process of integrin conformational change causes altered ligand binding, in this work we directly measured these three states in individual integrins in a physiologically based study.

  15. A dileucine-like sorting signal directs transport into an AP-3-dependent, clathrin-independent pathway to the yeast vacuole.

    PubMed Central

    Vowels, J J; Payne, G S

    1998-01-01

    Transport of yeast alkaline phosphatase (ALP) to the vacuole depends on the clathrin adaptor-like complex AP-3, but does not depend on proteins necessary for transport through pre-vacuolar endosomes. We have identified ALP sequences that direct sorting into the AP-3-dependent pathway using chimeric proteins containing residues from the ALP cytoplasmic domain fused to sequences from a Golgi-localized membrane protein, guanosine diphosphatase (GDPase). The full-length ALP cytoplasmic domain, or ALP amino acids 1-16 separated from the transmembrane domain by a spacer, directed GDPase chimeric proteins from the Golgi complex to the vacuole via the AP-3 pathway. Mutation of residues Leu13 and Val14 within the ALP cytoplasmic domain prevented AP-3-dependent vacuolar transport of both chimeric proteins and full-length ALP. This Leucine-Valine (LV)-based sorting signal targeted chimeric proteins and native ALP to the vacuole in cells lacking clathrin function. These results identify an LV-based sorting signal in the ALP cytoplasmic domain that directs transport into a clathrin-independent, AP-3-dependent pathway to the vacuole. The similarity of the ALP sorting signal to mammalian dileucine sorting motifs, and the evolutionary conservation of AP-3 subunits, suggests that dileucine-like signals constitute a core element for AP-3-dependent transport to lysosomal compartments in all eukaryotic cells. PMID:9564031

  16. AFM-IR: Technology and Applications in Nanoscale Infrared Spectroscopy and Chemical Imaging.

    PubMed

    Dazzi, Alexandre; Prater, Craig B

    2016-12-13

    Atomic force microscopy-based infrared spectroscopy (AFM-IR) is a rapidly emerging technique that provides chemical analysis and compositional mapping with spatial resolution far below conventional optical diffraction limits. AFM-IR works by using the tip of an AFM probe to locally detect thermal expansion in a sample resulting from absorption of infrared radiation. AFM-IR thus can provide the spatial resolution of AFM in combination with the chemical analysis and compositional imaging capabilities of infrared spectroscopy. This article briefly reviews the development and underlying technology of AFM-IR, including recent advances, and then surveys a wide range of applications and investigations using AFM-IR. AFM-IR applications that will be discussed include those in polymers, life sciences, photonics, solar cells, semiconductors, pharmaceuticals, and cultural heritage. In the Supporting Information , the authors provide a theoretical section that reviews the physics underlying the AFM-IR measurement and detection mechanisms.

  17. 3D Color Digital Elevation Map of AFM Sample

    NASA Technical Reports Server (NTRS)

    2008-01-01

    This color image is a three dimensional (3D) view of a digital elevation map of a sample collected by NASA's Phoenix Mars Lander's Atomic Force Microscope (AFM).

    The image shows four round pits, only 5 microns in depth, that were micromachined into the silicon substrate, which is the background plane shown in red. This image has been processed to reflect the levelness of the substrate.

    A Martian particle only one micrometer, or one millionth of a meter, across is held in the upper left pit.

    The rounded particle shown at the highest magnification ever seen from another world is a particle of the dust that cloaks Mars. Such dust particles color the Martian sky pink, feed storms that regularly envelop the planet and produce Mars' distinctive red soil.

    The particle was part of a sample informally called 'Sorceress' delivered to the AFM on the 38th Martian day, or sol, of the mission (July 2, 2008). The AFM is part of Phoenix's microscopic station called MECA, or the Microscopy, Electrochemistry, and Conductivity Analyzer.

    The AFM was developed by a Swiss-led consortium, with Imperial College London producing the silicon substrate that holds sampled particles.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  18. Probing the Double Layer: Effect of Image Forces on AFM

    PubMed Central

    Sachs, Frederick

    2006-01-01

    Force probes such as AFM tips or laser trap latex beads have a dielectric constant much less than that of the water that they displace. Thus when a probe approaches a charged surface under water it will be repelled simply based upon the image forces, and these can be of nN magnitude. PMID:16714346

  19. Structural investigations on native collagen type I fibrils using AFM

    SciTech Connect

    Strasser, Stefan; Zink, Albert; Janko, Marek; Heckl, Wolfgang M.; Thalhammer, Stefan . E-mail: stefan.thalhammer@gsf.de

    2007-03-02

    This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.

  20. Cantilever's behavior in the AC mode of an AFM

    SciTech Connect

    Nunes, V.B.; Zanette, S.I.; Caride, A.O.; Prioli, R.; Rivas, A.M.F

    2003-03-15

    In this paper, a model with a small number of parameters is used to simulate the motion of a cantilever in the AC mode of an atomic force microscope (AFM). The results elucidate the transition dependence-from noncontact to tapping operating mode-on the height of the contamination layer and on the stiffness of the sample.

  1. High-speed AFM images of thermal motion provide stiffness map of interfacial membrane protein moieties.

    PubMed

    Preiner, Johannes; Horner, Andreas; Karner, Andreas; Ollinger, Nicole; Siligan, Christine; Pohl, Peter; Hinterdorfer, Peter

    2015-01-14

    The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges.

  2. High-Speed AFM Images of Thermal Motion Provide Stiffness Map of Interfacial Membrane Protein Moieties

    PubMed Central

    2014-01-01

    The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges. PMID:25516527

  3. Scanning hall probe microscopy (SHPM) using quartz crystal AFM feedback.

    PubMed

    Dede, M; Urkmen, K; Girişen, O; Atabak, M; Oral, A; Farrer, I; Ritchie, D

    2008-02-01

    Scanning Hall Probe Microscopy (SHPM) is a quantitative and non-invasive technique for imaging localized surface magnetic field fluctuations such as ferromagnetic domains with high spatial and magnetic field resolution of approximately 50 nm and 7 mG/Hz(1/2) at room temperature. In the SHPM technique, scanning tunneling microscope (STM) or atomic force microscope (AFM) feedback is used to keep the Hall sensor in close proximity of the sample surface. However, STM tracking SHPM requires conductive samples; therefore the insulating substrates have to be coated with a thin layer of gold. This constraint can be eliminated with the AFM feedback using sophisticated Hall probes that are integrated with AFM cantilevers. However it is very difficult to micro fabricate these sensors. In this work, we have eliminated the difficulty in the cantilever-Hall probe integration process, just by gluing a Hall Probe chip to a quartz crystal tuning fork force sensor. The Hall sensor chip is simply glued at the end of a 32.768 kHz or 100 kHz Quartz crystal, which is used as force sensor. An LT-SHPM system is used to scan the samples. The sensor assembly is dithered at the resonance frequency using a digital Phase Locked Loop circuit and frequency shifts are used for AFM tracking. SHPM electronics is modified to detect AFM topography and the frequency shift, along with the magnetic field image. Magnetic domains and topography of an Iron Garnet thin film crystal, NdFeB demagnetised magnet and hard disk samples are presented at room temperature. The performance is found to be comparable with the SHPM using STM feedback.

  4. Possible enhancements of AFM spin-fluctuations in high-TC cuprates

    NASA Astrophysics Data System (ADS)

    Jarlborg, Thomas

    2009-03-01

    Ab-initio band calculations for high-TC cuprates, together with modelling based of a free electron like band, show a strong interaction between anti-ferromagnetic (AFM) spin waves and periodic lattice distortions as for phonons, even though this type of spin-phonon coupling (SPC) is underestimated in calculations using the local density approximation. The SPC has a direct influence on the properties of the HTC cuprates and it can explain many observations. The strongest effects are seen for modulated waves in the CuO bond direction, and a band gap is formed near the X,Y points, but unusal band dispersion (like ``waterfalls'') might also be induced below the Fermi energy (EF) in the diagonal direction. The band results are used to propose different ways of increasing AFM spin-fluctuations locally, and to have a higher density-of-states (DOS) at EF. Static potential modulations, via periodic distribution of dopants or lattice distortions, can be tuned to increase the DOS. This opens for possibilities to enhance coupling for spin fluctuations (λsf) and superconductivity. The exchange enhancement is in general increased near a surface, which suggests a tendency towards static spin configurations. The sensivity of the band results to corrections of the local density potential are discussed.

  5. A rapid, membrane-dependent pathway directs furrow formation through RalA in the early Drosophila embryo

    PubMed Central

    Holly, Ryan M.; Mavor, Lauren M.; Zuo, Zhongyuan; Blankenship, J. Todd

    2015-01-01

    Plasma membrane furrow formation is crucial in cell division and cytokinesis. Furrow formation in early syncytial Drosophila embryos is exceptionally rapid, with furrows forming in as little as 3.75 min. Here, we use 4D imaging to identify furrow formation, stabilization, and regression periods, and identify a rapid, membrane-dependent pathway that is essential for plasma membrane furrow formation in vivo. Myosin II function is thought to provide the ingression force for cytokinetic furrows, but the role of membrane trafficking pathways in guiding furrow formation is less clear. We demonstrate that a membrane trafficking pathway centered on Ras-like protein A (RalA) is required for fast furrow ingression in the early fly embryo. RalA function is absolutely required for furrow formation and initiation. In the absence of RalA and furrow function, chromosomal segregation is aberrant and polyploid nuclei are observed. RalA localizes to syncytial furrows, and mediates the movement of exocytic vesicles to the plasma membrane. Sec5, which is an exocyst complex subunit and localizes to ingressing furrows in wild-type embryos, becomes punctate and loses its cortical association in the absence of RalA function. Rab8 also fails to traffic to the plasma membrane and accumulates aberrantly in the cytoplasm in RalA disrupted embryos. RalA localization precedes F-actin recruitment to the furrow tip, suggesting that membrane trafficking might function upstream of cytoskeletal remodeling. These studies identify a pathway, which stretches from Rab8 to RalA and the exocyst complex, that mediates rapid furrow formation in early Drosophila embryos. PMID:26092850

  6. New paradigm in ethylene signaling: EIN2, the central regulator of the signaling pathway, interacts directly with the upstream receptors.

    PubMed

    Bisson, Melanie M A; Groth, Georg

    2011-01-01

    The membrane protein ETHYLENE INSENSITIVE2 (EIN2), which is supposed to act between the soluble serine/threonine kinase CTR1 and the EIN3/EIL family of transcription factors, is a central and most critical element of the ethylene signaling pathway in Arabidopsis. In a recent study, we have identified that EIN2 interacts tightly with all members of the Arabidopsis ethylene receptor family - proteins that mark the starting point of the signaling pathway. Our studies show consistently that the kinase domain of the receptors is essential for the formation of the EIN2-receptor complex. Furthermore, mutational analysis demonstrates that phosphorylation is a key mechanism in controlling the interaction of EIN2 and the ethylene receptors. Interaction studies in the presence of the ethylene agonist cyanide revealed a causal link between hormone binding and complex formation. In the presence of the plant hormone agonist the auto-kinase activity of the receptors is inhibited and the non-phosphorylated kinase domain of the receptors binds tightly to the carboxyl-terminal domain of EIN2. In the absence of cyanide inhibition of the auto-kinase activity is relieved and complex formation with the phosphorylated kinase domain of the receptors is reduced. Our data suggest a novel model on the integration of EIN2 in the ethylene signaling pathway.

  7. A Direct Link between Abscisic Acid Sensing and the Chromatin-Remodeling ATPase BRAHMA via Core ABA Signaling Pathway Components.

    PubMed

    Peirats-Llobet, Marta; Han, Soon-Ki; Gonzalez-Guzman, Miguel; Jeong, Cheol Woong; Rodriguez, Lesia; Belda-Palazon, Borja; Wagner, Doris; Rodriguez, Pedro L

    2016-01-04

    Optimal response to drought is critical for plant survival and will affect biodiversity and crop performance during climate change. Mitotically heritable epigenetic or dynamic chromatin state changes have been implicated in the plant response to the drought stress hormone abscisic acid (ABA). The Arabidopsis SWI/SNF chromatin-remodeling ATPase BRAHMA (BRM) modulates response to ABA by preventing premature activation of stress response pathways during germination. We show that core ABA signaling pathway components physically interact with BRM and post-translationally modify BRM by phosphorylation/dephosphorylation. Genetic evidence suggests that BRM acts downstream of SnRK2.2/2.3 kinases, and biochemical studies identified phosphorylation sites in the C-terminal region of BRM at SnRK2 target sites that are evolutionarily conserved. Finally, the phosphomimetic BRM(S1760D S1762D) mutant displays ABA hypersensitivity. Prior studies showed that BRM resides at target loci in the ABA pathway in the presence and absence of the stimulus, but is only active in the absence of ABA. Our data suggest that SnRK2-dependent phosphorylation of BRM leads to its inhibition, and PP2CA-mediated dephosphorylation of BRM restores the ability of BRM to repress ABA response. These findings point to the presence of a rapid phosphorylation-based switch to control BRM activity; this property could be potentially harnessed to improve drought tolerance in plants.

  8. Immune Cell Regulatory Pathways Unexplored as Host-Directed Therapeutic Targets for Mycobacterium tuberculosis: An Opportunity to Apply Precision Medicine Innovations to Infectious Diseases

    PubMed Central

    Mahon, Robert N.; Hafner, Richard

    2015-01-01

    The lack of novel antimicrobial drugs in development for tuberculosis treatment has provided an impetus for the discovery of adjunctive host-directed therapies (HDTs). Several promising HDT candidates are being evaluated, but major advancement of tuberculosis HDTs will require understanding of the master or “core” cell signaling pathways that control intersecting immunologic and metabolic regulatory mechanisms, collectively described as “immunometabolism.” Core regulatory pathways conserved in all eukaryotic cells include poly (ADP-ribose) polymerases (PARPs), sirtuins, AMP-activated protein kinase (AMPK), and mechanistic target of rapamycin (mTOR) signaling. Critical interactions of these signaling pathways with each other and their roles as master regulators of immunometabolic functions will be addressed, as well as how Mycobacterium tuberculosis is already known to influence various other cell signaling pathways interacting with them. Knowledge of these essential mechanisms of cell function regulation has led to breakthrough targeted treatment advances for many diseases, most prominently in oncology. Leveraging these exciting advances in precision medicine for the development of innovative next-generation HDTs may lead to entirely new paradigms for treatment and prevention of tuberculosis and other infectious diseases. PMID:26409283

  9. Understanding the TERS Effect with On-line Tunneling and Force Feedback Using Multiprobe AFM/NSOM with Raman Integration

    NASA Astrophysics Data System (ADS)

    Lewis, Aaron; Dekhter, Rimma; Hamra, Patricia; Bar-David, Yossi; Taha, Hesham

    Tip enhanced Raman scattering (TERS) has evolved in several directions over the past years. The data from this variety of methodologies has now accumulated to the point that there is a reasonable possibility of evolving an understanding of the underlying cause of the resulting effects that could be the origin of the various TERS enhancement processes. The objective of this presentation is to use the results thus far with atomic force microscopy (AFM) probes with noble metal coating, etching, transparent gold nanoparticles with and without a second nanoparticle [Wang and Schultz, ANALYST 138, 3150 (2013)] and tunneling feedback probes [R. Zhang et. al., NATURE 4 9 8, 8 2 (2013)]. We attempt at understanding this complex of results with AFM/NSOM multiprobe techniques. Results indicate that TERS is dominated by complex quantum interactions. This produces a highly confined and broadband plasmon field with all k vectors for effective excitation. Normal force tuning fork feedback with exposed tip probes provides an excellent means to investigate these effects with TERS probes that we have shown can circumvent the vexing problem of jump to contact prevalent in conventional AFM methodology and permit on-line switching between tunneling and AFM feedback modes of operation.

  10. High aspect ratio AFM Probe processing by helium-ion-beam induced deposition.

    PubMed

    Onishi, Keiko; Guo, Hongxuan; Nagano, Syoko; Fujita, Daisuke

    2014-11-01

    A Scanning Helium Ion Microscope (SHIM) is a high resolution surface observation instrument similar to a Scanning Electron Microscope (SEM) since both instruments employ finely focused particle beams of ions or electrons [1]. The apparent difference is that SHIMs can be used not only for a sub-nanometer scale resolution microscopic research, but also for the applications of very fine fabrication and direct lithography of surfaces at the nanoscale dimensions. On the other hand, atomic force microscope (AFM) is another type of high resolution microscopy which can measure a three-dimensional surface morphology by tracing a fine probe with a sharp tip apex on a specimen's surface.In order to measure highly uneven and concavo-convex surfaces by AFM, the probe of a high aspect ratio with a sharp tip is much more necessary than the probe of a general quadrangular pyramid shape. In this paper we report the manufacture of the probe tip of the high aspect ratio by ion-beam induced gas deposition using a nanoscale helium ion beam of SHIM.Gas of platinum organic compound was injected into the sample surface neighborhood in the vacuum chamber of SHIM. The decomposition of the gas and the precipitation of the involved metal brought up a platinum nano-object in a pillar shape on the normal commercial AFM probe tip. A SHIM system (Carl Zeiss, Orion Plus) equipped with the gas injection system (OmniProbe, OmniGIS) was used for the research. While the vacuum being kept to work, we injected platinum organic compound ((CH3)3(CH3C5H4)Pt) into the sample neighborhood and irradiated the helium ion beam with the shape of a point on the apex of the AFM probe tip. It is found that we can control the length of the Pt nano-pillar by irradiation time of the helium ion beam. The AFM probe which brought up a Pt nano-pillar is shown in Figure 1. It is revealed that a high-aspect-ratio Pt nano-pillar of ∼40nm diameter and up to ∼2000 nm length can be grown. In addition, for possible heating

  11. Ab initio study of CO2 capture mechanisms in aqueous monoethanolamine: reaction pathways for the direct interconversion of carbamate and bicarbonate.

    PubMed

    Matsuzaki, Yoichi; Yamada, Hidetaka; Chowdhury, Firoz A; Higashii, Takayuki; Onoda, Masami

    2013-09-26

    Ab initio molecular orbital calculations combined with the polarizable continuum model (PCM) formalism have been carried out for a comprehensive understanding of the mechanism of carbon dioxide (CO2) absorption by aqueous amine solutions. CO2 is captured by amines to generate carbamates and bicarbonate. We have examined the direct interconversion pathways of these two species (collectively represented by a reversible hydrolysis of carbamate) with the prototypical amine, monoethanolamine (MEA). We evaluate both a concerted and a stepwise mechanism for the neutral hydrolysis of MEA carbamate. Large activation energies (ca. 36 kcal/mol) and lack of increase in catalytic efficiency with the inclusion of additional water molecules are predicted in both the mechanisms. We also examined the mechanism of alkaline hydrolysis of MEA carbamate at high concentrations of amine (high pH). The addition of OH(-) ion to carbamate anion was theoretically not allowed due to the reduction in the nucleophilicity of the former as a result of microsolvation. We propose an alternative pathway for hydrolysis: a proton transfer from protonated MEA to carbamate to generate the carbamic acid that initially undergoes a nucleophilic addition of OH(-) and subsequent low-barrier reactions leading to the formation of bicarbonate and free MEA. On the basis of the calculated activation energies, this pathway would be the most efficient route for the direct interconversion of carbamate and bicarbonate without the intermediacy of the free CO2, while the actual contributions will be dependent on the concentrations of protonated MEA and OH(-) ions.

  12. IL-2 augments the therapeutic efficacy of adoptively transferred B cells which directly kill tumor cells via the CXCR4/CXCL12 and perforin pathways

    PubMed Central

    Chen, Xin; Xia, Leiming; Zhou, Li; Wang, Yi; Bao, Yangyi; Huang, Shiang; Ren, Xiubao; Lundy, Steven K.; Dai, Fu; Li, Qiao; Chang, Alfred E.

    2016-01-01

    We previously reported that antitumor B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. In this study, we defined additional mechanisms involved in B cell antitumor immunity. Administration of IL-2 significantly augmented the therapeutic efficacy of adoptively transferred tumor-draining lymph node (TDLN) B cells which express IL- 2R. Culture supernatant of purified B splenocytes harvested from the mice that received adoptive transfer of 4T1 TDLN B cells plus IL-2 administration produced larger amounts of IgG which bound to 4T1, resulting in 4T1 lysis. Furthermore, we detected CXCR4 expression on 4T1 TDLN B cells, and 4T1 tumor cells produced its ligand CXCL12. Transwell experiments demonstrated the chemoattraction of CXCR4-expressing 4T1 TDLN B cells towards CXCL12- producing 4T1 cells. Blockade of CXCR4 using a CXCR4-specific inhibitor, AMD3100, significantly reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Blockade of FasL and CXCR4 concurrently inhibited B cell-mediated direct killing of tumor cells in an additive manner, indicating that both Fas/FasL and CXCL12/CXCR4 pathways are involved in the direct killing of 4T1 cells by 4T1 TDLN B cells. TDLN B cells produced perforin. Additional transwell experiments showed that effector B cells could directly kill tumor cells in cell-cell contact via the Fas/FasL and CXCR4/CXCL12 pathways as well as perforin, while without cell contact, perforin secreted by B cells led to tumor cell cytotoxicity. These findings underscore the diversity of function by which B cells can play an important role in the host immune response to tumor. PMID:27528023

  13. Hydrodynamic effects of the tip movement on surface nanobubbles: a combined tapping mode, lift mode and force volume mode AFM study.

    PubMed

    Walczyk, Wiktoria; Hain, Nicole; Schönherr, Holger

    2014-08-28

    We report on an Atomic Force Microscopy (AFM) study of AFM tip-nanobubble interactions in experiments conducted on argon surface nanobubbles on HOPG (highly oriented pyrolytic graphite) in water in tapping mode, lift mode and Force Volume (FV) mode AFM. By subsequent data acquisition on the same nanobubbles in these three different AFM modes, we could directly compare the effect of different tip-sample interactions. The tip-bubble interaction strength was found to depend on the vertical and horizontal position of the tip on the bubble with respect to the bubble center. The interaction forces measured experimentally were in good agreement with the forces calculated using the dynamic interaction model. The strength of the hydrodynamic effect was also found to depend on the direction of the tip movement. It was more pronounced in the FV mode, in which the tip approaches the bubble from the top, than in the lift mode, in which the tip approaches the bubble from the side. This result suggests that the direction of tip movement influences the bubble deformation. The effect should be taken into account when nanobubbles are analysed by AFM in various scanning modes.

  14. Dopamine-dependent long-term depression is expressed in striatal spiny neurons of both direct and indirect pathways: implications for Parkinson's disease.

    PubMed

    Bagetta, Vincenza; Picconi, Barbara; Marinucci, Silvia; Sgobio, Carmelo; Pendolino, Valentina; Ghiglieri, Veronica; Fusco, Francesca R; Giampà, Carmen; Calabresi, Paolo

    2011-08-31

    Striatal medium spiny neurons (MSNs) are divided into two subpopulations exerting distinct effects on motor behavior. Transgenic mice carrying bacterial artificial chromosome (BAC) able to confer cell type-specific expression of enhanced green fluorescent protein (eGFP) for dopamine (DA) receptors have been developed to characterize differences between these subpopulations. Analysis of these mice, in contrast with original pioneering studies, showed that striatal long-term depression (LTD) was expressed in indirect but not in the direct pathway MSNs. To address this mismatch, we applied a new approach using combined BAC technology and receptor immunohistochemistry. We demonstrate that, in physiological conditions, DA-dependent LTD is expressed in both pathways showing that the lack of synaptic plasticity found in D(1) eGFP mice is associated to behavioral deficits. Our findings suggest caution in the use of this tool and indicate that the "striatal segregation" hypothesis might not explain all synaptic dysfunctions in Parkinson's disease.

  15. MicroRNA-140-5p inhibits hepatocellular carcinoma by directly targeting the unique isomerase Pin1 to block multiple cancer-driving pathways

    PubMed Central

    Yan, Xingxue; Zhu, Zhendong; Xu, Shenmin; Yang, Li-nan; Liao, Xin-Hua; Zheng, Min; Yang, Dayun; Wang, Jichuang; Chen, Dongmei; Wang, Long; Liu, Xiaolong; Liu, Jingfeng; Chen, Ruey-Hwa; Zhen Zhou, Xiao; Ping Lu, Kun; Liu, Hekun

    2017-01-01

    Hepatocellular carcinoma (HCC) is the second leading cause of cancer related-death. As a major common regulator of numerous cancer-driving pathways and a unique therapeutic target, the prolyl isomerase Pin1 is overexpressed in a majority of HCCs, whereas the mechanism underlying Pin1 overexpression remains elusive. Here we find that miR-140-5p inhibits HCC by directly targeting Pin1 to block multiple cancer-driving pathways. Bioinformatics analysis, miRNA binding and functional assays identify that miR-140-5p directly interacts with the 3′UTR of Pin1 and inhibits Pin1 translation. Furthermore, like stable Pin1 knockdown, moderate overexpression of miR-140-5p not only eliminates Pin1, but also inhibits cells growth and metastasis. Importantly, these effects of miR-140-5p are largely rescued by reconstitution of Pin1. Moreover, miR-140-5p inhibits multiple Pin1-dependent cancer pathways and suppresses tumor growth in mice. The clinical significance of these findings has been substantiated by the demonstrations that miR-140-5p is frequently down-regulated and inversely correlated with Pin1 overexpression in HCC tissues and cell lines. Given prevalent miR-140-5p downregulation in other cancers and major impact of Pin1 overexpression on activating numerous cancer-driving pathways including global miRNA downregulation, the miR-140-5p/Pin1 axis may play a major role in tumorigenesis and offer promising therapeutic targets for HCC and other cancers. PMID:28383568

  16. Angiosperms Are Unique among Land Plant Lineages in the Occurrence of Key Genes in the RNA-Directed DNA Methylation (RdDM) Pathway.

    PubMed

    Ma, Lu; Hatlen, Andrea; Kelly, Laura J; Becher, Hannes; Wang, Wencai; Kovarik, Ales; Leitch, Ilia J; Leitch, Andrew R

    2015-09-02

    The RNA-directed DNA methylation (RdDM) pathway can be divided into three phases: 1) small interfering RNA biogenesis, 2) de novo methylation, and 3) chromatin modification. To determine the degree of conservation of this pathway we searched for key genes among land plants. We used OrthoMCL and the OrthoMCL Viridiplantae database to analyze proteomes of species in bryophytes, lycophytes, monilophytes, gymnosperms, and angiosperms. We also analyzed small RNA size categories and, in two gymnosperms, cytosine methylation in ribosomal DNA. Six proteins were restricted to angiosperms, these being NRPD4/NRPE4, RDM1, DMS3 (defective in meristem silencing 3), SHH1 (SAWADEE homeodomain homolog 1), KTF1, and SUVR2, although we failed to find the latter three proteins in Fritillaria persica, a species with a giant genome. Small RNAs of 24 nt in length were abundant only in angiosperms. Phylogenetic analyses of Dicer-like (DCL) proteins showed that DCL2 was restricted to seed plants, although it was absent in Gnetum gnemon and Welwitschia mirabilis. The data suggest that phases (1) and (2) of the RdDM pathway, described for model angiosperms, evolved with angiosperms. The absence of some features of RdDM in F. persica may be associated with its large genome. Phase (3) is probably the most conserved part of the pathway across land plants. DCL2, involved in virus defense and interaction with the canonical RdDM pathway to facilitate methylation of CHH, is absent outside seed plants. Its absence in G. gnemon, and W. mirabilis coupled with distinctive patterns of CHH methylation, suggest a secondary loss of DCL2 following the divergence of Gnetales.

  17. Direct Pathway from Early/Recycling Endosomes to the Golgi Apparatus Revealed through the Study of Shiga Toxin B-fragment Transport

    PubMed Central

    Mallard, Frédéric; Antony, Claude; Tenza, Danièle; Salamero, Jean; Goud, Bruno; Johannes, Ludger

    1998-01-01

    Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37°C, ultrastructural studies on cryosections failed to detect B-fragment–specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor–containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN. PMID:9817755

  18. Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport.

    PubMed

    Mallard, F; Antony, C; Tenza, D; Salamero, J; Goud, B; Johannes, L

    1998-11-16

    Shiga toxin and other toxins of this family can escape the endocytic pathway and reach the Golgi apparatus. To synchronize endosome to Golgi transport, Shiga toxin B-fragment was internalized into HeLa cells at low temperatures. Under these conditions, the protein partitioned away from markers destined for the late endocytic pathway and colocalized extensively with cointernalized transferrin. Upon subsequent incubation at 37 degreesC, ultrastructural studies on cryosections failed to detect B-fragment-specific label in multivesicular or multilamellar late endosomes, suggesting that the protein bypassed the late endocytic pathway on its way to the Golgi apparatus. This hypothesis was further supported by the rapid kinetics of B-fragment transport, as determined by quantitative confocal microscopy on living cells and by B-fragment sulfation analysis, and by the observation that actin- depolymerizing and pH-neutralizing drugs that modulate vesicular transport in the late endocytic pathway had no effect on B-fragment accumulation in the Golgi apparatus. B-fragment sorting at the level of early/recycling endosomes seemed to involve vesicular coats, since brefeldin A treatment led to B-fragment accumulation in transferrin receptor-containing membrane tubules, and since B-fragment colocalized with adaptor protein type 1 clathrin coat components on early/recycling endosomes. Thus, we hypothesize that Shiga toxin B-fragment is transported directly from early/recycling endosomes to the Golgi apparatus. This pathway may also be used by cellular proteins, as deduced from our finding that TGN38 colocalized with the B-fragment on its transport from the plasma membrane to the TGN.

  19. Angiosperms Are Unique among Land Plant Lineages in the Occurrence of Key Genes in the RNA-Directed DNA Methylation (RdDM) Pathway

    PubMed Central

    Ma, Lu; Hatlen, Andrea; Kelly, Laura J.; Becher, Hannes; Wang, Wencai; Kovarik, Ales; Leitch, Ilia J.; Leitch, Andrew R.

    2015-01-01

    The RNA-directed DNA methylation (RdDM) pathway can be divided into three phases: 1) small interfering RNA biogenesis, 2) de novo methylation, and 3) chromatin modification. To determine the degree of conservation of this pathway we searched for key genes among land plants. We used OrthoMCL and the OrthoMCL Viridiplantae database to analyze proteomes of species in bryophytes, lycophytes, monilophytes, gymnosperms, and angiosperms. We also analyzed small RNA size categories and, in two gymnosperms, cytosine methylation in ribosomal DNA. Six proteins were restricted to angiosperms, these being NRPD4/NRPE4, RDM1, DMS3 (defective in meristem silencing 3), SHH1 (SAWADEE homeodomain homolog 1), KTF1, and SUVR2, although we failed to find the latter three proteins in Fritillaria persica, a species with a giant genome. Small RNAs of 24 nt in length were abundant only in angiosperms. Phylogenetic analyses of Dicer-like (DCL) proteins showed that DCL2 was restricted to seed plants, although it was absent in Gnetum gnemon and Welwitschia mirabilis. The data suggest that phases (1) and (2) of the RdDM pathway, described for model angiosperms, evolved with angiosperms. The absence of some features of RdDM in F. persica may be associated with its large genome. Phase (3) is probably the most conserved part of the pathway across land plants. DCL2, involved in virus defense and interaction with the canonical RdDM pathway to facilitate methylation of CHH, is absent outside seed plants. Its absence in G. gnemon, and W. mirabilis coupled with distinctive patterns of CHH methylation, suggest a secondary loss of DCL2 following the divergence of Gnetales. PMID:26338185

  20. Modulation of tumor cell stiffness and migration by type IV collagen through direct activation of integrin signaling pathway.

    PubMed

    Chen, Sheng-Yi; Lin, Jo-Shi; Yang, Bei-Chang

    2014-08-01

    Excessive collagen deposition plays a critical role in tumor progression and metastasis. To understand how type IV collagen affects mechanical stiffness and migration, low-collagen-IV-expressing transfectants of B16F10, U118MG, and Huh7 (denoted shCol cells) were established by the lentiviral-mediated delivery of small interfering RNA against type IV-α1 collagen (Col4A1). Although having similar growth rates, shCol cells showed a flatter morphology compared to that of the corresponding controls. Notably, knocking down the Col4A1 gene conferred the cells with higher levels of elasticity and lower motility. Exposure to blocking antibodies against human β1 integrin or α2β1 integrin or the pharmacological inhibition of Src and ERK activity by PP1 and U0126, respectively, effectively reduced cell motility and raised cell stiffness. Reduced Src and ERK activities in shCol cells indicate the involvement of a collagen IV/integrin signaling pathway. The forced expression of β1 integrin significantly stimulated Src and ERK phosphorylation, reduced cell stiffness, and accelerated cell motility. In an experimental metastasis assay using C57BL/6 mice, B16F10 shCol cells formed significantly fewer and smaller lung nodules, confirming the contribution of collagen to metastasis. In summary, the integrin signaling pathway activated in a tumor environment with collagen deposition is responsible for low cell elasticity and high metastatic ability.

  1. Distinct Argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline

    PubMed Central

    Gu, Weifeng; Shirayama, Masaki; Conte, Darryl; Vasale, Jessica; Batista, Pedro J.; Claycomb, Julie M.; Moresco, James J.; Youngman, Elaine; Keys, Jennifer; Stoltz, Matthew J.; Chen, Chun-Chieh G.; Chaves, Daniel A.; Duan, Shenghua E.; Kasschau, Kristen D.; Falgren, Noah; Yates, John R.; Mitani, Shohei; Carrington, James C.; Mello, Craig C.

    2009-01-01

    Summary Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that in the germ-line, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germ-line nuage structures called P-granules. WAGO-1 silences certain genes, transposons, pseudogenes and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest novel regulatory functions for small RNAs. PMID:19800275

  2. A pathway switch directs BAFF signaling to distinct NFκB transcription factors in maturing and proliferating B cells.

    PubMed

    Almaden, Jonathan V; Tsui, Rachel; Liu, Yi C; Birnbaum, Harry; Shokhirev, Maxim N; Ngo, Kim A; Davis-Turak, Jeremy C; Otero, Dennis; Basak, Soumen; Rickert, Robert C; Hoffmann, Alexander

    2014-12-24

    BAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences.

  3. AFM Manipulation of Viruses: Substrate Interactions and Mechanical Properties

    NASA Astrophysics Data System (ADS)

    Falvo, M. R.; Superfine, R.; Washburn, S.; Finch, M.; Taylor, R. M.; Chi, V.; Brooks, F. P.; Ferrari, F.; Samulski, R.

    1996-03-01

    Using an AFM tip as a manipulation tool, we have translated, rotated, and dissected individual Tobacco Mosaic Virus (TMV) and Adenovirus particles. We have implemented a teleoperation system which allows manual control of the relative tip-sample position while also allowing conventional AFM operation for imaging resulting structure. Using simple tip trajectories to bend the rod-shaped TMV, we observed a variety of resulting structures and mechanical failures. The distributed adhesive interaction between the virus and the sample surface, as well as the local tip-virus interaction affect the distortion in the shape of the virus. Experiments were performed in air as well as in liquid on graphite and Si substrates. The in-liquid experiments allow tuning of the environmental conditions, including osmolarity and pH, which are known to profoundly affect the virus structure. A continuum mechanical model relating mechanical properties to observations provides insight into the constraints for successful nondestructive manipulation.

  4. Nano-Bio-Mechanics of Neuroblastoma Cells Using AFM

    NASA Astrophysics Data System (ADS)

    Bastatas, Lyndon; Matthews, James; Kang, Min; Park, Soyeun

    2011-10-01

    We have conducted an in vitro study to determine the elastic moduli of neurobalstoma cell lines using atomic force microscopy. Using a panel of cell lines established from neuroblastoma patients at different stages of disease progress and treatment, we have investigated the differences in elastic moduli during a course of cancer progression and chemotherapy. The cells were grown on the hard substrates that are chemically functionalized to enhance adhesion. We have performed the AFM indentation experiments with different applied forces from the AFM probe. For the purpose of the comparison between cell lines, the indentations were performed only on cell centers. The obtained force-distance curves were analyzed using the Hertz model in order to extract the elastic moduli. We have found that the elastic moduli of human neuroblastoma cells significantly varied during the disease progression. We postulate that the observed difference might be affected by the treatment and chemotherapy.

  5. BOREAS AFM-5 Level-1 Upper Air Network Data

    NASA Technical Reports Server (NTRS)

    Barr, Alan; Hrynkiw, Charmaine; Newcomer, Jeffrey A. (Editor); Hall, Forrest G. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-5 team collected and processed data from the numerous radiosonde flights during the project. The goals of the AFM-05 team were to provide large-scale definition of the atmosphere by supplementing the existing Atmospheric Environment Service (AES) aerological network, both temporally and spatially. This data set includes basic upper-air parameters collected from the network of upper-air stations during the 1993, 1994, and 1996 field campaigns over the entire study region. The data are contained in tabular ASCII files. The level-1 upper-air network data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files also are available on a CD-ROM (see document number 20010000884).

  6. Automated assembly of holder chips to AFM probes

    NASA Astrophysics Data System (ADS)

    Reinhart, Gunther; Jacob, Dirk; Fouchier, Marc

    2001-10-01

    At the Belgian institute IMEC techniques for the production of electrically conductive atomic force microscope (AFM) probes are developed. To facilitate handling of the fragile probes, holder chips are required. The assembly of such holder chips, which can be split up into the application of solder paste, the positioning of the holder chip and the soldering of the chip, is a crucial manufacturing step, that, until now, was performed manually for economic reasons. With the help of a modular micro assembly tool, developed by the Institute for Machine Tools and Industrial Management (iwb) of the Technische Universitaet Muenchen, an economical automated assembly of the holder chips was developed. Thanks to our integrated sensor technology, even the automated assembly onto the extremely fragile membranes of moulded AFM probes was possible. In particular, the dispensing process of the solder paste onto the membranes was improved by the integration of a non-contact sensor for the needle clearance.

  7. Leading Change: Transitioning the AFMS into a High Reliability Organization

    DTIC Science & Technology

    2016-02-16

    AIR WAR COLLEGE AIR UNIVERSITY LEADING CHANGE: TRANSITIONING THE AFMS INTO A HIGH RELIABILTY ORGANIZATION by Robert K. Bogart...academic research paper are those of the author and do not reflect the official policy or position of the US government, the Department of Defense, or Air ...University. In accordance with Air Force Instruction 51-303, it is not copyrighted, but is the property of the United States government. iii

  8. Investigation of biopolymer networks by means of AFM

    NASA Astrophysics Data System (ADS)

    Keresztes, Z.; Rigó, T.; Telegdi, J.; Kálmán, E.

    Natural hydrogel alginate was investigated by means of atomic force microscopy (AFM) to gain microscale information on the morphological and rheological properties of the biopolymer network cross-linked by various cations. Local rheological properties of the gels measured by force spectroscopy gave correlation between increasing ion selectivity and increasing polymer elasticity. Adhesive forces acting between the surface of the gel and the probe, and also the intrinsic rheological properties of bulk polymers affect the microscopical image formation.

  9. Adiabatic Compression Sensitivity of AF-M315E

    DTIC Science & Technology

    2015-07-01

    the development of green rocket propellants . The Air Force Research Laboratory’s (AFRL) monopropellant, AF-M315E, has been selected for...art rocket fuels and propellants . A known quantity of liquid propellant is placed in a metal U-tube and held isothermally in a preheated mixture of... Propellant Infusion Mission (GPIM) program. As the propulsion system developed by Aerojet- Rocketdyne for this propellant advances in maturity, studies

  10. Quantitative nano-mechanics of biological cells with AFM

    NASA Astrophysics Data System (ADS)

    Sokolov, Igor

    2013-03-01

    The importance of study of living cells is hard to overestimate. Cell mechanics is a relatively young, yet not a well-developed area. Besides just a fundamental interest, large practical need has emerged to measure cell mechanics quantitatively. Recent studies revealed a significant correlation between stiffness of biological cells and various human diseases, such as cancer, malaria, arthritis, and even aging. However, really quantitative studies of mechanics of biological cells are virtually absent. It is not even clear if the cell, being a complex and heterogeneous object, can be described by the elastic modulus at all. Atomic force microscopy (AFM) is a natural instrument to study properties of cells in their native environments. Here we will demonstrate that quantitative measurements of elastic modulus of cells with AFM are possible. Specifically, we will show that the ``cell body'' (cell without ``brush'' surface layer, a non-elastic layer surrounding cells) typically demonstrates the response of a homogeneous elastic medium up to the deformation of 10-20%, but if and only if a) the cellular brush layer is taken into account, b) rather dull AFM probes are used. This will be justified with the help of the strong condition of elastic behavior of material: the elastic modulus is shown to be independent on the indentation depth. We will also demonstrate that an attempt either to ignore the brush layer or to use sharp AFM probes will result in the violation of the strong condition, which implies impossibility to use the concept of the elastic modulus to describe cell mechanics in such experiments. Examples of quantitative measurements of the Young's modulus of the cell body and the cell brush parameters will be given for various cells. Address when submitting: Clarkson University, Potsdam, NY 13699

  11. Biophysical properties of cardiomyocyte surface explored by multiparametric AFM.

    PubMed

    Smolyakov, Georges; Cauquil, Marie; Severac, Childerick; Lachaize, Véronique; Guilbeau-Frugier, Céline; Sénard, Jean-Michel; Galés, Céline; Dague, Etienne

    2017-03-02

    PeakForce Quantitative Nanomechanical Mapping (PeakForce QNM) multiparametric AFM mode was adapted to qualitative and quantitative study of the lateral membrane of cardiomyocytes (CMs), extending this powerful mode to the study of soft cells. On living CM, PeakForce QNM depicted the crests and hollows periodic alternation of cell surface architecture previously described using AFM Force Volume (FV) mode. PeakForce QNM analysis provided better resolution in terms of pixel number compared to FV mode and reduced acquisition time, thus limiting the consequences of spontaneous living adult CM dedifferentiation once isolated from the cardiac tissue. PeakForce QNM mode on fixed CMs clearly visualized subsarcolemmal mitochondria (SSM) and their loss following formamide treatment, concomitant with the interfibrillar mitochondria climbing up and forming heaps at the cell surface. Interestingly, formamide-promoted SSM loss allowed visualization of the sarcomeric apparatus ultrastructure below the plasma membrane. High PeakForce QNM resolution led to better contrasted mechanical maps than FV mode and provided correlation between adhesion, dissipation, mechanical and topographical maps. Modified hydrophobic AFM tip enhanced contrast on adhesion and dissipation maps and suggested that CM surface crests and hollows exhibit distinct chemical properties. Finally, two-dimensional Fast Fourier Transform to objectively quantify AFM maps allowed characterization of periodicity of both sarcomeric Z-line and M-band. Overall, this study validated PeakForce QNM as a valuable and innovative mode for the exploration of living and fixed CMs. In the future, it could be applied to depict cell membrane architectural, mechanical and chemical defects as well as sarcomeric abnormalities associated with cardiac diseases.

  12. Nonclassical antigen-processing pathways are required for MHC class II-restricted direct tumor recognition by NY-ESO-1-specific CD4(+) T cells.

    PubMed

    Matsuzaki, Junko; Tsuji, Takemasa; Luescher, Immanuel; Old, Lloyd J; Shrikant, Protul; Gnjatic, Sacha; Odunsi, Kunle

    2014-04-01

    Tumor antigen-specific CD4(+) T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4(+) T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 protein pulsed on DP04(+) target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4(+) T cells more efficiently recognized the short 8-9-mer peptides than the non-tumor-recognizing CD4(+) T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomal degradation and transporter-associated with antigen-processing-mediated peptide transport, were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologic inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4(+) T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment.

  13. Nanoscale rippling on polymer surfaces induced by AFM manipulation

    PubMed Central

    2015-01-01

    Summary Nanoscale rippling induced by an atomic force microscope (AFM) tip can be observed after performing one or many scans over the same area on a range of materials, namely ionic salts, metals, and semiconductors. However, it is for the case of polymer films that this phenomenon has been widely explored and studied. Due to the possibility of varying and controlling various parameters, this phenomenon has recently gained a great interest for some technological applications. The advent of AFM cantilevers with integrated heaters has promoted further advances in the field. An alternative method to heating up the tip is based on solvent-assisted viscoplastic deformations, where the ripples develop upon the application of a relatively low force to a solvent-rich film. An ensemble of AFM-based procedures can thus produce nanoripples on polymeric surfaces quickly, efficiently, and with an unprecedented order and control. However, even if nanorippling has been observed in various distinct modes and many theoretical models have been since proposed, a full understanding of this phenomenon is still far from being achieved. This review aims at summarizing the current state of the art in the perspective of achieving control over the rippling process on polymers at a nanoscale level. PMID:26733086

  14. Near-Field Spectroscopy with Nanoparticles Deposited by AFM

    NASA Technical Reports Server (NTRS)

    Anderson, Mark S.

    2008-01-01

    An alternative approach to apertureless near-field optical spectroscopy involving an atomic-force microscope (AFM) entails less complexity of equipment than does a prior approach. The alternative approach has been demonstrated to be applicable to apertureless near-field optical spectroscopy of the type using an AFM and surface enhanced Raman scattering (SERS), and is expected to be equally applicable in cases in which infrared or fluorescence spectroscopy is used. Apertureless near-field optical spectroscopy is a means of performing spatially resolved analyses of chemical compositions of surface regions of nanostructured materials. In apertureless near-field spectroscopy, it is common practice to utilize nanostructured probe tips or nanoparticles (usually of gold) having shapes and dimensions chosen to exploit plasmon resonances so as to increase spectroscopic-signal strengths. To implement the particular prior approach to which the present approach is an alternative, it is necessary to integrate a Raman spectrometer with an AFM and to utilize a special SERS-active probe tip. The resulting instrumentation system is complex, and the tasks of designing and constructing the system and using the system to acquire spectro-chemical information from nanometer-scale regions on a surface are correspondingly demanding.

  15. Tissue section AFM: In situ ultrastructural imaging of native biomolecules

    PubMed Central

    Graham, Helen K.; Hodson, Nigel W.; Hoyland, Judith A.; Millward-Sadler, Sarah J.; Garrod, David; Scothern, Anthea; Griffiths, Christopher E.M.; Watson, Rachel E.B.; Cox, Thomas R.; Erler, Janine T.; Trafford, Andrew W.; Sherratt, Michael J.

    2010-01-01

    Conventional approaches for ultrastructural high-resolution imaging of biological specimens induce profound changes in bio-molecular structures. By combining tissue cryo-sectioning with non-destructive atomic force microscopy (AFM) imaging we have developed a methodology that may be applied by the non-specialist to both preserve and visualize bio-molecular structures (in particular extracellular matrix assemblies) in situ. This tissue section AFM technique is capable of: i) resolving nm–µm scale features of intra- and extracellular structures in tissue cryo-sections; ii) imaging the same tissue region before and after experimental interventions; iii) combining ultrastructural imaging with complimentary microscopical and micromechanical methods. Here, we employ this technique to: i) visualize the macro-molecular structures of unstained and unfixed fibrillar collagens (in skin, cartilage and intervertebral disc), elastic fibres (in aorta and lung), desmosomes (in nasal epithelium) and mitochondria (in heart); ii) quantify the ultrastructural effects of sequential collagenase digestion on a single elastic fibre; iii) correlate optical (auto fluorescent) with ultrastructural (AFM) images of aortic elastic lamellae. PMID:20144712

  16. Fabrication and characterization of mesoscale protein patterns using atomic force microscopy (AFM)

    NASA Astrophysics Data System (ADS)

    Gao, Pei

    2011-07-01

    A versatile AFM local oxidation lithography was developed for fabricating clean protein patterns ranging from nanometer to sub-millimeter scale on octadecyltrichlorosilane (OTS) layer of Si (100) wafer. This protein patterning method can generate bio-active protein pattern with a clean background without the need of the anti-fouling the surface or repetitive rinsing. As a model system, lysozyme protein patterns were investigated through their binding reactions with antibodies and aptamers by AFM. Polyclonal anti-lysozyme antibodies and anti-lysozyme aptamer are found to preferentially bind to the lysozyme molecules on the edge of a protein pattern before their binding to the interior ones. It was also demonstrated that the topography of the immobilized protein pattern affects the antibody binding direction. We found that the anti-lysozyme antibodies binding to the edge lysozyme molecules on the half-buried pattern started from the top but the binding on the extruded pattern started from the side because of their different spatial accessibility. In addition, after incubating lysozyme pattern with anti-lysozyme aptamer in buffer solution for enough long time, some fractal-shaped aptamer fibers with 1-6nm high and up to tens of micrometers long were formed by the self-assembling of aptamer molecules on the surface. The aptamer fibers anchor specifically on the edge of protein patterns, which originates from the biospecific recognition between the aptamer and its target protein. Once these edge-bound fibers have formed, they can serve as scaffolds for further assembly processes. We used these aptamer fibers as templates to fabricate palladium and streptavidin nanowires, which anchored on the pattern edges and never cross over or collapse over each other. The aptamer fiber scaffold potentially can lead to an effective means to fabricate and interface nanowires to existing surface patterns. KEYWORDS: Atomic Force Microscopy (AFM), Protein Patterns, Lysozyme, Aptamer

  17. Direct and indirect pathways to lamina I in the medulla oblongata and spinal cord of the cat

    NASA Technical Reports Server (NTRS)

    Holstege, Gert

    1988-01-01

    The pathways to lamina I in the medulla oblongata and spinal cord of the cat were traced using horse-radish-peroxidase (HRP) and autoradiographic techniques. The HRP results indicated that several neuronal cell groups in the brain stem and hypothalamus project to the spinal cord throughout its total length. The autoradiographic tracing results demonstrated that the strongest projections to lamina I are derived from the following four areas: the caudal nucleus raphe magnus (NRM), the ventral part of the caudal pontine and NRM, the contralaterally projecting lateral pontine or paralemniscal tegmentum, and the paraventricular nucleus of the hypothalamus. In addition, a limited, especially at lumbosacral levels, distinct projection to lamina I was found to originate in the most caudal part of the medullary tegmentum.

  18. Pathway Targeted Immunotherapy: Rationale and Evidence of Durable Clinical Responses with a Novel, EGF-directed Agent for Advanced NSCLC.

    PubMed

    Rosell, Rafael; Neninger, Elia; Nicolson, Marianne; Huber, Rudolf M; Thongprasert, Sumitra; Parikh, Purvish M; D'Hondt, Erik

    2016-11-01

    Abnormalities in the epidermal growth factor (EGF) and EGFR pathway promote progression of NSCLC. Immunization with EGF vaccine induces specific, neutralizing anti-EGF antibodies that prevent binding of the ligand to its receptor. This concept of pathway targeted immunotherapy (PTI) was validated in vitro by dose-related suppression of EGFR, Akt, and Erk1/2 phosphorylation in cell lines with different mutations. A randomized phase II trial showed improved overall survival (OS) in subgroups with advanced NSCLC showing a clear immunologic response. By per-protocol analysis of the ensuing phase IIb trial, patients receiving EGF PTI survived 3 months longer than controls (12.43 versus 9.43 months; hazard ratio = 0.77 [95% confidence interval, 0.61-0.98]). These data were confirmed in a larger trial showing an OS benefit over control of >3 months. The variable most strongly correlated with efficacy was circulating EGF at enrolment. Patients with serum EGF levels >250 pg/mL benefited most from treatment with EGF PTI. Of 188 patients tested, 94 were above this biomarker threshold. The OS benefit from active versus control treatment was 6.7 months. More than 15% of patients had responses for >5 years. Long-term survivors are seen in all EGF PTI trials. Treatment is well-tolerated, induces high anti-EGF antibody titers, reduces levels of circulating serum EGF, achieves durable responses, and significantly prolongs OS. A threshold of 250 pg/mL has been set to enrich the study population in the ongoing pivotal trial. This biomarker-guided study in an enriched population of patients with both squamous and nonsquamous stage IV NSCLC aims to replicate the favorable efficacy/tolerability balance of earlier studies.

  19. To Serve the Future Hour. An Anthology on New Directions for Nursing. Pathways to Practice, Vol. 2.

    ERIC Educational Resources Information Center

    Reitt, Barbara B., Ed.

    Nurses today are faced with the problem of choosing from a proliferating array of alternatives, both when they decide what type of nursing education to pursue and when they must choose between the many occupational directions they face on graduation from nursing school. Recognizing these recent developments in both education and health, the…

  20. Quality Child Care Supports the Achievement of Low-Income Children: Direct and Indirect Pathways Through Caregiving and the Home Environment

    PubMed Central

    McCartney, Kathleen; Dearing, Eric; Taylor, Beck A.; Bub, Kristen L.

    2009-01-01

    Existing studies of child care have not been able to determine whether higher quality child care protects children from the effects of poverty, whether poverty and lower quality child care operate as dual risk factors, or whether both are true. The objective of the current study was to test two pathways through which child care may serve as a naturally occurring intervention for low-income children: a direct pathway through child care quality to child outcomes, and an indirect pathway through improvements in the home environment. Children were observed in their homes and child care settings at 6, 15, 24, and 36 months. An interaction between family income-to-needs ratio and child care quality predicted School Readiness, Receptive Language, and Expressive Language, as well as improvements in the home environment. Children from low-income families profited from observed learning supports in the form of sensitive care and stimulation of cognitive development, and their parents profited from unobserved informal and formal parent supports. Policy implications are discussed. PMID:19578561

  1. Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein*

    PubMed Central

    Takahashi-Niki, Kazuko; Kato-Ose, Izumi; Murata, Hiroaki; Maita, Hiroshi; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2015-01-01

    DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf. PMID:26048984

  2. Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein.

    PubMed

    Takahashi-Niki, Kazuko; Kato-Ose, Izumi; Murata, Hiroaki; Maita, Hiroshi; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2015-07-17

    DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf.

  3. PDE10A inhibitors stimulate or suppress motor behavior dependent on the relative activation state of the direct and indirect striatal output pathways

    PubMed Central

    Megens, Anton A H P; Hendrickx, Herman M R; Mahieu, Michel M A; Wellens, Annemie L Y; de Boer, Peter; Vanhoof, Greet

    2014-01-01

    The enzyme phosphodiesterase 10A (PDE10A) regulates the activity of striatal, medium spiny neurons (MSNs), which are divided into a behaviorally stimulating, Gs-coupled D1 receptor-expressing “direct” pathway and a behaviorally suppressant, Gi-coupled D2 receptor-expressing “indirect” pathway. Activating both pathways, PDE10A inhibitors (PDE10AIs) combine functional characteristics of D2 antagonists and D1 agonists. While the effects of PDE10AIs on spontaneous and stimulated behavior have been extensively reported, the present study investigates their effects on suppressed behavior under various conditions of reduced dopaminergic neurotransmission: blockade of D1 receptors with SCH-23390, blockade of D2 receptors with haloperidol, or depletion of dopamine with RO-4-1284 or reserpine. In rats, PDE10AIs displayed relatively low cataleptic activity per se. After blocking D1 receptors, however, they induced pronounced catalepsy at low doses close to those required for inhibition of apomorphine-induced behavior; slightly higher doses resulted in behavioral stimulant effects, counteracting the catalepsy. PDE10AIs also counteracted catalepsy and related behaviors induced by D2 receptor blockade or dopamine depletion; catalepsy was replaced by behavioral stimulant effects under the latter but not the former condition. Similar interactions were observed at the level of locomotion in mice. At doses close to those inhibiting d-amphetamine-induced hyperlocomotion, PDE10AIs reversed hypolocomotion induced by D1 receptor blockade or dopamine depletion but not hypolocomotion induced by D2 receptor blockade. It is concluded that PDE10AIs stimulate or inhibit motor behavior dependent on the relative activation state of the direct and indirect striatal output pathways. PMID:25505601

  4. Crystal structures of Boro-AFm and sBoro-AFt phases

    SciTech Connect

    Champenois, Jean-Baptiste; Cau Dit Coumes, Celine; Leroux, Fabrice; Mercier, Cyrille; Revel, Bertrand; Damidot, Denis

    2012-10-15

    Crystal structures of boron-containing AFm (B-AFm) and AFt (B-AFt) phases have been solved ab-initio and refined from X-ray powder diffraction. {sup 11}B NMR and Raman spectroscopies confirm the boron local environment in both compounds: three-fold coordinated in B-AFm corresponding to HBO{sub 3}{sup 2-} species, and four-fold coordinated in B-AFt corresponding to B (OH){sub 4}{sup -} species. B-AFm crystallizes in the rhombohedral R3{sup Macron }c space group and has the 3CaO{center_dot}Al{sub 2}O{sub 3}{center_dot}CaHBO{sub 3}{center_dot}12H{sub 2}O (4CaO{center_dot}Al{sub 2}O{sub 3}{center_dot}1/2B{sub 2}O{sub 3}{center_dot}12.5H{sub 2}O, C{sub 4}AB{sub 1/2}H{sub 12.5}) general formulae with planar trigonal HBO{sub 3}{sup 2-} anions weakly bonded at the centre of the interlayer region. One HBO{sub 3}{sup 2-} anion is statistically distributed with two weakly bonded water molecules on the same crystallographic site. B-AFt crystallizes in the trigonal P3cl space group and has the 3CaO{center_dot}Al{sub 2}O{sub 3}{center_dot}Ca(OH){sub 2}{center_dot}2Ca(B (OH){sub 4}){sub 2}{center_dot}24H{sub 2}O (6CaO{center_dot}Al{sub 2}O{sub 3}{center_dot}2B{sub 2}O{sub 3}{center_dot}33H{sub 2}O, C{sub 6}AB{sub 2}H{sub 33}) general formulae with tetrahedral B (OH){sub 4}{sup -} anions located in the channel region of the structure. All tetrahedral anions are oriented in a unique direction, leading to a hexagonal c lattice parameter about half that of ettringite.

  5. A review of the application of atomic force microscopy (AFM) in food science and technology.

    PubMed

    Liu, Shaoyang; Wang, Yifen

    2011-01-01

    Atomic force microscopy (AFM) is a powerful nanoscale analysis technique used in food area. This versatile technique can be used to acquire high-resolution sample images and investigate local interactions in air or liquid surroundings. In this chapter, we explain the principles of AFM and review representative applications of AFM in gelatin, casein micelle, carrageenan, gellan gum, starch, and interface. We elucidate new knowledge revealed with AFM as well as ways to use AFM to obtain morphology and rheology information in different food fields.

  6. PbSe nanocrystal growth as nanocubes and nanorods on peptide nanotubes via different directed-assembly pathways.

    PubMed

    Shi, Menglu; Su, Wei; Matsui, Hiroshi

    2010-11-01

    Pb-binding TAR-1 peptides (Ile-Ser-Leu-Leu-His-Ser-Thr) were covalently conjugated on a bolaamphiphile peptide nanotube substrate and the precursors of PbSe were incubated at room temperature. This resulted in the growth of highly crystalline PbSe nanocubes on this biomimetic cylindrical substrate. The growth mechanism to generate nanocubes occurs via the directed self-assembly of nanoparticles and then nanoparticle fusion. The peptide conformation and the cylindrical peptide nanotube substrate play important roles in the mesoscopic crystallization of PbSe nanocubes. Changing the buffer for the peptide immobilization process from 2-(N-morpholino)ethanesulfonic acid to phosphate induces a transformation in the nanocrystal shape from nanocube to nanorods. The conformational change of the TAR-1 peptide on the nanotubes due to the change in the buffer seems to be responsible for aggregating intermediate nanoparticles in different directions for the directed fusion and mesoscopic crystallization of PbSe into the different shapes.

  7. The Brisbane Longitudinal Twin Study Pathways to Cannabis Use, Abuse and Dependence Project: Current status, preliminary results and future directions

    PubMed Central

    Gillespie, NA; Henders, AK; Davenport, TA; Hermens, DF; Wright, MJ; Martin, NG; Hickie, IB

    2013-01-01

    We describe the data being collected from the Brisbane Longitudinal Twin Study (BLTS) in Australia as part of the US National Institute on Drug Abuse (NIDA) funded project Pathways to Cannabis Use, Abuse and Dependence. The history, recruitment, assessment and retention of twin families in this project are described in detail along with preliminary findings and plans for future research. The goal of this NIDA project is to make a significant contribution to the discovery of quantitative trait loci (QTL) influencing cannabis use disorders. Although the focus is cannabis use, abuse and dependence in young adults, measures of comorbid illicit drug use disorders are also being collected. In addition, a variety of internalizing and externalizing disorders are being assessed, funded by support from the Australian National Health and Medical Research Council. Because these same twins have participated in numerous twin studies since 1992, future plans will include linking different phenotypes to investigate relationships between drug use, psychiatric disorders and psychological phenotypes within cross-sectional and longitudinal or developmental frameworks. PMID:23187020

  8. Spin Dynamics and Quantum Tunneling in Fe8 Nanomagnet and in AFM Rings by NMR

    SciTech Connect

    Ho-Baek, Seung

    2004-01-01

    In this thesis, our main interest has been to investigate the spin dynamics and quantum tunneling in single molecule magnets (SMMs), For this we have selected two different classes of SMMs: a ferrimagnetic total high spin S = 10 cluster Fe8 and antiferromagnetic (AFM) ring-type clusters. For Fe8, our efforts have been devoted to the investigation of the quantum tunneling of magnetization in the very low temperature region. The most remarkable experimental finding in Fe8 is that the nuclear spin-lattice relaxation rate (1/T{sub l}) at low temperatures takes place via strong collision mechanism, and thus it allows to measure directly the tunneling rate vs T and H for the first time. For AFM rings, we have shown that 1/T{sub l} probes the thermal fluctuations of the magnetization in the intermediate temperature range. We find that the fluctuations are dominated by a single characteristic frequency which has a power law T-dependence indicative of fluctuations due to electron-acoustic phonon interactions.

  9. Solvent-mediated repair and patterning of surfaces by AFM

    SciTech Connect

    Elhadj, S; Chernov, A; De Yoreo, J

    2007-10-30

    A tip-based approach to shaping surfaces of soluble materials with nanometer-scale control is reported. The proposed method can be used, for example, to eliminate defects and inhomogeneities in surface shape, repair mechanical or laser-induced damage to surfaces, or perform 3D lithography on the length scale of an AFM tip. The phenomenon that enables smoothing and repair of surfaces is based on the transport of material from regions of high- to low-curvature within the solution meniscus formed in a solvent-containing atmosphere between the surface in question and an AFM tip scanned over the surface. Using in situ AFM measurements of the kinetics of surface remodeling on KDP (KH{sub 2}PO{sub 4}) crystals in humid air, we show that redistribution of solute material during relaxation of grooves and mounds is driven by a reduction in surface free energy as described by the Gibbs-Thomson law. We find that the perturbation from a flat interface evolves according to the diffusion equation where the effective diffusivity is determined by the product of the surface stiffness and the step kinetic coefficient. We also show that, surprisingly, if the tip is instead scanned over or kept stationary above an atomically flat area of the surface, a convex structure is formed with a diameter that is controlled by the dimensions of the meniscus, indicating that the presence of the tip and meniscus reduces the substrate chemical potential beneath that of the free surface. This allows one to create nanometer-scale 3D structures of arbitrary shape without the removal of substrate material or the use of extrinsic masks or chemical compounds. Potential applications of these tip-based phenomena are discussed.

  10. Image Analysis and Length Estimation of Biomolecules Using AFM

    PubMed Central

    Sundstrom, Andrew; Cirrone, Silvio; Paxia, Salvatore; Hsueh, Carlin; Kjolby, Rachel; Gimzewski, James K.; Reed, Jason; Mishra, Bud

    2014-01-01

    There are many examples of problems in pattern analysis for which it is often possible to obtain systematic characterizations, if in addition a small number of useful features or parameters of the image are known a priori or can be estimated reasonably well. Often, the relevant features of a particular pattern analysis problem are easy to enumerate, as when statistical structures of the patterns are well understood from the knowledge of the domain. We study a problem from molecular image analysis, where such a domain-dependent understanding may be lacking to some degree and the features must be inferred via machine-learning techniques. In this paper, we propose a rigorous, fully automated technique for this problem. We are motivated by an application of atomic force microscopy (AFM) image processing needed to solve a central problem in molecular biology, aimed at obtaining the complete transcription profile of a single cell, a snapshot that shows which genes are being expressed and to what degree. Reed et al. (“Single molecule transcription profiling with AFM,” Nanotechnology, vol. 18, no. 4, 2007) showed that the transcription profiling problem reduces to making high-precision measurements of biomolecule backbone lengths, correct to within 20–25 bp (6–7.5 nm). Here, we present an image processing and length estimation pipeline using AFM that comes close to achieving these measurement tolerances. In particular, we develop a biased length estimator on trained coefficients of a simple linear regression model, biweighted by a Beaton–Tukey function, whose feature universe is constrained by James–Stein shrinkage to avoid overfitting. In terms of extensibility and addressing the model selection problem, this formulation subsumes the models we studied. PMID:22759526

  11. Optical fiber fluorescence spectroscopy for detecting AFM1 in milk

    NASA Astrophysics Data System (ADS)

    Mignani, A. G.; Cucci, C.; Ciaccheri, L.; Dall'Asta, C.; Galaverna, G.; Dossena, A.; Marchelli, R.

    2008-04-01

    Fluorescence spectroscopy carried out by means of optical fibers was used for the rapid screening of M1 aflatoxin in milk, enabling the detection of concentrations up to the legal limit, which is 50 ppt. A compact fluorometric device equipped with a LED source, a miniaturized spectrometer, and optical fibers for illumination/detection of the measuring micro-cell was tested for measuring threshold values of AFM1 in pre-treated milk samples. Multivariate processing of the spectral data made it possible to obtain a preliminary screening at the earlier stages of the industrial process, as well as to discard contaminated milk stocks before their inclusion in the production chain.

  12. High-speed AFM probe with micromachined membrane tip

    NASA Astrophysics Data System (ADS)

    Kim, Byungki; Kwak, Byung Hyung; Jamil, Faize

    2008-08-01

    This paper presents a micromachined silicon membrane type AFM tip designed to move nearly 1µm by electrostatic force. Since the tip can be vibrated in small amplitude with AC voltage input and can be displaced up to 1μm by DC voltage input, an additional piezo actuator is not required for scanning of submicron features. The micromachined membrane tips are designed to have 100 kHz ~ 1 MHz resonant frequency. Displacement of the membrane tip is measured by an optical interferometer using a micromachined diffraction grating on a quartz wafer which is positioned behind the membrane tip.

  13. Theoretical modelling of AFM for bimetallic tip-substrate interactions

    NASA Technical Reports Server (NTRS)

    Bozzolo, Guillermo; Ferrante, John

    1991-01-01

    Recently, a new technique for calculating the defect energetics of alloys based on Equivalent Crystal Theory was developed. This new technique successfully predicts the bulk properties for binary alloys as well as segregation energies in the dilute limit. The authors apply this limit for the calculation of energy and force as a function of separation of an atomic force microscope (AFM) tip and substrate. The study was done for different combinations of tip and sample materials. The validity of the universality discovered for the same metal interfaces is examined for the case of different metal interactions.

  14. The Advancing State of AF-M315E Technology

    NASA Technical Reports Server (NTRS)

    Masse, Robert; Spores, Ronald A.; McLean, Chris

    2014-01-01

    The culmination of twenty years of applied research in hydroxyl ammonium nitrate (HAN)-based monopropellants, the NASA Space Technology mission Directorate's (STMD) Green Propellant Infusion Mission (GPIM) will achieve the first on-orbit demonstration of an operational AF-M315E green propellant propulsion system by the end of 2015. Following an contextual overview of the completed flight design of the GPIM propellant storage and feed system, results of first operation of a flight-representative heavyweight 20-N engineering model thruster (to be conducted in mid-2014) are presented with performance comparisons to prior lab model (heavyweight) test articles.

  15. Asymmetries in sensory pathways from skin to motoneurons on each side of the body determine the direction of an avoidance response in hatchling Xenopus tadpoles.

    PubMed

    Zhao, F Y; Burton, B G; Wolf, E; Roberts, A

    1998-01-15

    1. When swimming is initiated by tail stimulation in hatchling Xenopus tadpoles, the first trunk contraction is usually on the opposite side and directs the animal away from the stimulus. We have investigated how asymmetries in the skin sensory pathways mediate this response. 2. In alpha-bungarotoxin-immobilized tadpoles, intracellular recordings were made of responses to ipsilateral (ISS) and contralateral skin stimulation (CSS) in thirty-two presumed motoneurons. ISS evokes an inhibitory postsynaptic potential (IPSP) followed by an excitatory postsynaptic potential (EPSP) whereas CSS only evokes an EPSP. Blocking the short latency IPSP evoked by ISS with strychnine reduced the difference in spike latency on the two sides but spikes still occurred first to CSS. 3. Motoneuron EPSPs evoked by ISS and CSS were therefore recorded during microperfusion of strychnine to block the short latency IPSP. We found: (a) the CSS-EPSPs have lower threshold, larger amplitude at a given intensity of stimulus, faster rising phase, and shorter latencies than those of ISS-EPSPs; (b) the ISS-EPSP onset latencies were longer than CSS-EPSPs and became shorter as the stimulus intensity increased while those of CSS-EPSPs remained little changed. At high stimulus intensities, EPSPs caused by CSS and ISS became similar; and (c) onset latencies of ISS-EPSPs had higher variance than those of CSS-EPSPs. However, this difference was reduced as the stimulus intensity was increased. 4. Since motoneuron EPSP onset latencies varied with stimulus intensity, we proposed that the pathway from the opposite side had stronger synapses from afferents to sensory interneurons. To test this proposal we built a neuronal population model of the spinal pathway from skin afferents, via sensory interneurons to ipsilateral and contralateral motoneurons incorporating this asymmetry. Inhibition was omitted from the model. 5. Simulated motoneuron EPSPs in response to skin stimulation on each side of the body showed

  16. AFM-Based Single Molecule Techniques: Unraveling the Amyloid Pathogenic Species

    PubMed Central

    Ruggeri, Francesco Simone; Habchi, Johnny; Cerreta, Andrea; Dietler, Giovanni

    2016-01-01

    Background A wide class of human diseases and neurodegenerative disorders, such as Alzheimer’s disease, is due to the failure of a specific peptide or protein to keep its native functional conformational state and to undergo a conformational change into a misfolded state, triggering the formation of fibrillar cross-β sheet amyloid aggregates. During the fibrillization, several coexisting species are formed, giving rise to a highly heterogeneous mixture. Despite its fundamental role in biological function and malfunction, the mechanism of protein self-assembly and the fundamental origins of the connection between aggregation, cellular toxicity and the biochemistry of neurodegeneration remains challenging to elucidate in molecular detail. In particular, the nature of the specific state of proteins that is most prone to cause cytotoxicity is not established. Methods: In the present review, we present the latest advances obtained by Atomic Force Microscopy (AFM) based techniques to unravel the biophysical properties of amyloid aggregates at the nanoscale. Unraveling amyloid single species biophysical properties still represents a formidable experimental challenge, mainly because of their nanoscale dimensions and heterogeneous nature. Bulk techniques, such as circular dichroism or infrared spectroscopy, are not able to characterize the heterogeneity and inner properties of amyloid aggregates at the single species level, preventing a profound investigation of the correlation between the biophysical properties and toxicity of the individual species. Conclusion: The information delivered by AFM based techniques could be central to study the aggregation pathway of proteins and to design molecules that could interfere with amyloid aggregation delaying the onset of misfolding diseases. PMID:27189600

  17. Amyloid β peptide directly impairs pineal gland melatonin synthesis and melatonin receptor signaling through the ERK pathway.

    PubMed

    Cecon, Erika; Chen, Min; Marçola, Marina; Fernandes, Pedro A C; Jockers, Ralf; Markus, Regina P

    2015-06-01

    Melatonin is the hormone produced by the pineal gland known to regulate physiologic rhythms and to display immunomodulatory and neuroprotective properties. It has been reported that Alzheimer disease patients show impaired melatonin production and altered expression of the 2 G protein-coupled melatonin receptors (MTRs), MT₁ and MT₂, but the underlying mechanisms are not known. Here we evaluated whether this dysfunction of the melatonergic system is directly caused by amyloid β peptides (Aβ(1-40) and Aβ(1-42)). Aβ treatment of rat pineal glands elicited an inflammatory response within the gland, evidenced by the up-regulation of 52 inflammatory genes, and decreased the production of melatonin up to 75% compared to vehicle-treated glands. Blocking NF-κB activity prevented this effect. Exposure of HEK293 cells stably expressing recombinant MT₁ or MT₂ receptors to Aβ lead to a 40% reduction in [(125)I]iodomelatonin binding to MT₁. ERK1/2 activation triggered by MTRs, but not by the β₂-adrenergic receptor, was markedly impaired by Aβ in HEK293 transfected cells, as well as in primary rat endothelial cells expressing endogenous MTRs. Our data reveal the melatonergic system as a new target of Aβ, opening new perspectives to Alzheimer disease diagnosis and therapeutic intervention.

  18. The Dynamics of Individual Nucleosomes Controls the Chromatin Condensation Pathway: Direct Atomic Force Microscopy Visualization of Variant Chromatin

    PubMed Central

    Montel, Fabien; Menoni, Hervé; Castelnovo, Martin; Bednar, Jan; Dimitrov, Stefan; Angelov, Dimitar; Faivre-Moskalenko, Cendrine

    2009-01-01

    Abstract Chromatin organization and dynamics is studied at scales ranging from single nucleosome to nucleosomal array by using a unique combination of biochemical assays, single molecule imaging technique, and numerical modeling. We show that a subtle modification in the nucleosome structure induced by the histone variant H2A.Bbd drastically modifies the higher order organization of the nucleosomal arrays. Importantly, as directly visualized by atomic force microscopy, conventional H2A nucleosomal arrays exhibit specific local organization, in contrast to H2A.Bbd arrays, which show “beads on a string” structure. The combination of systematic image analysis and theoretical modeling allows a quantitative description relating the observed gross structural changes of the arrays to their local organization. Our results suggest strongly that higher-order organization of H1-free nucleosomal arrays is determined mainly by the fluctuation properties of individual nucleosomes. Moreover, numerical simulations suggest the existence of attractive interactions between nucleosomes to provide the degree of compaction observed for conventional chromatin fibers. PMID:19619469

  19. SU-8 hollow cantilevers for AFM cell adhesion studies

    NASA Astrophysics Data System (ADS)

    Martinez, Vincent; Behr, Pascal; Drechsler, Ute; Polesel-Maris, Jérôme; Potthoff, Eva; Vörös, Janos; Zambelli, Tomaso

    2016-05-01

    A novel fabrication method was established to produce flexible, transparent, and robust tipless hollow atomic force microscopy (AFM) cantilevers made entirely from SU-8. Channels of 3 μm thickness and several millimeters length were integrated into 12 μm thick and 40 μm wide cantilevers. Connected to a pressure controller, the devices showed high sealing performance with no leakage up to 6 bars. Changing the cantilever lengths from 100 μm to 500 μm among the same wafer allowed the targeting of various spring constants ranging from 0.5 to 80 N m-1 within a single fabrication run. These hollow polymeric AFM cantilevers were operated in the optical beam deflection configuration. To demonstrate the performance of the device, single-cell force spectroscopy experiments were performed with a single probe detaching in a serial protocol more than 100 Saccharomyces cerevisiae yeast cells from plain glass and glass coated with polydopamine while measuring adhesion forces in the sub-nanoNewton range. SU-8 now offers a new alternative to conventional silicon-based hollow cantilevers with more flexibility in terms of complex geometric design and surface chemistry modification.

  20. Iron oxide mineral-water interface reactions studied by AFM

    SciTech Connect

    Hawley, M.E.; Rogers, P.S.Z.

    1994-07-01

    Natural iron mineral surfaces have been examined in air by atomic force (AFM) and scanning tunneling (STM) microscopies. A number of different surface features were found to be characteristic of the native surface. Even surfaces freshly exposed by crushing larger crystals were found to have a pebbly surface texture caused by the presence of thin coatings of what might be surface precipitates. This finding is interpreted as evidence for previous exposure to water, probably through an extensive network of microfractures. Surface reactions on the goethite crystals were studied by AFM at size resolutions ranging from microns to atomic resolution before, during, and after reaction with distilled water and 0.lN HCl. Immediate and extensive surface reconfiguration occurred on contact with water. In one case, after equilibration with water for 3 days, surface reprecipitation, etching and pitting were observed. Atomic resolution images taken under water were found to be disordered. The result of surface reaction was generally to increase the surface area substantially through the extension of surface platelet arrays, present prior to reaction. This work is being done in support of the site characterization project at Yucca Mountain.

  1. AFM analysis of bleaching effects on dental enamel microtopography

    NASA Astrophysics Data System (ADS)

    Pedreira de Freitas, Ana Carolina; Espejo, Luciana Cardoso; Botta, Sergio Brossi; Teixeira, Fernanda de Sa; Luz, Maria Aparecida A. Cerqueira; Garone-Netto, Narciso; Matos, Adriana Bona; Salvadori, Maria Cecilia Barbosa da Silveira

    2010-02-01

    The purpose of this in vitro study was to test a new methodology to evaluate the effects of 35% hydrogen peroxide agent on the microtopography of sound enamel using an atomic force microscope (AFM). The buccal sound surfaces of three extracted human lower incisors were used, without polishing the surfaces to maintain them with natural morphology. These unpolished surfaces were subjected to bleaching procedure with 35% hydrogen peroxide that consisted of 4 applications of the bleaching agent on enamel surfaces for 10 min each application. Surface images were obtained in a 15 μm × 15 μm area using an AFM. The roughness (Ra and RMS) and the power spectral density (PSD) were obtained before and after the bleaching treatment. As results we could inquire that the PSD analyses were very suitable to identifying the morphological changes on the surfaces, while the Ra and RMS parameters were insufficient to represent the morphological alterations promoted by bleaching procedure on enamel. The morphological wavelength in the range of visible light spectrum (380-750 nm) was analyzed, showing a considerable increase of the PSD with the bleaching treatment.

  2. Pathogen identification using peptide nanotube biosensors and impedance AFM

    NASA Astrophysics Data System (ADS)

    Maccuspie, Robert I.

    Pathogen identification at highly sensitive levels is crucial to meet urgent needs in fighting the spread of disease or detecting bioterrorism events. Toward that end, a new method for biosensing utilizing fluorescent antibody nanotubes is proposed. Fundamental studies on the self-assembly of these peptide nanotubes are performed, as are applications of aligning these nanotubes on surfaces. As biosensors, these nanotubes incorporate recognition units with antibodies at their ends and fluorescent signaling units at their sidewalls. When viral pathogens were mixed with these antibody nanotubes in solution, the nanotubes rapidly aggregated around the viruses. The size of the aggregates increased as the concentration of viruses increased, as detected by flow cytometry on the order of attomolar concentrations by changes in fluorescence and light scattering intensities. This enabled determination of the concentrations of viruses at trace levels (102 to 106 pfu/mL) within 30 minutes from the receipt of samples to the final quantitative data analysis, as demonstrated on Adenovirus, Herpes Simplex Virus, Influenza, and Vaccinia virus. As another separate approach, impedance AFM is used to study the electrical properties of individual viruses and nanoparticles used as model systems. The design, development, and implementation of the impedance AFM for an Asylum Research platform is described, as well as its application towards studying the impedance of individual nanoparticles as a model system for understanding the fundamental science of how the life cycle of a virus affects its electrical properties. In combination, these approaches fill a pressing need to quantify viruses both rapidly and sensitively.

  3. Kinetics and Mechanisms of Thiol–Disulfide Exchange Covering Direct Substitution and Thiol Oxidation-Mediated Pathways

    PubMed Central

    2013-01-01

    Abstract Significance: Disulfides are important building blocks in the secondary and tertiary structures of proteins, serving as inter- and intra-subunit cross links. Disulfides are also the major products of thiol oxidation, a process that has primary roles in defense mechanisms against oxidative stress and in redox regulation of cell signaling. Although disulfides are relatively stable, their reduction, isomerisation, and interconversion as well as their production reactions are catalyzed by delicate enzyme machineries, providing a dynamic system in biology. Redox homeostasis, a thermodynamic parameter that determines which reactions can occur in cellular compartments, is also balanced by the thiol–disulfide pool. However, it is the kinetic properties of the reactions that best represent cell dynamics, because the partitioning of the possible reactions depends on kinetic parameters. Critical Issues: This review is focused on the kinetics and mechanisms of thiol–disulfide substitution and redox reactions. It summarizes the challenges and advances that are associated with kinetic investigations in small molecular and enzymatic systems from a rigorous chemical perspective using biological examples. The most important parameters that influence reaction rates are discussed in detail. Recent Advances and Future Directions: Kinetic studies of proteins are more challenging than small molecules, and quite often investigators are forced to sacrifice the rigor of the experimental approach to obtain the important kinetic and mechanistic information. However, recent technological advances allow a more comprehensive analysis of enzymatic systems via using the systematic kinetics apparatus that was developed for small molecule reactions, which is expected to provide further insight into the cell's machinery. Antioxid. Redox Signal. 18, 1623–1641. PMID:23075118

  4. Novel pathways for injury from offshore oil spills: direct, sublethal and indirect effects of the Deepwater Horizon oil spill on pelagic Sargassum communities.

    PubMed

    Powers, Sean P; Hernandez, Frank J; Condon, Robert H; Drymon, J Marcus; Free, Christopher M

    2013-01-01

    The pelagic brown alga Sargassum forms an oasis of biodiversity and productivity in an otherwise featureless ocean surface. The vast pool of oil resulting from the Deepwater Horizon oil spill came into contact with a large portion of the Gulf of Mexico's floating Sargassum mats. Aerial surveys performed during and after the oil spill show compelling evidence of loss and subsequent recovery of Sargassum. Expanding on the trends observed in the aerial surveys, we conducted a series of mesocosm experiments to test the effect of oil and dispersants on the vertical position and weight of the Sargassum complex (Sargassum natans and S. fluitans), as well as on the dissolved oxygen concentrations surrounding the algae. Dispersant and dispersed-oil had significant effects on the vertical position of both species of Sargassum over a period of 72 hours. Similarly, dissolved oxygen concentrations were lowest in dispersant and dispersed-oil treatments, respectively. Cumulatively, our findings suggest three pathways for oil-spill related injury: (1) Sargassum accumulated oil on the surface exposing animals to high concentrations of contaminants; (2) application of dispersant sank Sargassum, thus removing the habitat and potentially transporting oil and dispersant vertically; and (3) low oxygen surrounded the habitat potentially stressing animals that reside in the alga. These pathways represent direct, sublethal, and indirect effects of oil and dispersant release that minimize the ecosystem services provided by floating Sargassum - the latter two effects are rarely considered in assessing impacts of oil spills or response procedures.

  5. Water oxidation with mononuclear ruthenium(II) polypyridine complexes involving a direct Ru(IV)═O pathway in neutral and alkaline media.

    PubMed

    Badiei, Yosra M; Polyansky, Dmitry E; Muckerman, James T; Szalda, David J; Haberdar, Rubabe; Zong, Ruifa; Thummel, Randolph P; Fujita, Etsuko

    2013-08-05

    The catalytic water oxidation mechanism proposed for many single-site ruthenium complexes proceeds via the nucleophilic attack of a water molecule on the Ru(V)═O species. In contrast, Ru(II) complexes containing 4-t-butyl-2,6-di-1',8'-(naphthyrid-2'-yl)-pyridine (and its bisbenzo-derivative), an equatorial water, and two axial 4-picolines follow the thermodynamically more favorable "direct pathway" via [Ru(IV)═O](2+), which avoids the higher oxidation state [Ru(V)═O](3+) in neutral and basic media. Our experimental and theoretical results that focus on the pH-dependent onset catalytic potentials indicative of a PCET driven low-energy pathway for the formation of products with an O-O bond (such as [Ru(III)-OOH](2+) and [Ru(IV)-OO](2+)) at an applied potential below the Ru(V)═O/Ru(IV)═O couple clearly support such a mechanism. However, in the cases of [Ru(tpy)(bpy)(OH2)](2+) and [Ru(tpy)(bpm)(OH2)](2+), the formation of the Ru(V)═O species appears to be required before O-O bond formation. The complexes under discussion provide a unique functional model for water oxidation that proceeds by four consecutive PCET steps in neutral and alkaline media.

  6. Novel Pathways for Injury from Offshore Oil Spills: Direct, Sublethal and Indirect Effects of the Deepwater Horizon Oil Spill on Pelagic Sargassum Communities

    PubMed Central

    Powers, Sean P.; Hernandez, Frank J.; Condon, Robert H.; Drymon, J. Marcus; Free, Christopher M.

    2013-01-01

    The pelagic brown alga Sargassum forms an oasis of biodiversity and productivity in an otherwise featureless ocean surface. The vast pool of oil resulting from the Deepwater Horizon oil spill came into contact with a large portion of the Gulf of Mexico’s floating Sargassum mats. Aerial surveys performed during and after the oil spill show compelling evidence of loss and subsequent recovery of Sargassum. Expanding on the trends observed in the aerial surveys, we conducted a series of mesocosm experiments to test the effect of oil and dispersants on the vertical position and weight of the Sargassum complex (Sargassum natans and S. fluitans), as well as on the dissolved oxygen concentrations surrounding the algae. Dispersant and dispersed-oil had significant effects on the vertical position of both species of Sargassum over a period of 72 hours. Similarly, dissolved oxygen concentrations were lowest in dispersant and dispersed-oil treatments, respectively. Cumulatively, our findings suggest three pathways for oil-spill related injury: (1) Sargassum accumulated oil on the surface exposing animals to high concentrations of contaminants; (2) application of dispersant sank Sargassum, thus removing the habitat and potentially transporting oil and dispersant vertically; and (3) low oxygen surrounded the habitat potentially stressing animals that reside in the alga. These pathways represent direct, sublethal, and indirect effects of oil and dispersant release that minimize the ecosystem services provided by floating Sargassum – the latter two effects are rarely considered in assessing impacts of oil spills or response procedures. PMID:24086378

  7. Two Components of the RNA-Directed DNA Methylation Pathway Associate with MORC6 and Silence Loci Targeted by MORC6 in Arabidopsis

    PubMed Central

    Liu, Zhang-Wei; Zhou, Jin-Xing; Huang, Huan-Wei; Li, Yong-Qiang; Shao, Chang-Rong; Li, Lin; Cai, Tao; Chen, She

    2016-01-01

    The SU(VAR)3-9 homolog SUVH9 and the double-stranded RNA-binding protein IDN2 were thought to be components of an RNA-directed DNA methylation (RdDM) pathway in Arabidopsis. We previously found that SUVH9 interacts with MORC6 but how the interaction contributes to transcriptional silencing remains elusive. Here, our genetic analysis indicates that SUVH2 and SUVH9 can either act in the same pathway as MORC6 or act synergistically with MORC6 to mediate transcriptional silencing. Moreover, we demonstrate that IDN2 interacts with MORC6 and mediates the silencing of a subset of MORC6 target loci. Like SUVH2, SUVH9, and IDN2, other RdDM components including Pol IV, Pol V, RDR2, and DRM2 are also required for transcriptional silencing at a subset of MORC6 target loci. MORC6 was previously shown to mediate transcriptional silencing through heterochromatin condensation. We demonstrate that the SWI/SNF chromatin-remodeling complex components SWI3B, SWI3C, and SWI3D interact with MORC6 as well as with SUVH9 and then mediate transcriptional silencing. These results suggest that the RdDM components are involved not only in DNA methylation but also in MORC6-mediated heterochromatin condensation. This study illustrates how DNA methylation is linked to heterochromatin condensation and thereby enhances transcriptional silencing at methylated genomic regions. PMID:27171427

  8. Voluntary exercise during extinction of auditory fear conditioning reduces the relapse of fear associated with potentiated activity of striatal direct pathway neurons.

    PubMed

    Mika, Agnieszka; Bouchet, Courtney A; Bunker, Preston; Hellwinkel, Justin E; Spence, Katie G; Day, Heidi E W; Campeau, Serge; Fleshner, Monika; Greenwood, Benjamin N

    2015-11-01

    Relapse of previously extinguished fear presents a significant, pervasive obstacle to the successful long-term treatment of anxiety and trauma-related disorders. Thus, identification of a novel means to enhance fear extinction to stand the passage of time and generalize across contexts is of the utmost importance. Acute bouts of exercise can be used as inexpensive, noninvasive treatment strategies to reduce anxiety, and have been shown to enhance memory for extinction when performed in close temporal proximity to the extinction session. However, it is unclear whether acute exercise can be used to prevent relapse of fear, and the neural mechanisms underlying this potential effect are unknown. The current study therefore examined whether acute exercise during extinction of auditory fear can protect against the later relapse of fear. Male F344 rats lacking an extended history of wheel running were conditioned to fear a tone CS and subsequently extinguished within either a freely mobile running wheel, a locked wheel, or a control context lacking a wheel. Rats exposed to fear extinction within a freely mobile wheel ran during fear extinction, and demonstrated reduced fear as well as attenuated corticosterone levels during re-exposure to the extinguished CS during the relapse test in a novel context 1week later. Examination of cfos mRNA patterns elicited by re-exposure to the extinguished CS during the relapse test revealed that acute exercise during extinction decreased activation of brain circuits classically involved in driving fear expression and interestingly, increased activity within neurons of the direct striatal pathway involved in reward signaling. These data suggest that exercise during extinction reduces relapse through a mechanism involving the direct pathway of the striatum. It is suggested that a positive affective state could become associated with the CS during exercise during extinction, thus resulting in a relapse-resistant extinction memory.

  9. Voluntary exercise during extinction of auditory fear conditioning reduces the relapse of fear associated with potentiated activity of striatal direct pathway neurons

    PubMed Central

    Mika, Agnieszka; Bouchet, Courtney A.; Bunker, Preston; Hellwinkel, Justin E.; Spence, Katie G.; Day, Heidi E.W.; Campeau, Serge; Fleshner, Monika

    2015-01-01

    Relapse of previously extinguished fear presents a significant, pervasive obstacle to the successful long-term treatment of anxiety and trauma-related disorders. Thus, identification of a novel means to enhance fear extinction to stand the passage of time and generalize across contexts is of the utmost importance. Acute bouts of exercise can be used as inexpensive, noninvasive treatment strategies to reduce anxiety, and have been shown to enhance memory for extinction when performed in close temporal proximity to the extinction session. However, it is unclear whether acute exercise can be used to prevent relapse of fear, and the neural mechanisms underlying this potential effect are unknown. The current study therefore examined whether acute exercise during extinction of auditory fear can protect against the later relapse of fear. Male, F344 rats lacking an extended history of wheel running were conditioned to fear a tone CS and subsequently extinguished within either a freely mobile running wheel, a locked wheel, or a control context lacking a wheel. Rats exposed to fear extinction within a freely mobile wheel ran during fear extinction, and demonstrated reduced fear as well as attenuated corticosterone levels during re-exposure to the extinguished CS during the relapse test in a novel context 1 week later. Examination of cfos mRNA patterns elicited by re-exposure to the extinguished CS during the relapse test revealed that acute exercise during extinction decreased activation of brain circuits classically involved in driving fear expression and interestingly, increased activity within neurons of the direct striatal pathway involved in reward signaling. These data suggest that exercise during extinction reduces relapse through a mechanism involving the direct pathway of the striatum. It is suggested that a positive affective state could become associated with the CS during exercise during extinction, thus resulting in a relapse-resistant extinction memory. PMID

  10. The importance of correcting for variable probe-sample interactions in AFM-IR spectroscopy: AFM-IR of dried bacteria on a polyurethane film.

    PubMed

    Barlow, Daniel E; Biffinger, Justin C; Cockrell-Zugell, Allison L; Lo, Michael; Kjoller, Kevin; Cook, Debra; Lee, Woo Kyung; Pehrsson, Pehr E; Crookes-Goodson, Wendy J; Hung, Chia-Suei; Nadeau, Lloyd J; Russell, John N

    2016-08-02

    AFM-IR is a combined atomic force microscopy-infrared spectroscopy method that shows promise for nanoscale chemical characterization of biological-materials interactions. In an effort to apply this method to quantitatively probe mechanisms of microbiologically induced polyurethane degradation, we have investigated monolayer clusters of ∼200 nm thick Pseudomonas protegens Pf-5 bacteria (Pf) on a 300 nm thick polyether-polyurethane (PU) film. Here, the impact of the different biological and polymer mechanical properties on the thermomechanical AFM-IR detection mechanism was first assessed without the additional complication of polymer degradation. AFM-IR spectra of Pf and PU were compared with FTIR and showed good agreement. Local AFM-IR spectra of Pf on PU (Pf-PU) exhibited bands from both constituents, showing that AFM-IR is sensitive to chemical composition both at and below the surface. One distinct difference in local AFM-IR spectra on Pf-PU was an anomalous ∼4× increase in IR peak intensities for the probe in contact with Pf versus PU. This was attributed to differences in probe-sample interactions. In particular, significantly higher cantilever damping was observed for probe contact with PU, with a ∼10× smaller Q factor. AFM-IR chemical mapping at single wavelengths was also affected. We demonstrate ratioing of mapping data for chemical analysis as a simple method to cancel the extreme effects of the variable probe-sample interactions.

  11. Intrinsically High-Q Dynamic AFM Imaging in Liquid with a Significantly Extended Needle Tip

    PubMed Central

    Minary-Jolandan, Majid; Tajik, Arash; Wang, Ning; Yu, Min-Feng

    2012-01-01

    Atomic force microscope (AFM) probe with a long and rigid needle tip was fabricated and studied for high Q factor dynamic (tapping mode) AFM imaging of samples submersed in liquid. The extended needle tip over a regular commercially-available tapping mode AFM cantilever was sufficiently long to keep the AFM cantilever from submersed in liquid, which significantly minimized the hydrodynamic damping involved in dynamic AFM imaging of samples in liquid. Dynamic AFM imaging of samples in liquid at an intrinsic Q factor of over 100 and an operation frequency of over 200 kHz was demonstrated. The method has the potential to be extended to acquire viscoelastic materials properties and provide truly gentle imaging of soft biological samples in physiological environments. PMID:22595833

  12. An AFM study of calcite dissolution in concentrated electrolyte solutions

    NASA Astrophysics Data System (ADS)

    Ruiz Agudo, E.; Putnis, C. V.; Putnis, A.; Rodriguez-Navarro, C.

    2009-04-01

    Calcite-solution interactions are of a paramount importance in a range of processes such as the removal of heavy metals, carbon dioxide sequestration, landscape modeling, weathering of building stone and biomineralization. Water in contact with minerals often carries significant amounts of solutes; additionally, their concentration may vary due to evaporation and condensation. It is well known that calcite dissolution is affected dramatically by the presence of such solutes. Here we present investigations on the dissolution of calcite in the presence of different electrolytes. Both bulk (batch reactors) experiments and nanoscale (in situ AFM) techniques are used to study the dissolution of calcite in a range of solutions containing alkaly cations balanced by halide anions. Previous works have indicated that the ionic strength has little influence in calcite dissolution rates measured from bulk experiments (Pokrovsky et al. 2005; Glendhill and Morse, 2004). Contrary to these results, our quantitative analyses of AFM observations show an enhancement of the calcite dissolution rate with increasing electrolyte concentration. Such an effect is concentration-dependent and it is most evident in concentrated solutions. AFM experiments have been carried out in a fluid cell using calcite cleavage surfaces in contact with solutions of simple salts of the alkaly metals and halides at different undersaturations with respect to calcite to try to specify the effect of the ionic strength on etch pit spreading rate and calcite dissolution rate. These results show that the presence of soluble salts may critically affect the weathering of carbonate rocks in nature as well as the decay of carbonate stone in built cultural heritage. References: Pokrosky, O.S.; Golubev, S.V.; Schott, J. Dissolution kinetics of calcite, dolomite and magnesite at 25°C and 0 to 50 atm pCO2. Chemical Geology, 2005, 217 (3-4) 239-255. Glendhill, D.K.; Morse, J.W. Dissolution kinetics of calcite in Na

  13. Dispersion and Fixation of Adeno-Associated Virus with Glutaraldehyde for Afm Studies

    NASA Astrophysics Data System (ADS)

    Wang, Peng; Wang, Xinyan; Yang, Haijun; Lü, Junhong

    Sample preparation is an important procedure for atomic force microscope (AFM) studies. However, flexible virus particles have a tendency to aggregate together and are easily compressed during sample preparation or by AFM tip that subsequently hamper studying of virus by AFM. Herein, low concentration chemical reagent of glutaraldehyde (2%, v/v) is pre-mixed in virus suspension that facilitates the dispersion and observation of recombinant serotype 2 adeno-associated virus particles deposited on mica surface with little deformation.

  14. Acquisition of a Modular, Multi-laser, Raman-AFM Instrument for Multdisciplinary Research

    DTIC Science & Technology

    2015-04-28

    vapor deposition on copper foils. The four lasers range from the blue to 785 nm and provides a unique handle to determine excitation dependence of...Acquisition of a Modular, Multi- laser , Raman- AFM Instrument for Multdisciplinary Research A four- laser , confocal Raman/Atomic Force Scanning... laser , Raman-AFM Instrument for Multdisciplinary Research Report Title A four- laser , confocal Raman/Atomic Force Scanning microscope (Raman-AFM

  15. Ultrastructural evidence for a direct excitatory pathway from the nucleus retroambiguus to lateral longissimus and quadratus lumborum motoneurons in the female golden hamster.

    PubMed

    Gerrits, Peter O; Mouton, Leonora J; de Weerd, Henk; Georgiadis, Janniko R; Krukerink, Marco; Holstege, Gert

    2004-12-20

    During mating, the female golden hamster displays a stereotyped specific receptive posture, characterized by lordosis of the back, elevation of the tail, and extension of the legs. Muscles involved in this posture are thought to be iliopsoas, cutaneus trunci, lateral longissimus (LL), and quadratus lumborum (QL). Lesion studies in rats suggest that mating behavior is controlled by the mesencephalic periaqueductal gray (PAG). The PAG does not project directly to the motoneurons innervating the muscles involved in mating, but is thought to make use of the nucleus retroambiguus (NRA) as relay. The NRA is located ventrolaterally in the most caudal medulla, and projects directly to iliopsoas and cutaneus trunci motoneuronal cell groups. The question is whether this is also true for LL and QL muscles. Retrograde HRP tracing experiments revealed that LL and QL motoneurons are located medially in the ventral horn of the T12-L6 and T13-L4 segments, respectively. A subsequent ultrastructural study combined wheatgerm agglutinin-conjugated horseradish peroxidase injections in the NRA with cholera-toxin B-subunit injections in LL and QL muscles. The results revealed monosynaptic contacts between anterogradely labeled NRA-fiber terminals with retrogradely labeled dendrites of both LL and QL motoneurons. Almost all these terminals had asymmetrical synapses and contained spherical vesicles, suggesting an excitatory function of this NRA-motoneuronal pathway. These results correspond with the hypothesis that in hamster the PAG-NRA-motoneuronal projection not only involves motoneurons of iliopsoas and cutaneus trunci but also of LL and QL.

  16. Bubble colloidal AFM probes formed from ultrasonically generated bubbles.

    PubMed

    Vakarelski, Ivan U; Lee, Judy; Dagastine, Raymond R; Chan, Derek Y C; Stevens, Geoffrey W; Grieser, Franz

    2008-02-05

    Here we introduce a simple and effective experimental approach to measuring the interaction forces between two small bubbles (approximately 80-140 microm) in aqueous solution during controlled collisions on the scale of micrometers to nanometers. The colloidal probe technique using atomic force microscopy (AFM) was extended to measure interaction forces between a cantilever-attached bubble and surface-attached bubbles of various sizes. By using an ultrasonic source, we generated numerous small bubbles on a mildly hydrophobic surface of a glass slide. A single bubble picked up with a strongly hydrophobized V-shaped cantilever was used as the colloidal probe. Sample force measurements were used to evaluate the pure water bubble cleanliness and the general consistency of the measurements.

  17. Visualization of internal structure of banana starch granule through AFM.

    PubMed

    Peroni-Okita, Fernanda H G; Gunning, A Patrick; Kirby, Andrew; Simão, Renata A; Soares, Claudinéia A; Cordenunsi, Beatriz R

    2015-09-05

    Atomic force microscopy (AFM) is a high resolution technique for studying the external and internal structures of starch granules. For this purpose granules were isolated from bananas and embedded in a non-penetrating resin. To achieve image contrast of the ultrastructure, the face of the cut blocks were wetted in steam and force modulation mode imaging was used. Images of starch from green bananas showed large variation of height across the granule due to a locational specific absorption of water and swelling of amorphous regions; the data reveal that the center of the granules are structurally different and have different viscoelastic properties. Images of starches from ripe bananas showed an even greater different level of organization: absence of growth rings around the hilum; the central region of the granule is richer in amylose; very porous surface with round shaped dark structures; the size of blocklets are larger than the green fruits.

  18. AFM, SEM and TEM Studies on Porous Anodic Alumina

    PubMed Central

    2010-01-01

    Porous anodic alumina (PAA) has been intensively studied in past decade due to its applications for fabricating nanostructured materials. Since PAA’s pore diameter, thickness and shape vary too much, a systematical study on the methods of morphology characterization is meaningful and essential for its proper development and utilization. In this paper, we present detailed AFM, SEM and TEM studies on PAA and its evolvements with abundant microstructures, and discuss the advantages and disadvantages of each method. The sample preparation, testing skills and morphology analysis are discussed, especially on the differentiation during characterizing complex cross-sections and ultrasmall nanopores. The versatility of PAAs is also demonstrated by the diversity of PAAs’ microstructure. PMID:20672104

  19. AFM, SEM and TEM Studies on Porous Anodic Alumina

    NASA Astrophysics Data System (ADS)

    Zhu, Yuan Yuan; Ding, Gu Qiao; Ding, Jian Ning; Yuan, Ning Yi

    2010-04-01

    Porous anodic alumina (PAA) has been intensively studied in past decade due to its applications for fabricating nanostructured materials. Since PAA’s pore diameter, thickness and shape vary too much, a systematical study on the methods of morphology characterization is meaningful and essential for its proper development and utilization. In this paper, we present detailed AFM, SEM and TEM studies on PAA and its evolvements with abundant microstructures, and discuss the advantages and disadvantages of each method. The sample preparation, testing skills and morphology analysis are discussed, especially on the differentiation during characterizing complex cross-sections and ultrasmall nanopores. The versatility of PAAs is also demonstrated by the diversity of PAAs’ microstructure.

  20. SPR and AFM study of engineered biomolecule immobilisation techniques.

    PubMed

    Craig, Ian; McLaughlin, James A

    2006-01-01

    A comparative study into two novel and diverse schemes designed to improve immobilization of biomolecules for biosensing purposes is presented. In the first method a silicon rich matrix is created using PECVD. The second method involves creating nano-patterns on the sensor surface to create a large number of surface discontinuities to which the proteins will bind preferentially. The basic theory of SPR is provided to show the importance of the surface sensitive nature of this optical transduction technique. The present work suggests that both may prove both for SPR and other biosensing applications. Of the two schemes proposed, the results for nano-patterning seem to suggest that it is promoting better surface attachment of biomolecules. The results of SPR and AFM studies are presented that have shown that each of these schemes promotes improved binding of various proteins.

  1. An improved measurement of dsDNA elasticity using AFM

    NASA Astrophysics Data System (ADS)

    Nguyen, Thi-Huong; Lee, Sang-Myung; Na, Kyounghwan; Yang, Sungwook; Kim, Jinseok; Yoon, Eui-Sung

    2010-02-01

    The mechanical properties of a small fragment (30 bp) of an individual double-stranded deoxyribonucleic acid (dsDNA) in water have been investigated by atomic force microscopy (AFM). We have stretched three systems including ssDNA, double-fixed dsDNA (one strand of the dsDNA molecules was biotinylated at the 3'-end and thiolated at the 5'-end, this was reversed for the other complementary strand) and single-fixed dsDNA (one strand of the dsDNA molecules was biotinylated at the 3'-end and thiolated at the 5'-end, whereas the other complementary strand was biotinylated at only the 5'-end). The achieved thiolation and biotinylation were to bind ds- or ssDNA to the gold surface and streptavidin-coated AFM tip, respectively. Analysis of the force versus displacement (F-D) curves from tip-DNA-substrate systems shows that the pull-off length (Lo) and stretch length (δ) from the double-fixed system were shorter than those observed in the ssDNA and the single-fixed system. The obtained stretch force (Fst) from the single-fixed dsDNA was much greater than that from the ssDNA even though it was about 10 pN greater than the one obtained in the double-fixed system. As a result, the Young's modulus of the double-fixed dsDNA was greater than that of the single-fixed dsDNA and the ssDNA. A more reliable stiffness of the dsDNA was observed via the double-fixed system, since there is no effect of the unpaired molecules during stretching, which always occurred in the single-fixed system. The unpaired molecules were also observed by comparing the stiffness of ssDNA and single-fixed dsDNA in which the end of one strand was left free.

  2. AFM tip effect on a thin liquid film.

    PubMed

    Ledesma-Alonso, R; Legendre, D; Tordjeman, Ph

    2013-06-25

    We study the interaction between an AFM probe and a liquid film deposited over a flat substrate. We investigate the effects of the physical and geometrical parameters, with a special focus on the film thickness E, the probe radius R, and the distance D between the probe and the free surface. Deformation profiles have been calculated from the numerical simulations of the Young-Laplace equation by taking into account the probe/liquid and the liquid/substrate interactions, characterized by the Hamaker constants, Hpl and Hls. We demonstrate that the deformation of a shallow film is determined by a particular characteristic length λF = (2πγE(4)/Hls)(1/2), resulting from the balance between the capillary force (γ is the surface tension) and the van der Waals liquid/substrate attraction. For the case of a bulk liquid, the extent of the interface deformation is simply controlled by the capillary length λC = (γ/Δρg)(1/2). These trends point out two asymptotic regimes, which in turn are bounded by two characteristic film thicknesses Eg = (Hls/2πΔρg)(1/4) and Eγ = (R(2)Hls/2πγ)(1/4). For E > Eg, the bulk behavior is recovered, and for E < Eγ, we show the existence of a particular shallow film regime in which a localized tip effect is observed. This tip effect is characterized by the small magnitude of the deformation and an important restriction of its radial extent λF localized below the probe. In addition, we have found that the film thickness has a significant effect on the threshold separation distance Dmin below which the irreversible jump-to-contact process occurs: Dmin is probe radius-dependent for the bulk whereas it is film-thickness-dependent for shallow films. These results have an important impact on the optimal AFM scanning conditions.

  3. Communication between thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component active centers: evidence for a "direct pathway" between the 4'-aminopyrimidine N1' atoms.

    PubMed

    Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-04-09

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4'-aminopyrimidine N1' atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu(571), Glu(235), and Glu(237)) and Arg(606) resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. 1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. 2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. 3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. 4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu(235) makes no direct contact with the cofactor. The role of the conserved Glu(571) residue in both catalysis and cofactor orientation is revealed by the combined results for the first time.

  4. Measurement of a CD and sidewall angle artifact with two-dimensional CD AFM metrology

    NASA Astrophysics Data System (ADS)

    Dixson, Ronald G.; Sullivan, Neal T.; Schneir, Jason; McWaid, Thomas H.; Tsai, Vincent W.; Prochazka, Jerry; Young, Michael

    1996-05-01

    Despite the widespread acceptance of SEM metrology in semiconductor manufacturing, there is no SEM CD standard currently available. Producing such a standard is challenging because SEM CD measurements are not only a function of the linewidth, but also dependent on the line material, sidewall roughness, sidewall angle, line height, substrate material, and the proximity of other objects. As the presence of AFM metrology in semiconductor manufacturing increases, the history of SEM CD metrology raises a number of questions about the prospect of AFM CD artifacts. Is an AFM CD artifact possible? What role would it play in the manufacturing environment? Although AFM has some important advantages over SEM, such as relative insensitivity to material differences, the throughput and reliability of most AFM instruments is not yet at the level necessary to support in-line CD metrology requirements. What, then, is the most useful relationship between AFM and SEM metrology? As a means of addressing some of these questions, we have measured the CD and sidewall angle of 1.2 micrometer oxy-nitride line on Si using three different techniques: optical microscopy (with modeling), AFM, and cross sectional TEM. Systematic errors in the AFM angle measurements were reduced by using a rotational averaging technique that we describe. We found good agreement with uncertainties below 30 nm (2 sigma) for the CD measurement and 1.0 degrees (2 sigma) for the sidewall angles. Based upon these results we suggest a measurement procedure which will yield useful AFM CD artifacts. We consider the possibility that AFMs, especially when used with suitable CD artifacts, can effectively support SEM CD metrology. This synergistic relationship between the AFM and SEM represents an emerging paradigm that has also been suggested by a number of others.

  5. Structure and Dynamics of Four-way DNA Junctions Dynamics Revealed by Single-Molecule AFM

    NASA Astrophysics Data System (ADS)

    Lyubchenko, Yuri

    2004-03-01

    For-way DNA junctions (Holliday junctions) are critical intermediates for homologous, site-specific recombination, DNA repair and replication. A wealth of structural information is available for immobile four-way junctions. However, these data cannot give the answer on the mechanism of branch migration, the major property of the Holliday junction. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal, exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration. Note that it was the first time when the dynamics of the four-way DNA junction capable of branch migration had been analyzed. We applied time-lapse atomic force microscopy (single molecule dynamics AFM) to image directly loosely bound DNA at liquid-surface interface. These experiments show that mobile Holliday junctions adopt an unfolded conformation during branch migration. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction. The analysis of the Holliday junctions dynamics at conditions limiting branch migration revealed a broad movement of the arms suggesting that the range of mobility of these junctions is much wider than detected before. Further applications of the time-lapse AFM approach in attempt to resolve the subpopulations of the junctions conformers and the prospects for analyses of dynamics of complex biological systems will be discussed.

  6. Hematite/silver nanoparticle bilayers on mica--AFM, SEM and streaming potential studies.

    PubMed

    Morga, Maria; Adamczyk, Zbigniew; Oćwieja, Magdalena; Bielańska, Elżbieta

    2014-06-15

    Bilayers of hematite/silver nanoparticles were obtained in the self-assembly process and thoroughly characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM), and in situ streaming potential measurements. The hematite nanoparticles, forming a supporting layer, were 22 nm in diameter, exhibiting an isoelectric point at pH 8.9. The silver nanoparticles, used to obtain an external layer, were 29 nm in diameter, and remained negative within the pH range 3 to 11. In order to investigate the particle deposition, mica sheets were used as a model solid substrate. The coverage of the supporting layer was adjusted by changing the bulk concentration of the hematite suspension and the deposition time. Afterward, silver nanoparticle monolayers of controlled coverage were deposited under the diffusion-controlled transport. The coverage of bilayers was determined by a direct enumeration of deposited particles from SEM micrographs and AFM images. Additionally, the formation of the hematite/silver bilayers was investigated by streaming potential measurements carried out under in situ conditions. The effect of the mica substrate and the coverage of a supporting layer on the zeta potential of bilayers was systematically studied. It was established that for the coverage exceeding 0.20, the zeta potential of bilayers was independent on the substrate and the supporting layer coverage. This behavior was theoretically interpreted in terms of the 3D electrokinetic model. Beside significance for basic sciences, these measurements allowed to develop a robust method of preparing nanoparticle bilayers of controlled properties, having potential applications in catalytic processes.

  7. Proceedings of the 2010 AFMS Medical Research Symposium. Volume 5. Nursing Track: Abstracts and Presentations

    DTIC Science & Technology

    2011-03-15

    will include hematocrit, hemoglobin , mean corpuscle volume, iron , total iron binding capacity, Ferritin , and soluble transferring receptor. The...Iraq/Afghanistan ........ 2  Iron Status of Deployed Military Members...2010 AFMS Medical Research Symposium Volume 5 Nursing 8 Proceedings of the 2010 AFMS Medical Research Symposium Volume 5 Nursing 9 Iron

  8. miR-506 acts as a tumor suppressor by directly targeting the hedgehog pathway transcription factor Gli3 in human cervical cancer.

    PubMed

    Wen, S-Y; Lin, Y; Yu, Y-Q; Cao, S-J; Zhang, R; Yang, X-M; Li, J; Zhang, Y-L; Wang, Y-H; Ma, M-Z; Sun, W-W; Lou, X-L; Wang, J-H; Teng, Y-C; Zhang, Z-G

    2015-02-05

    Although significant advances have recently been made in the diagnosis and treatment of cervical carcinoma, the long-term survival rate for advanced cervical cancer remains low. Therefore, an urgent need exists to both uncover the molecular mechanisms and identify potential therapeutic targets for the treatment of cervical cancer. MicroRNAs (miRNAs) have important roles in cancer progression and could be used as either potential therapeutic agents or targets. miR-506 is a component of an X chromosome-linked miRNA cluster. The biological functions of miR-506 have not been well established. In this study, we found that miR-506 expression was downregulated in approximately 80% of the cervical cancer samples examined and inversely correlated with the expression of Ki-67, a marker of cell proliferation. Gain-of-function and loss-of-function studies in human cervical cancer, Caski and SiHa cells, demonstrated that miR-506 acts as a tumor suppressor by inhibiting cervical cancer growth in vitro and in vivo. Further studies showed that miR-506 induced cell cycle arrest at the G1/S transition, and enhanced apoptosis and chemosensitivity of cervical cancer cell. We subsequently identified Gli3, a hedgehog pathway transcription factor, as a direct target of miR-506 in cervical cancer. Furthermore, Gli3 silencing recapitulated the effects of miR-506, and reintroduction of Gli3 abrogated miR-506-induced cell growth arrest and apoptosis. Taken together, we conclude that miR-506 exerts its anti-proliferative function by directly targeting Gli3. This newly identified miR-506/Gli3 axis provides further insight into the pathogenesis of cervical cancer and indicates a potential novel therapeutic agent for the treatment of cervical cancer.

  9. An uncleaved signal peptide directs the Malus xiaojinensis iron transporter protein Mx IRT1 into the ER for the PM secretory pathway.

    PubMed

    Zhang, Peng; Tan, Song; Berry, James O; Li, Peng; Ren, Na; Li, Shuang; Yang, Guang; Wang, Wei-Bing; Qi, Xiao-Ting; Yin, Li-Ping

    2014-11-07

    Malus xiaojinensis iron-regulated transporter 1 (Mx IRT1) is a highly effective inducible iron transporter in the iron efficient plant Malus xiaojinensis. As a multi-pass integral plasma membrane (PM) protein, Mx IRT1 is predicted to consist of eight transmembrane domains, with a putative N-terminal signal peptide (SP) of 1-29 amino acids. To explore the role of the putative SP, constructs expressing Mx IRT1 (with an intact SP) and Mx DsIRT1 (with a deleted SP) were prepared for expression in Arabidopsis and in yeast. Mx IRT1 could rescue the iron-deficiency phenotype of an Arabidopsis irt1 mutant, and complement the iron-limited growth defect of the yeast mutant DEY 1453 (fet3fet4). Furthermore, fluorescence analysis indicated that a chimeric Mx IRT1-eGFP (enhanced Green Fluorescent Protein) construct was translocated into the ER (Endoplasmic reticulum) for the PM sorting pathway. In contrast, the SP-deleted Mx DsIRT1 could not rescue either of the mutant phenotypes, nor direct transport of the GFP signal into the ER. Interestingly, immunoblot analysis indicated that the SP was not cleaved from the mature protein following transport into the ER. Taken together, data presented here provides strong evidence that an uncleaved SP determines ER-targeting of Mx IRT1 during the initial sorting stage, thereby enabling the subsequent transport and integration of this protein into the PM for its crucial role in iron uptake.

  10. Long-Lasting Visuo-Vestibular Mismatch in Freely-Behaving Mice Reduces the Vestibulo-Ocular Reflex and Leads to Neural Changes in the Direct Vestibular Pathway

    PubMed Central

    Carcaud, Julie; França de Barros, Filipa; Eugène, Daniel; Reveret, Lionel; Moore, Lee E.; Vidal, Pierre-Paul

    2017-01-01

    Abstract Calibration of the vestibulo-ocular reflex (VOR) depends on the presence of visual feedback. However, the cellular mechanisms associated with VOR modifications at the level of the brainstem remain largely unknown. A new protocol was designed to expose freely behaving mice to a visuo-vestibular mismatch during a 2-week period. This protocol induced a 50% reduction of the VOR. In vivo pharmacological experiments demonstrated that the VOR reduction depends on changes located outside the flocculus/paraflocculus complex. The cellular mechanisms associated with the VOR reduction were then studied in vitro on brainstem slices through a combination of vestibular afferent stimulation and patch-clamp recordings of central vestibular neurons. The evoked synaptic activity demonstrated that the efficacy of the synapses between vestibular afferents and central vestibular neurons was decreased. In addition, a long-term depression protocol failed to further decrease the synapse efficacy, suggesting that the VOR reduction might have occurred through depression-like mechanisms. Analysis of the intrinsic membrane properties of central vestibular neurons revealed that the synaptic changes were supplemented by a decrease in the spontaneous discharge and excitability of a subpopulation of neurons. Our results provide evidence that a long-lasting visuo-vestibular mismatch leads to changes in synaptic transmission and intrinsic properties of central vestibular neurons in the direct VOR pathway. Overall, these results open new avenues for future studies on visual and vestibular interactions conducted in vivo and in vitro. PMID:28303261

  11. Muscle-specific variations in use-dependent crossed-facilitation of corticospinal pathways mediated by transcranial direct current (DC) stimulation.

    PubMed

    Carson, Richard G; Kennedy, Niamh C; Linden, Mark A; Britton, Lisa

    2008-08-22

    The tendency for contractions of muscles in the upper limb to give rise to increases in the excitability of corticospinal projections to the homologous muscles of the opposite limb is well known. Although the suppression of this tendency is integral to tasks of daily living, its exploitation may prove to be critical in the rehabilitation of acquired hemiplegias. Transcranial direct current (DC) stimulation induces changes in cortical excitability that outlast the period of application. We present evidence that changes in the reactivity of the corticospinal pathway induced by DC stimulation of the motor cortex interact systematically with those brought about by contraction of the muscles of the ipsilateral limb. During the application of flexion torques (up to 50% of maximum) applied at the left wrist, motor evoked potentials (MEPs) were evoked in the quiescent muscles of the right arm by magnetic stimulation of the left motor cortex (M1). The MEPs were obtained prior to and following 10 min of anodal, cathodal or sham DC stimulation of left M1. Cathodal stimulation counteracted increases in the crossed-facilitation of projections to the (right) wrist flexors that otherwise occurred as a result of repeated flexion contractions at the left wrist. In addition, cathodal stimulation markedly decreased the excitability of corticospinal projections to the wrist extensors of the right limb. Thus changes in corticospinal excitability induced by DC stimulation can be shaped (i.e. differentiated by muscle group) by focal contractions of muscles in the limb ipsilateral to the site of stimulation.

  12. An Uncleaved Signal Peptide Directs the Malus xiaojinensis Iron Transporter Protein Mx IRT1 into the ER for the PM Secretory Pathway

    PubMed Central

    Zhang, Peng; Tan, Song; Berry, James O.; Li, Peng; Ren, Na; Li, Shuang; Yang, Guang; Wang, Wei-Bing; Qi, Xiao-Ting; Yin, Li-Ping

    2014-01-01

    Malus xiaojinensis iron-regulated transporter 1 (Mx IRT1) is a highly effective inducible iron transporter in the iron efficient plant Malus xiaojinensis. As a multi-pass integral plasma membrane (PM) protein, Mx IRT1 is predicted to consist of eight transmembrane domains, with a putative N-terminal signal peptide (SP) of 1–29 amino acids. To explore the role of the putative SP, constructs expressing Mx IRT1 (with an intact SP) and Mx DsIRT1 (with a deleted SP) were prepared for expression in Arabidopsis and in yeast. Mx IRT1 could rescue the iron-deficiency phenotype of an Arabidopsis irt1 mutant, and complement the iron-limited growth defect of the yeast mutant DEY 1453 (fet3fet4). Furthermore, fluorescence analysis indicated that a chimeric Mx IRT1-eGFP (enhanced Green Fluorescent Protein) construct was translocated into the ER (Endoplasmic reticulum) for the PM sorting pathway. In contrast, the SP-deleted Mx DsIRT1 could not rescue either of the mutant phenotypes, nor direct transport of the GFP signal into the ER. Interestingly, immunoblot analysis indicated that the SP was not cleaved from the mature protein following transport into the ER. Taken together, data presented here provides strong evidence that an uncleaved SP determines ER-targeting of Mx IRT1 during the initial sorting stage, thereby enabling the subsequent transport and integration of this protein into the PM for its crucial role in iron uptake. PMID:25387073

  13. Mechanical Characterization of Photo-crosslinked, Thermoresponsive Hydrogel Thin Films via AFM Nanoindentation

    NASA Astrophysics Data System (ADS)

    Le, Thao; Aidala, Katherine; Hayward, Ryan

    2014-03-01

    Thin hydrogel films with patterned swelling are known to buckle into programmed three-dimensional shapes, offering approaches to fabricate reversibly self-folding micro-devices for actuators and drug delivery devices. To precisely control the shapes adopted, it is important to quantitatively understand the relationship between swelling and mechanical properties. Furthermore, to understand the buckling pathways and the mechanical responses of the swelled materials, it is also important to identify how the gels undergo stress relaxation. However, the low moduli, high water contents, and micrometer-scale thicknesses of these materials have so far made mechanical characterization difficult. In this study, we use an AFM nanoindentation technique to characterize the mechanical properties of photo-crosslinked, thermoresponsive poly(N-isopropylacrylamide) hydrogel thin films. Simultaneously, we conduct stress relaxation experiments at microscopic indentation lengths to differentiate between the effects of viscoelastic and poroelastic response mechanisms. This research was funded by the Army Research Office through W911NF-11-1-0080 and the NSF Materials Research Science and Engineering Center at the University of Massachusetts through DMR-0820506.

  14. BOREAS AFM-12 1-km AVHRR Seasonal Land Cover Classification

    NASA Technical Reports Server (NTRS)

    Steyaert, Lou; Hall, Forrest G.; Newcomer, Jeffrey A. (Editor); Knapp, David E. (Editor); Loveland, Thomas R.; Smith, David E. (Technical Monitor)

    2000-01-01

    The Boreal Ecosystem-Atmosphere Study (BOREAS) Airborne Fluxes and Meteorology (AFM)-12 team's efforts focused on regional scale Surface Vegetation and Atmosphere (SVAT) modeling to improve parameterization of the heterogeneous BOREAS landscape for use in larger scale Global Circulation Models (GCMs). This regional land cover data set was developed as part of a multitemporal one-kilometer Advanced Very High Resolution Radiometer (AVHRR) land cover analysis approach that was used as the basis for regional land cover mapping, fire disturbance-regeneration, and multiresolution land cover scaling studies in the boreal forest ecosystem of central Canada. This land cover classification was derived by using regional field observations from ground and low-level aircraft transits to analyze spectral-temporal clusters that were derived from an unsupervised cluster analysis of monthly Normalized Difference Vegetation Index (NDVI) image composites (April-September 1992). This regional data set was developed for use by BOREAS investigators, especially those involved in simulation modeling, remote sensing algorithm development, and aircraft flux studies. Based on regional field data verification, this multitemporal one-kilometer AVHRR land cover mapping approach was effective in characterizing the biome-level land cover structure, embedded spatially heterogeneous landscape patterns, and other types of key land cover information of interest to BOREAS modelers.The land cover mosaics in this classification include: (1) wet conifer mosaic (low, medium, and high tree stand density), (2) mixed coniferous-deciduous forest (80% coniferous, codominant, and 80% deciduous), (3) recent visible bum, vegetation regeneration, or rock outcrops-bare ground-sparsely vegetated slow regeneration bum (four classes), (4) open water and grassland marshes, and (5) general agricultural land use/ grasslands (three classes). This land cover mapping approach did not detect small subpixel-scale landscape

  15. AFM imaging of ligand binding to platelet integrin alphaIIbbeta3 receptors reconstituted into planar lipid bilayers.

    PubMed

    Hussain, Mohammad A; Agnihotri, Aashiish; Siedlecki, Christopher A

    2005-07-19

    The platelet integrin alphaIIbbeta3 plays a key role in platelet adhesion, activation, and aggregation at the subendothelium and at protein-coated synthetic biomaterials. In this study, interactions between alphaIIbbeta3 and both protein and peptide ligands for the receptor were imaged under physiological conditions by high-resolution atomic force microscopy (AFM). To directly image the ligand-receptor interactions, alphaIIbbeta3 receptors were reconstituted into a supported lipid bilayer formed on a mica surface in the AFM fluid cell assembly and subsequently activated with Mn2+. Fibrinogen, the natural protein ligand for the integrin, as well as a nanogold-labeled peptide ligand (an RGD-containing heptamer) were infused into the AFM fluid cell, incubated with the reconstituted and activated receptors, and imaged under buffer. Height images illustrating topographical features showed the integrin reconstituted in the bilayer. Fibrinogen molecules binding to the receptors were easily observed in the height images, with fibrinogen showing its characteristic trinodular structure and occasionally bridging integrin receptors. Fibrinogen was observed to bind to integrins at the D-domain consistent with the location of the gamma-chain dodecapeptide, while fibrinogen bridging integrins bound to receptors on opposite sides of the protein consistent with a 2-fold axis of symmetry. Peptide ligands were not visible in height images; however, phase images that map the mechanical properties detected the nanogold labels and demonstrated the presence of peptide ligands bound to the receptors. The results demonstrate the ability of this high-resolution microscopy technique to directly visualize single ligand/receptor interactions in a dynamic and physiologically relevant environment, and establish a framework for future fundamental studies of single protein/receptor interactions during normal pathological processes as well as biomaterial surface-induced thrombosis.

  16. Sensing Positive versus Negative Reward Signals through Adenylyl Cyclase-Coupled GPCRs in Direct and Indirect Pathway Striatal Medium Spiny Neurons

    PubMed Central

    Nair, Anu G.; Eriksson, Olivia; Vincent, Pierre

    2015-01-01

    importance of interaction between acetylcholine, dopamine, and adenosine signals via adenylyl-cyclase coupled GPCRs in shaping the dopamine-dependent cAMP/PKA signaling in striatal neurons. Specifically, a dopamine peak and an acetylcholine dip must interact, via D1 and M4 receptor, and a dopamine dip must interact with adenosine tone, via D2 and A2a receptor, in direct and indirect pathway neurons, respectively, to have any significant downstream PKA activation. PMID:26468202

  17. New insights into morphology of high performance BHJ photovoltaics revealed by high resolution AFM.

    PubMed

    Wang, Dong; Liu, Feng; Yagihashi, Noritoshi; Nakaya, Masafumi; Ferdous, Sunzida; Liang, Xiaobin; Muramatsu, Atsushi; Nakajima, Ken; Russell, Thomas P

    2014-10-08

    Direct imaging of the bulk heterojunction (BHJ) thin film morphology in polymer-based solar cells is essential to understand device function and optimize efficiency. The morphology of the BHJ active layer consists of bicontinuous domains of the donor and acceptor materials, having characteristic length scales of several tens of nanometers, that reduces charge recombination, enhances charge separation, and enables electron and hole transport to their respective electrodes. Direct imaging of the morphology from the molecular to macroscopic level, though, is lacking. Though transmission electron tomography provides a 3D, real-space image of the morphology, quantifying the structure is not possible. Here we used high-resolution atomic force microscopy (AFM) in the tapping and nanomechanical modes to investigate the BHJ active layer morphology that, when combined with Ar(+) etching, provided unique insights with unparalleled spatial resolution. PCBM was seen to form a network that interpenetrated into the fibrillar network of the hole-conducting polymer, both being imbedded in a mixture of the two components. The free surface was found to be enriched with polymer crystals having a "face-on" orientation and the morphology at the anode interface was markedly different.

  18. The PDZ Protein Canoe/AF-6 Links Ras-MAPK, Notch and Wingless/Wnt Signaling Pathways by Directly Interacting with Ras, Notch and Dishevelled

    PubMed Central

    Carmena, Ana; Speicher, Stephan; Baylies, Mary

    2006-01-01

    Over the past few years, it has become increasingly apparent that signal transduction pathways are not merely linear cascades; they are organized into complex signaling networks that require high levels of regulation to generate precise and unique cell responses. However, the underlying regulatory mechanisms by which signaling pathways cross-communicate remain poorly understood. Here we show that the Ras-binding protein Canoe (Cno)/AF-6, a PDZ protein normally associated with cellular junctions, is a key modulator of Wingless (Wg)/Wnt, Ras-Mitogen Activated Protein Kinase (MAPK) and Notch (N) signaling pathways cross-communication. Our data show a repressive effect of Cno/AF-6 on these three signaling pathways through physical interactions with Ras, N and the cytoplasmic protein Dishevelled (Dsh), a key Wg effector. We propose a model in which Cno, through those interactions, actively coordinates, at the membrane level, Ras-MAPK, N and Wg signaling pathways during progenitor specification. PMID:17183697

  19. Signaling pathway via TNF-alpha/NF-kappaB in intestinal epithelial cells may be directly involved in colitis-associated carcinogenesis.

    PubMed

    Onizawa, Michio; Nagaishi, Takashi; Kanai, Takanori; Nagano, Ken-ichi; Oshima, Shigeru; Nemoto, Yasuhiro; Yoshioka, Atsushi; Totsuka, Teruji; Okamoto, Ryuichi; Nakamura, Tetsuya; Sakamoto, Naoya; Tsuchiya, Kiichiro; Aoki, Kazuhiro; Ohya, Keiichi; Yagita, Hideo; Watanabe, Mamoru

    2009-04-01

    Treatment with anti-TNF-alpha MAb has been accepted as a successful maintenance therapy for patients with inflammatory bowel diseases (IBD). Moreover, it has been recently reported that blockade of TNF receptor (TNFR) 1 signaling in infiltrating hematopoietic cells may prevent the development of colitis-associated cancer (CAC). However, it remains unclear whether the TNF-alpha signaling in epithelial cells is involved in the development of CAC. To investigate this, we studied the effects of anti-TNF-alpha MAb in an animal model of CAC by administration of azoxymethane (AOM) followed by sequential dextran sodium sulfate (DSS) ingestion. We observed that the NF-kappaB pathway is activated in colonic epithelia from DSS-administered mice in association with upregulation of TNFR2 rather than TNFR1. Immunoblot analysis also revealed that the TNFR2 upregulation accompanied by the NF-kappaB activation is further complicated in CAC tissues induced in AOM/DSS-administered mice compared with the nontumor area. Such NF-kappaB activity in the epithelial cells is significantly suppressed by the treatment of MP6-XT22, an anti-TNF-alpha MAb. Despite inability to reduce the severity of colitis, sequential administration of MP6-XT22 reduced the numbers and size of tumors in association with the NF-kappaB inactivation. Taken together, present studies suggest that the TNFR2 signaling in intestinal epithelial cells may be directly involved in the development of CAC with persistent colitis and imply that the maintenance therapy with anti-TNF-alpha MAb may prevent the development of CAC in patients with long-standing IBD.

  20. Cocaine Withdrawal Impairs mGluR5-Dependent Long-Term Depression in Nucleus Accumbens Shell Neurons of Both Direct and Indirect Pathways.

    PubMed

    Huang, Chiung-Chun; Liang, Ying-Ching; Lee, Cheng-Che; Hsu, Kuei-Sen

    2015-12-01

    We previously reported that animals withdrawn from repeated cocaine exposure exhibited a selective deficit in the ability to elicit metabotropic glutamate receptor 5 (mGluR5)-dependent long-term depression (LTD) in the nucleus accumbens (NAc) shell. To determine whether such impairment occurs in the NAc in a cell-type-specific manner, we used bacterial artificial chromosome (BAC) transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of gene regulatory elements for the dopamine D1 receptor (Drd1) or dopamine D2 receptor (Drd2) to identify distinct subpopulations of medium spiny neurons (MSNs). We found that bath application of group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) reliably induced LTD in both NAc shell and core MSNs of wild-type, hemizygous Drd1-eGFP, and Drd2-eGFP mice. Confirming our previous results, cocaine withdrawal selectively impaired DHPG-LTD in NAc shell Drd1-expressing direct and Drd2-expressing indirect pathway MSNs. We also found that the expression of DHPG-LTD in NAc MSNs was not affected by the Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 1-naphthyl acetyl spermine. Furthermore, systemic administration of mGluR5-negative allosteric modulator fenobam before the daily injection of cocaine preserved mGluR5 function and significantly reduced the expression of cocaine-induced behavioral sensitization. These results reveal that withdrawal from repeated cocaine exposure may result in the impairment of NAc mGluR5-LTD in a subregion- but not cell-type-specific manner and suggests that pharmacological antagonism of mGluR5 may represent a potential strategy for reducing cocaine-induced addictive behaviors.

  1. Tip Characterization Method using Multi-feature Characterizer for CD-AFM

    PubMed Central

    Orji, Ndubuisi G.; Itoh, Hiroshi; Wang, Chumei; Dixson, Ronald G.; Walecki, Peter S.; Schmidt, Sebastian W.; Irmer, Bernd

    2016-01-01

    In atomic force microscopy (AFM) metrology, the tip is a key source of uncertainty. Images taken with an AFM show a change in feature width and shape that depends on tip geometry. This geometric dilation is more pronounced when measuring features with high aspect ratios, and makes it difficult to obtain absolute dimensions. In order to accurately measure nanoscale features using an AFM, the tip dimensions should be known with a high degree of precision. We evaluate a new AFM tip characterizer, and apply it to critical dimension AFM (CD-AFM) tips used for high aspect ratio features. The characterizer is made up of comb-shaped lines and spaces, and includes a series of gratings that could be used as an integrated nanoscale length reference. We also demonstrate a simulation method that could be used to specify what range of tip sizes and shapes the characterizer can measure. Our experiments show that for non re-entrant features, the results obtained with this characterizer are consistent to 1 nm with the results obtained by using widely accepted but slower methods that are common practice in CD-AFM metrology. A validation of the integrated length standard using displacement interferometry indicates a uniformity of better than 0.75%, suggesting that the sample could be used as highly accurate and SI traceable lateral scale for the whole evaluation process. PMID:26720439

  2. AFM nanoscale indentation in air of polymeric and hybrid materials with highly different stiffness

    NASA Astrophysics Data System (ADS)

    Suriano, Raffaella; Credi, Caterina; Levi, Marinella; Turri, Stefano

    2014-08-01

    In this study, nanomechanical properties of a variety of polymeric materials was investigated by means of AFM. In particular, selecting different AFM probes, poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS) bulk samples, sol-gel hybrid thin films and hydrated hyaluronic acid hydrogels were indented in air to determine the elastic modulus. The force-distance curves and the indentation data were found to be greatly affected by the cantilever stiffness and by tip geometry. AFM indentation tests show that the choice of the cantilever spring constant and of tip shape is crucially influenced by elastic properties of samples. When adhesion-dominated interactions occur between the tip and the surface of samples, force-displacement curves reveal that a suitable functionalization of AFM probes allows the control of such interactions and the extraction of Young' modulus from AFM curves that would be otherwise unfeasible. By applying different mathematical models depending on AFM probes and materials under investigation, the values of Young's modulus were obtained and compared to those measured by rheological and dynamic mechanical analysis or to literature data. Our results show that a wide range of elastic moduli (10 kPa-10 GPa) can be determined by AFM in good agreement with those measured by conventional macroscopic measurements.

  3. Direct measurement of electrostatic fields using single Teflon nanoparticle attached to AFM tip

    PubMed Central

    2013-01-01

    Abstract A single 210-nm Teflon nanoparticle (sTNP) was attached to the vertex of a silicon nitride (Si3N4) atomic force microscope tip and charged via contact electrification. The charged sTNP can then be considered a point charge and used to measure the electrostatic field adjacent to a parallel plate condenser using 30-nm gold/20-nm titanium as electrodes. This technique can provide a measurement resolution of 250/100 nm along the X- and Z-axes, and the minimum electrostatic force can be measured within 50 pN. PACS 07.79.Lh, 81.16.-c, 84.37. + q PMID:24314111

  4. XPS and AFM Study of GaAs Surface Treatment

    SciTech Connect

    Contreras-Guerrero, R.; Wallace, R. M.; Aguirre-Francisco, S.; Herrera-Gomez, A.; Lopez-Lopez, M.

    2008-11-13

    Obtaining smooth and atomically clean surfaces is an important step in the preparation of a surface for device manufacturing. In this work different processes are evaluated for cleaning a GaAs surface. A good surface cleaning treatment is that which provides a high level of uniformity and controllability of the surface. Different techniques are useful as cleaning treatments depending on the growth process to be used. The goal is to remove the oxygen and carbon contaminants and then form a thin oxide film to protect the surface, which is easy to remove later with thermal desorption mechanism like molecular beam epitaxy (MBE) with minimal impact to the surface. In this study, atomic force microscopy (AFM), x-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS) were used to characterize the structure of the surface, the composition, as well as detect oxygen and carbon contaminant on the GaAs surface. This study consists in two parts. The first part the surface was subjected to different chemical treatments. The chemical solutions were: (a)H{sub 2}SO{sub 4}:H{sub 2}O{sub 2}:H{sub 2}O(4:1:100), (b) HCl: H{sub 2}O(1:3), (c)NH{sub 4}OH 29%. The treatments (a) and (b) reduced the oxygen on the surface. Treatment (c) reduces carbon contamination. In the second part we made MOS devices on the surfaces treated. They were characterized by CV and IV electrical measurements. They show frequency dispersion.

  5. AF-M315E Propulsion System Advances and Improvements

    NASA Technical Reports Server (NTRS)

    Masse, Robert K.; Allen, May; Driscoll, Elizabeth; Spores, Ronald A.; Arrington, Lynn A.; Schneider, Steven J.; Vasek, Thomas E.

    2016-01-01

    Even as for the GR-1 awaits its first on-orbit demonstration on the planned 2017 launch of NASA's Green Propulsion Infusion Mission (GPIM) program, ongoing efforts continue to advance the technical state-of-the-art through improvements in the performance, life capability, and affordability of both Aerojet Rocketdyne's 1-N-class GR-1 and 20-N-class GR-22 green monopropellant thrusters. Hot-fire testing of a design upgrade of the GR-22 thruster successfully demonstrated resolution of a life-limiting thermo-structural issue encountered during prototype testing on the GPIM program, yielding both an approximately 2x increase in demonstrating life capability, as well as fundamental insights relating to how ionic liquid thrusters operate, thruster scaling, and operational factors affecting catalyst bed life. Further, a number of producibility improvements, related to both materials and processes and promising up to 50% unit cost reduction, have been identified through a comprehensive Design for Manufacturing and Assembly (DFMA) assessment activity recently completed at Aerojet Rocketdyne. Focused specifically on the GR-1 but applicable to the common-core architecture of both thrusters, ongoing laboratory (heavyweight) thruster testing being conducted under a Space Act Agreement at NASA Glenn Research Center has already validated a number of these proposed manufacturability upgrades, additionally achieving a greater than 40% increase in thruster life. In parallel with technical advancements relevant to conventional large spacecraft, a joint effort between NASA and Aerojet Rocketdyne is underway to prepare 1-U CubeSat AF-M315E propulsion module for first flight demonstration in 2018.

  6. The formation of liquid bridge in different operating modes of AFM

    NASA Astrophysics Data System (ADS)

    Wei, Zheng; Sun, Yan; Ding, WenXuan; Wang, ZaiRan

    2016-09-01

    The liquid bridge is one of the principal factors that cause artifacts in ambient-pressure atomic force microscope (AFM) images. Additionally, it is the main component of the adhesion force in ambient conditions. To understand the AFM imaging mechanism and the sample characteristics, it is essential to study the liquid bridge. This study interprets the physical mechanism involved in liquid bridge formation, which is composed of three different physical processes: the squeezing process, capillary condensation, and liquid film flow. We discuss the contributions of these three mechanisms to the volume and the capillary force of the liquid bridge in different AFM operation modes.

  7. Wetting properties of AFM probes by means of contact angle measurement

    NASA Astrophysics Data System (ADS)

    Tao, Zhenhua; Bhushan, Bharat

    2006-09-01

    An atomic force microscopy (AFM) based technique was developed to measure the wetting properties of probe tips. By advancing and receding the AFM tip across the water surface, the meniscus force between the tip and the liquid was measured at the tip-water separation. The water contact angle was determined from the meniscus force. The obtained contact angle results were compared with that by the sessile drop method. It was found that the AFM based technique provided higher contact angle values than the sessile drop method. The mechanisms responsible for the difference are discussed.

  8. Probing Cytoskeletal Structures by Coupling Optical Superresolution and AFM Techniques for a Correlative Approach

    PubMed Central

    Chacko, Jenu Varghese; Zanacchi, Francesca Cella; Diaspro, Alberto

    2013-01-01

    In this article, we describe and show the application of some of the most advanced fluorescence superresolution techniques, STED AFM and STORM AFM microscopy towards imaging of cytoskeletal structures, such as microtubule filaments. Mechanical and structural properties can play a relevant role in the investigation of cytoskeletal structures of interest, such as microtubules, that provide support to the cell structure. In fact, the mechanical properties, such as the local stiffness and the elasticity, can be investigated by AFM force spectroscopy with tens of nanometers resolution. Force curves can be analyzed in order to obtain the local elasticity (and the Young's modulus calculation by fitting the force curves from every pixel of interest), and the combination with STED/STORM microscopy integrates the measurement with high specificity and yields superresolution structural information. This hybrid modality of superresolution-AFM working is a clear example of correlative multimodal microscopy. PMID:24027190

  9. Nanogap based graphene coated AFM tips with high spatial resolution, conductivity and durability.

    PubMed

    Lanza, Mario; Gao, Teng; Yin, Zixuan; Zhang, Yanfeng; Liu, Zhongfan; Tong, Yuzhen; Shen, Ziyong; Duan, Huiling

    2013-11-21

    After one decade of analyzing the intrinsic properties of graphene, interest into the development of graphene-based devices and micro electromechanical systems is increasing. Here, we fabricate graphene-coated atomic force microscope tips by growing the graphene on copper foil and transferring it onto the apex of a commercially available AFM tip. The resulting tip exhibits surprising enhanced resolution in nanoscale electrical measurements. By means of topographic AFM maps and statistical analyses we determine that this superior performance may be related to the presence of a nanogap between the graphene and the tip apex, which reduces the tip radius and tip-sample contact area. In addition, the graphene-coated tips show a low tip-sample interaction, high conductivity and long life times. The novel fabrication-friendly tip could improve the quality and reliability of AFM experiments, while reducing the cost of AFM-based research.

  10. Adiabatic Compression Sensitivity of AF-M315E (Briefing Charts)

    DTIC Science & Technology

    2015-07-27

    dynamic response • Waterhammer effect Distribution A: Approved for public release; distribution unlimited Hydroxyethylhydrazinium Nitrate ...Hydroxylammonium Nitrate (HEHN) (HAN) [ ]-NO3 + [ ]HOCH2CH2N2H4 [ ]-+[ ]NH3OH NO3 AF-M315E

  11. Human chorionic gonadotropin suppresses human breast cancer cell growth directly via p53-mediated mitochondrial apoptotic pathway and indirectly via ovarian steroid secretion.

    PubMed

    Yuri, Takashi; Kinoshita, Yuichi; Emoto, Yuko; Yoshizawa, Katsuhiko; Tsubura, Airo

    2014-03-01

    The tumor-suppressive effects of human chorionic gonadotropin (hCG) against human breast cancer cells were examined. In cell viability assays, hCG inhibited the growth of three human breast cancer cell lines (estrogen receptor (ER)-positive KPL-1 and MCF-7, and ER-negative MKL-F cells), and the growth inhibition activity of hCG was most pronounced against KPL-1 cells (luteinizing hormone/chorionic gonadotropin receptor (LHCGR)-positive and luminal-A subtype). In hCG-treated KPL-1 cells, immunoblotting analysis revealed the expression of tumor suppressor protein p53 peaking at 12 h following treatment, followed by cleavage of caspase-9 and caspase-3 at 24 h and 48 h, respectively. KPL-1-transplanted athymic mice were divided into 3 groups: a sham-treated group that received an inoculation of KPL-1 cells at 6 weeks of age followed by daily intraperitoneal (i.p.) injection of saline; an in vitro hCG-treated KPL-1 group that received an inoculation of KPL-1 cells pre-treated with 100 IU/ml hCG in vitro for 48 h at 6 weeks of age, followed by daily i.p. injection of saline; and an in vivo hCG-treated group that received an KPL-1 cell inoculation at 6 weeks of age, followed by daily i.p. injection of 100 IU hCG. The daily injections of saline or hCG continued until the end of the experiment when mice reached 11 weeks of age. KPL-1 tumor growth was retarded in in vitro and in vivo hCG-treated mice compared to sham-treated controls, and the final tumor volume and tumor weight tended to be suppressed in the in vitro hCG-treated group and were significantly suppressed in the in vivo hCG-treated group. In vivo 100-IU hCG injections for 5 weeks elevated serum estradiol levels (35.7 vs. 23.5 pg/ml); thus, the mechanisms of hCG action may be directly coordinated via the p53-mediated mitochondrial apoptotic pathway and indirectly through ovarian steroid secretion that elevates estrogen levels. It is thus concluded that hCG may be an attractive agent for treating human breast

  12. Comparison of the ability of quantitative parameters to differentiate surface texture of Atomic Force Microscope (AFM) images

    NASA Astrophysics Data System (ADS)

    Niedzielski, Bethany; Caragianis Broadbridge, Christine; DaPonte, John S.; Gherasimova, Maria

    2010-01-01

    The purpose of this study was to compare the ability of several texture analysis parameters to differentiate textured samples from a smooth control on images obtained with an Atomic Force Microscope (AFM). Surface roughness plays a major role in the realm of material science, especially in integrated electronic devices. As these devices become smaller and smaller, new materials with better electrical properties are needed. New materials with smoother surface morphology have been found to have superior electrical properties than their rougher counterparts. Therefore, in many cases surface texture is indicative of the electrical properties that material will have. Physical vapor deposition techniques such as Jet Vapor Deposition and Molecular Beam Epitaxy are being utilized to synthesize these materials as they have been found to create pure and uniform thin layers. For the current study, growth parameters were varied to produce a spectrum of textured samples. The focus of this study was the image processing techniques associated with quantifying surface texture. As a result of the limited sample size, there was no attempt to draw conclusions about specimen processing methods. The samples were imaged using an AFM in tapping mode. In the process of collecting images, it was discovered that roughness data was much better depicted in the microscope's "height" mode as opposed to "equal area" mode. The AFM quantified the surface texture of each image by returning RMS roughness and the first order histogram statistics of mean roughness, standard deviation, skewness, and kurtosis. Color images from the AFM were then processed on an off line computer running NIH ImageJ with an image texture plug in. This plug in produced another set of first order statistics computed from each images' histogram as well as second order statistics computed from each images' cooccurrence matrix. The second order statistics, which were originally proposed by Haralick, include contrast, angular

  13. A Multistage Pathway for Human Prion Protein Aggregation in Vitro: From Multimeric Seeds to β-Oligomers and Nonfibrillar Structures

    PubMed Central

    Cho, Kang R.; Yu, Shuiliang; Yin, Shaoman; Plomp, Marco; Qiu, S. Roger; Lakshminarayanan, Rajamani; Moradian-Oldak, Janet; Sy, Man-Sun

    2015-01-01

    Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt—Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of β-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3–4 monomers quickly appeared. Oligomers of ∼11–12 monomers then formed through direct interaction of seeds, rather than by subsequent monomer attachment. All larger aggregates formed through association of these β-oligomers. Although both WT and 10OR exhibited identical aggregation mechanisms, the latter oligomerized faster due to lower solubility and, hence, thermodynamic stability. This novel aggregation pathway has implications for prion diseases as well as others caused by protein aggregation. PMID:21534611

  14. BOREAS AFM-1 NOAA/ATDD Long-EZ Aircraft Flux data Over the SSA

    NASA Technical Reports Server (NTRS)

    Crawford, Timothy L.; Baldocchi, Dennis; Hall, Forrest G. (Editor); Knapp, David E. (Editor); Gunter, Laureen; Dumas, Ed; Smith, David E. (Technical Monitor)

    2000-01-01

    This data set contains measurements from the Airborne Flux and Meteorology (AFM)-1 National Oceanographic and Atmospheric Administration/Atmospheric Turbulence and Diffusion Division (NOAA/ATDD) Long-EZ Aircraft collected during the 1994 Intensive Field Campaigns (IFCs) at the southern study area (SSA). These measurements were made from various instruments mounted on the aircraft. The data that were collected include aircraft altitude, wind direction, wind speed, air temperature, potential temperature, water mixing ratio, U and V components of wind velocity, static pressure, surface radiative temperature, downwelling and upwelling total radiation, downwelling and upwelling longwave radiation, net radiation, downwelling and upwelling photosynthectically active radiation (PAR), greenness index, CO2 concentration, O3 concentration, and CH4 concentration. There are also various columns that indicate the standard deviation, skewness, kurtosis, and trend of some of these data. The data are stored in tabular ASCII files. The NOAA/ATDD Long-EZ aircraft flux data are available from the Earth Observing System Data and Information System (EOSDIS) Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC). The data files are available on a CD-ROM (see document number 20010000884).

  15. Surface characterisation of two strains of Staphylococcus epidermidis with different slime-production by AFM

    NASA Astrophysics Data System (ADS)

    Méndez-Vilas, A.; Gallardo-Moreno, A. M.; González-Martín, M. L.; Calzado-Montero, R.; Nuevo, M. J.; Bruque, J. M.; Pérez-Giraldo, C.

    2004-11-01

    Slime-producer Staphylococcus epidermidis is one opportunistic bacteria directly related to biomaterial infections inside the human body. The characterisation of the bacterial surface is crucial when trying to control its adhesion process and prevent the biofilm formation. This work aims to analyse the microscopic and submicroscopic surface structure of two strains of S. epidermidis with different slime production, as well as mapping the surface interaction forces. Atomic force microscopy (AFM) shows that S. epidermidis ATCC35984 is covered by a granular-like film, highly compacted with the presence of repeated "holes". However, S. epidermidis ATCC35983 only shows a partial coverage by a less compacted granular-like film, mainly located in the inter-cellular zones. Both films are related to the slime of the two strains studied. As regards to the adhesion forces, results show a greater adhesion of the tip to the slime covering S. epidermidis ATCC35984, than that covering the surface of S. epidermidis ATCC35983. In addition, the adhesion to the free-slime zones of the last strain was higher than to the slime-covered parts.

  16. Measuring the viscoelastic creep of soft samples by step response AFM.

    PubMed

    Yango, Achu; Schäpe, Jens; Rianna, Carmela; Doschke, Holger; Radmacher, Manfred

    2016-10-12

    We have measured the creep response of soft gels and cells after applying a step in loading force with atomic force microscopy (AFM). By analysing the creep response data using the standard linear solid model, we can quantify the viscous and elastic properties of these soft samples independently. Cells, in comparison with gels of similar softness, are much more viscous, as has been qualitatively observed in conventional force curve data before. Here, we quantify the spring constant and the viscous damping coefficient from the creep response data. We propose two different modes for applying a force step: (1) indirectly by increasing the sample height or (2) directly by employing magnetic cantilevers. Both lead to similar results, whereas the latter seems to be better defined since it resembles closely a constant strain mode. The former is easier to implement in most instruments, and thus may be preferable from a practical point of view. Creep analysis by step response is much more appropriate to analyse the viscoelastic response of soft samples like cells than the usually used force curve analysis.

  17. Porous titania films fabricated via sol gel rout - Optical and AFM characterization

    NASA Astrophysics Data System (ADS)

    Karasiński, Paweł; Gondek, Ewa; Drewniak, Sabina; Kajzer, Anita; Waczyńska-Niemiec, Natalia; Basiaga, Marcin; Izydorczyk, Weronika; Kouari, Youssef E. L.

    2016-06-01

    Mesoporous titania films of low refractive index ∼1.72 and thickness within the range of 57-96 nm were fabricated via sol-gel rout and dip-coating technique on a soda-lime glass substrate. Tetrabutylorthotitanate Ti(OBu)4 was used as a titania precursor. High porosity and consequently low refractive index were achieved using the polyethylene glycol (PEG 1100) as a template. Based on transmittance, using Tauc's relations, the optical energy band gaps and the Urbach energy were determined. The research shows that in the fabricated titania films there are two types of optical energy band gaps, connected with direct and indirect electron transitions and brought about by the presence of amorphous and crystalline phase respectively. Based on the quantum size effect, the diameters of nanocrystals versus film thickness were determined. AFM studies of the titania films have demonstrated that there are changes of surface morphology taking place with the change of thickness. We have demonstrated that the surface morphology of titania films has influence on wettability.

  18. AFM/TIRF force clamp measurements of neurosecretory vesicle tethers reveal characteristic unfolding steps

    PubMed Central

    Harris, Mark C.; Cislo, Dillon; Lenz, Joan S.; Umbach, Christopher

    2017-01-01

    Although several proteins have been implicated in secretory vesicle tethering, the identity and mechanical properties of the components forming the physical vesicle-plasma membrane link remain unknown. Here we present the first experimental measurements of nanomechanical properties of secretory vesicle-plasma membrane tethers using combined AFM force clamp and TIRF microscopy on membrane sheets from PC12 cells expressing the vesicle marker ANF-eGFP. Application of pulling forces generated tether extensions composed of multiple steps with variable length. The frequency of short (<10 nm) tether extension events was markedly higher when a fluorescent vesicle was present at the cantilever tip and increased in the presence of GTPγS, indicating that these events reflect specifically the properties of vesicle-plasma membrane tethers. The magnitude of the short tether extension events is consistent with extension lengths expected from progressive unfolding of individual helices of the exocyst complex, supporting its direct role in forming the physical vesicle-plasma membrane link. PMID:28323853

  19. AFM characterization of spin coated carboxylated polystyrene nanospheres/xyloglucan layers on mica and silicon.

    PubMed

    Lubambo, Adriana F; Lucyszyn, Neoli; Petzhold, Cesar L; Sierakowski, Maria-R; Schreiner, Wido H; Saul, Cyro K

    2013-03-01

    Self-assembled nano-arrays have a potential application as solid-phase diagnostics in many biomedical devices. The easiness of its production is directly connected to manufacture cost reduction. In this work, we present self-assembled structures starting from spin coated thin films of carboxylated polystyrene (PSC) and xyloglucan (XG) mixtures on both mica and silicon substrates. AFM images showed PSC nanospheres on top of a homogeneous layer of XG, for both substrates. The average nanosphere diameter fluctuated for a constant speed and it was likely to be independent of the component proportions on the mixture within a range of 30-50% (v/v) PSC. It was also observed that the largest diameters were found at the center of the sample and the smallest at the border. The detected nanospheres were also more numerous at the border. This behavior presents a similarity to spin coated colloidal dispersions. We observed that the average nanosphere diameter on mica substrates was bigger than the nanosphere diameters obtained on top of silicon substrates, under the same conditions. This result seems to be possibly connected to different mixture-surface interactions.

  20. Leveraging Air Force Medical Service (AFMS) Senior Leadership Corps Diversity to Improve Efficiency

    DTIC Science & Technology

    2013-04-01

    commanders and AFMS senior leadership; • Set a single PME standard for AFMS officers; • Shift provider billets to patient care roles and establish...single PME standard, and by realigning human resources to increase clinical currency, medical readiness and resource efficiency. Some structural...organizational entity. Like running a surgical service or a medical service. . . . It’s much bigger than that, because you’re dealing with finance and

  1. Surface Microstructure of Mo(C)N Coatings Investigated by AFM

    NASA Astrophysics Data System (ADS)

    Kuznetsova, T.; Zubar, T.; Chizhik, S.; Gilewicz, A.; Lupicka, O.; Warcholinski, B.

    2016-12-01

    MoCN coatings have been formed by cathodic arc evaporation using the mixture of acetylene and nitrogen and pure molybdenum target. The surface structure, in conjunction with x-ray data, was analyzed by atomic force microscopy (AFM). The AFM results show differently shaped grain forms on the surface of coatings investigated. The increase in carbon in chemical coatings composition results in the reduction in surface grain size and the increase in roughness of the coatings.

  2. Attaching single biomolecules selectively to the apex of AFM tips for measuring specific interactions.

    PubMed

    Gu, Jianhua; Xiao, Zhongdang; Yam, Chi-Ming; Qin, Guoting; Deluge, Maxence; Boutet, Sabine; Cai, Chengzhi

    2005-11-01

    We present a general approach for preparing well-defined AFM tips for probing single target molecules. We demonstrated that carboxylic acid groups could be generated by electrochemical oxidation selectively at the apex of an AFM tip that is coated with a monolayer of oligo(ethylene glycol) derivatives for resisting nonspecific interactions. These carboxylic acid groups were used as handles to tether only one ligand molecule, such as biotin, to the tip apex for measurement of specific interactions with biomolecules.

  3. Using XAFS, EDAX and AFM in comparative study of various natural and synthetic emeralds

    NASA Astrophysics Data System (ADS)

    Parikh, P.; Saini, N. L.; Dalela, S.; Bhardwaj, D. M.; Fernandes, S.; Gupta, R. P.; Garg, K. B.

    2003-01-01

    We have performed XAFS, EDAX and AFM studies on some natural and synthetic emeralds. While the XAFS results yield information on changes in the valence of the Cr ion and the n-n distance the AFM is used to determine the areal atomic density on surface of the crystals. It is a pilot study to explore if the three techniques can offer a possible way of distinguishing between the natural and synthetic emeralds and the results are promising.

  4. AFM Quantitative Morphological Analysis Of The Step Bunching Instability Formed On GaAs(110) During H-assisted MBE

    NASA Astrophysics Data System (ADS)

    Crespillo, M. L.; Tejedor, P.

    2007-04-01

    Power Spectral Density (PSD) analysis of Atomic Force Microscopy (AFM) images has been applied to study the effect of H-assisted surface oxide cleaning on the step bunching and Bales-Zangwill instabilities that develop during homoepitaxial growth from molecular beams of Ga and As4 on vicinal GaAs(110) substrates at high temperatures and high As:Ga flux ratios, leading to the formation of a characteristic ripple pattern along the [001] tilt direction. As growth proceeds in the presence of chemisorbed H, step bunching gradually vanishes and the ripple pattern breaks up into an array of self-organized nanowires running along the [11¯0] step edge direction.

  5. X-AFm stabilization as a mechanism of bypassing conversion phenomena in calcium aluminate cements

    SciTech Connect

    Falzone, Gabriel; Balonis, Magdalena; Sant, Gaurav

    2015-06-15

    Phase conversion phenomena are often observed in calcium aluminate cements (CACs), when the water-rich hydrates (e.g., CAH{sub 10}, C{sub 2}AH{sub 8}) formed at early ages, at temperatures ≤ 30 °C, expel water in time to form more compact, less water-rich structures (C{sub 3}AH{sub 6}). The phase conversions follow a path regulated by the thermodynamic stabilities (solubilities) of phases. Based on this premise, it is proposed that conversion phenomena in CACs can be bypassed by provoking the precipitation of phases more preferred than those typically encountered along the conversion pathway. Therefore, X-AFm formation (where in this case, X = NO{sub 3}{sup −}) triggered by the sequential addition of calcium nitrate (Ca(NO{sub 3}){sub 2} = CN) additives is identified as a new means of bypassing conversion. A multi-method approach comprising X-ray diffraction (XRD), thermal analytics, and evaluations of the compressive strength is applied to correlate phase balances and properties of CAC systems cured at 25 °C and 45 °C. The results highlight the absence of the C{sub 3}AH{sub 6} phase across all systems and the curing conditions considered, with enhanced strengths being noted, when sufficient quantities of CN are added. The experimental outcomes are supported by insights gained from thermodynamic calculations which highlight thermodynamic selectivity as a means of regulating and controlling the evolutions of solid phase balances using inorganic salts in CACs, and more generally in cementing material systems.

  6. Hydrocarbons in phlogopite from Kasenyi kamafugitic rocks (SW Uganda): cross-correlated AFM, confocal microscopy and Raman imaging

    NASA Astrophysics Data System (ADS)

    Moro, Daniele; Valdrè, Giovanni; Mesto, Ernesto; Scordari, Fernando; Lacalamita, Maria; Ventura, Giancarlo Della; Bellatreccia, Fabio; Scirè, Salvatore; Schingaro, Emanuela

    2017-01-01

    This study presents a cross-correlated surface and near surface investigation of two phlogopite polytypes from Kasenyi kamafugitic rocks (SW Uganda) by means of advanced Atomic Force Microscopy (AFM), confocal microscopy and Raman micro-spectroscopy. AFM revealed comparable nanomorphology and electrostatic surface potential for the two mica polytypes. A widespread presence of nano-protrusions located on the mica flake surface was also observed, with an aspect ratio (maximum height/maximum width) from 0.01 to 0.09. Confocal microscopy showed these features to range from few nm to several μm in dimension, and shapes from perfectly circular to ellipsoidic and strongly elongated. Raman spectra collected across the bubbles showed an intense and convolute absorption in the range 3000–2800 cm‑1, associated with weaker bands at 1655, 1438 and 1297 cm‑1, indicating the presence of fluid inclusions consisting of aliphatic hydrocarbons, alkanes and cycloalkanes, with minor amounts of oxygenated compounds, such as carboxylic acids. High-resolution Raman images provided evidence that these hydrocarbons are confined within the bubbles. This work represents the first direct evidence that phlogopite, a common rock-forming mineral, may be a possible reservoir for hydrocarbons.

  7. Hydrocarbons in phlogopite from Kasenyi kamafugitic rocks (SW Uganda): cross-correlated AFM, confocal microscopy and Raman imaging.

    PubMed

    Moro, Daniele; Valdrè, Giovanni; Mesto, Ernesto; Scordari, Fernando; Lacalamita, Maria; Ventura, Giancarlo Della; Bellatreccia, Fabio; Scirè, Salvatore; Schingaro, Emanuela

    2017-01-18

    This study presents a cross-correlated surface and near surface investigation of two phlogopite polytypes from Kasenyi kamafugitic rocks (SW Uganda) by means of advanced Atomic Force Microscopy (AFM), confocal microscopy and Raman micro-spectroscopy. AFM revealed comparable nanomorphology and electrostatic surface potential for the two mica polytypes. A widespread presence of nano-protrusions located on the mica flake surface was also observed, with an aspect ratio (maximum height/maximum width) from 0.01 to 0.09. Confocal microscopy showed these features to range from few nm to several μm in dimension, and shapes from perfectly circular to ellipsoidic and strongly elongated. Raman spectra collected across the bubbles showed an intense and convolute absorption in the range 3000-2800 cm(-1), associated with weaker bands at 1655, 1438 and 1297 cm(-1), indicating the presence of fluid inclusions consisting of aliphatic hydrocarbons, alkanes and cycloalkanes, with minor amounts of oxygenated compounds, such as carboxylic acids. High-resolution Raman images provided evidence that these hydrocarbons are confined within the bubbles. This work represents the first direct evidence that phlogopite, a common rock-forming mineral, may be a possible reservoir for hydrocarbons.

  8. Hydrocarbons in phlogopite from Kasenyi kamafugitic rocks (SW Uganda): cross-correlated AFM, confocal microscopy and Raman imaging

    PubMed Central

    Moro, Daniele; Valdrè, Giovanni; Mesto, Ernesto; Scordari, Fernando; Lacalamita, Maria; Ventura, Giancarlo Della; Bellatreccia, Fabio; Scirè, Salvatore; Schingaro, Emanuela

    2017-01-01

    This study presents a cross-correlated surface and near surface investigation of two phlogopite polytypes from Kasenyi kamafugitic rocks (SW Uganda) by means of advanced Atomic Force Microscopy (AFM), confocal microscopy and Raman micro-spectroscopy. AFM revealed comparable nanomorphology and electrostatic surface potential for the two mica polytypes. A widespread presence of nano-protrusions located on the mica flake surface was also observed, with an aspect ratio (maximum height/maximum width) from 0.01 to 0.09. Confocal microscopy showed these features to range from few nm to several μm in dimension, and shapes from perfectly circular to ellipsoidic and strongly elongated. Raman spectra collected across the bubbles showed an intense and convolute absorption in the range 3000–2800 cm−1, associated with weaker bands at 1655, 1438 and 1297 cm−1, indicating the presence of fluid inclusions consisting of aliphatic hydrocarbons, alkanes and cycloalkanes, with minor amounts of oxygenated compounds, such as carboxylic acids. High-resolution Raman images provided evidence that these hydrocarbons are confined within the bubbles. This work represents the first direct evidence that phlogopite, a common rock-forming mineral, may be a possible reservoir for hydrocarbons. PMID:28098185

  9. Tunable Semicrystalline Thin Film Cellulose Substrate for High-Resolution, In-Situ AFM Characterization of Enzymatic Cellulose Degradation.

    PubMed

    Ganner, Thomas; Roŝker, Stephanie; Eibinger, Manuel; Kraxner, Johanna; Sattelkow, Jürgen; Rattenberger, Johannes; Fitzek, Harald; Chernev, Boril; Grogger, Werner; Nidetzky, Bernd; Plank, Harald

    2015-12-23

    In the field of enzymatic cellulose degradation, fundamental interactions between different enzymes and polymorphic cellulose materials are of essential importance but still not understood in full detail. One technology with the potential of direct visualization of such bioprocesses is atomic force microscopy (AFM) due to its capability of real-time in situ investigations with spatial resolutions down to the molecular scale. To exploit the full capabilities of this technology and unravel fundamental enzyme-cellulose bioprocesses, appropriate cellulose substrates are decisive. In this study, we introduce a semicrystalline-thin-film-cellulose (SCFTC) substrate which fulfills the strong demands on such ideal cellulose substrates by means of (1) tunable polymorphism via variable contents of homogeneously sized cellulose nanocrystals embedded in an amorphous cellulose matrix; (2) nanoflat surface topology for high-resolution and high-speed AFM; and (3) fast, simple, and reproducible fabrication. The study starts with a detailed description of SCTFC preparation protocols including an in-depth material characterization. In the second part, we demonstrate the suitability of SCTFC substrates for enzymatic degradation studies by combined, individual, and sequential exposure to TrCel6A/TrCel7A cellulases (Trichoderma reesei) to visualize synergistic effects down to the nanoscale.

  10. AFM review study on pox viruses and living cells.

    PubMed

    Ohnesorge, F M; Hörber, J K; Häberle, W; Czerny, C P; Smith, D P; Binnig, G

    1997-10-01

    Single living cells were studied in growth medium by atomic force microscopy at a high--down to one image frame per second--imaging rate over time periods of many hours, stably producing hundreds of consecutive scans with a lateral resolution of approximately 30-40 nm. The cell was held by a micropipette mounted onto the scanner-piezo as shown in Häberle, W., J. K. H. Hörber, and G. Binnig. 1991. Force microscopy on living cells. J. Vac. Sci. Technol. B9:1210-0000. To initiate specific processes on the cell surface the cells had been infected with pox viruses as reported earlier and, most likely, the liberation of a progeny virion by the still-living cell was observed, hence confirming and supporting earlier results (Häberle, W., J. K. H. Hörber, F. Ohnesorge, D. P. E. Smith, and G. Binnig. 1992. In situ investigations of single living cells infected by viruses. Ultramicroscopy. 42-44:1161-0000; Hörber, J. K. H., W. Häberle, F. Ohnesorge, G. Binnig, H. G. Liebich, C. P. Czerny, H. Mahnel, and A. Mayr. 1992. Investigation of living cells in the nanometer regime with the atomic force microscope. Scanning Microscopy. 6:919-930). Furthermore, the pox viruses used were characterized separately by AFM in an aqueous environment down to the molecular level. Quasi-ordered structural details were resolved on a scale of a few nm where, however, image distortions and artifacts due to multiple tip effects are probably involved--just as in very high resolution (<15-20 nm) images on the cells. Although in a very preliminary manner, initial studies on the mechanical resonance properties of a single living (noninfected) cell, held by the micropipette, have been performed. In particular, frequency response spectra were recorded that indicate elastic properties and enough stiffness of these cells to make the demonstrated rapid scanning of the imaging tip plausible. Measurements of this kind, especially if they can be proven to be cell-type specific, may perhaps have a large

  11. A novel pathway for amyloids self-assembly in aggregates at nanomolar concentration mediated by the interaction with surfaces

    PubMed Central

    Banerjee, Siddhartha; Hashemi, Mohtadin; Lv, Zhengjian; Maity, Sibaprasad; Rochet, Jean-Christophe; Lyubchenko, Yuri L.

    2017-01-01

    A limitation of the amyloid hypothesis in explaining the development of neurodegenerative diseases is that the level of amyloidogenic polypeptide in vivo is below the critical concentration required to form the aggregates observed in post-mortem brains. We discovered a novel, on-surface aggregation pathway of amyloidogenic polypeptide that eliminates this long-standing controversy. We applied atomic force microscope (AFM) to demonstrate directly that on-surface aggregation takes place at a concentration at which no aggregation in solution is observed. The experiments were performed with the full-size Aβ protein (Aβ42), a decapeptide Aβ(14-23) and α-synuclein; all three systems demonstrate a dramatic preference of the on-surface aggregation pathway compared to the aggregation in the bulk solution. Time-lapse AFM imaging, in solution, show that over time, oligomers increase in size and number and release in solution, suggesting that assembled aggregates can serve as nuclei for aggregation in bulk solution. Computational modeling performed with the all-atom MD simulations for Aβ(14-23) peptide shows that surface interactions induce conformational transitions of the monomer, which facilitate interactions with another monomer that undergoes conformational changes stabilizing the dimer assembly. Our findings suggest that interactions of amyloidogenic polypeptides with cellular surfaces play a major role in determining disease onset. PMID:28358113

  12. Biomechanical properties of murine meniscus surface via AFM-based nanoindentation.

    PubMed

    Li, Qing; Doyran, Basak; Gamer, Laura W; Lu, X Lucas; Qin, Ling; Ortiz, Christine; Grodzinsky, Alan J; Rosen, Vicki; Han, Lin

    2015-06-01

    This study aimed to quantify the biomechanical properties of murine meniscus surface. Atomic force microscopy (AFM)-based nanoindentation was performed on the central region, proximal side of menisci from 6- to 24-week old male C57BL/6 mice using microspherical tips (Rtip≈5µm) in PBS. A unique, linear correlation between indentation depth, D, and response force, F, was found on menisci from all age groups. This non-Hertzian behavior is likely due to the dominance of tensile resistance by the collagen fibril bundles on meniscus surface that are mostly aligned along the circumferential direction. The indentation resistance was calculated as both the effective modulus, Eind, via the isotropic Hertz model, and the effective stiffness, Sind = dF/dD. Values of Sind and Eind were found to depend on indentation rate, suggesting the existence of poro-viscoelasticity. These values do not significantly vary with anatomical sites, lateral versus medial compartments, or mouse age. In addition, Eind of meniscus surface (e.g., 6.1±0.8MPa for 12 weeks of age, mean±SEM, n=13) was found to be significantly higher than those of meniscus surfaces in other species, and of murine articular cartilage surface (1.4±0.1MPa, n=6). In summary, these results provided the first direct mechanical knowledge of murine knee meniscus tissues. We expect this understanding to serve as a mechanics-based benchmark for further probing the developmental biology and osteoarthritis symptoms of meniscus in various murine models.

  13. Quality Child Care Supports the Achievement of Low-Income Children: Direct and Indirect Pathways through Caregiving and the Home Environment

    ERIC Educational Resources Information Center

    McCartney, Kathleen; Dearing, Eric; Taylor, Beck A.; Bub, Kristen L.

    2007-01-01

    Existing studies of child care have not been able to determine whether higher quality child care protects children from the effects of poverty, whether poverty and lower quality child care operate as dual risk factors, or whether both are true. The objective of the current study was to test two pathways through which child care may serve as a…

  14. A rapid and automated relocation method of an AFM probe for high-resolution imaging.

    PubMed

    Zhou, Peilin; Yu, Haibo; Shi, Jialin; Jiao, Niandong; Wang, Zhidong; Wang, Yuechao; Liu, Lianqing

    2016-09-30

    The atomic force microscope (AFM) is one of the most powerful tools for high-resolution imaging and high-precision positioning for nanomanipulation. The selection of the scanning area of the AFM depends on the use of the optical microscope. However, the resolution of an optical microscope is generally no larger than 200 nm owing to wavelength limitations of visible light. Taking into consideration the two determinants of relocation-relative angular rotation and positional offset between the AFM probe and nano target-it is therefore extremely challenging to precisely relocate the AFM probe to the initial scan/manipulation area for the same nano target after the AFM probe has been replaced, or after the sample has been moved. In this paper, we investigate a rapid automated relocation method for the nano target of an AFM using a coordinate transformation. The relocation process is both simple and rapid; moreover, multiple nano targets can be relocated by only identifying a pair of reference points. It possesses a centimeter-scale location range and nano-scale precision. The main advantages of this method are that it overcomes the limitations associated with the resolution of optical microscopes, and that it is label-free on the target areas, which means that it does not require the use of special artificial markers on the target sample areas. Relocation experiments using nanospheres, DNA, SWCNTs, and nano patterns amply demonstrate the practicality and efficiency of the proposed method, which provides technical support for mass nanomanipulation and detection based on AFM for multiple nano targets that are widely distributed in a large area.

  15. Development of a 3D-AFM for true 3D measurements of nanostructures

    NASA Astrophysics Data System (ADS)

    Dai, Gaoliang; Häßler-Grohne, Wolfgang; Hüser, Dorothee; Wolff, Helmut; Danzebrink, Hans-Ulrich; Koenders, Ludger; Bosse, Harald

    2011-09-01

    The development of advanced lithography requires highly accurate 3D metrology methods for small line structures of both wafers and photomasks. Development of a new 3D atomic force microscopy (3D-AFM) with vertical and torsional oscillation modes is introduced in this paper. In its configuration, the AFM probe is oscillated using two piezo actuators driven at vertical and torsional resonance frequencies of the cantilever. In such a way, the AFM tip can probe the surface with a vertical and a lateral oscillation, offering high 3D probing sensitivity. In addition, a so-called vector approach probing (VAP) method has been applied. The sample is measured point-by-point using this method. At each probing point, the tip is approached towards the surface until the desired tip-sample interaction is detected and then immediately withdrawn from the surface. Compared to conventional AFMs, where the tip is kept continuously in interaction with the surface, the tip-sample interaction time using the VAP method is greatly reduced and consequently the tip wear is reduced. Preliminary experimental results show promising performance of the developed system. A measurement of a line structure of 800 nm height employing a super sharp AFM tip could be performed with a repeatability of its 3D profiles of better than 1 nm (p-v). A line structure of a Physikalisch-Technische Bundesanstalt photomask with a nominal width of 300 nm has been measured using a flared tip AFM probe. The repeatability of the middle CD values reaches 0.28 nm (1σ). A long-term stability investigation shows that the 3D-AFM has a high stability of better than 1 nm within 197 measurements taken over 30 h, which also confirms the very low tip wear.

  16. Wnt3a stimulates Mepe, matrix extracellular phosphoglycoprotein, expression directly by the activation of the canonical Wnt signaling pathway and indirectly through the stimulation of autocrine Bmp-2 expression.

    PubMed

    Cho, Young-Dan; Kim, Woo-Jin; Yoon, Won-Joon; Woo, Kyung-Mi; Baek, Jeong-Hwa; Lee, Gene; Kim, Gwan-Shik; Ryoo, Hyun-Mo

    2012-06-01

    Matrix extracellular phosphoglycoprotein (MEPE) is a specific marker of mineralizing osteoblasts and osteocytes. Canonical BMP and Wnt signaling pathways are two of the strongest paracrine signals stimulating osteogenesis. Our previous results indicated that Mepe expression is stimulated by the BMP-2-signaling pathway. The specific aim of this study addressed whether Mepe expression is also controlled by Wnt signaling, and whether there is a cross-regulation between two major osteogenic signaling pathways. Treatment with Wnt3a, a canonical Wnt signaling stimulator, strongly enhanced Mepe mRNA expression. Knock-down of β-catenin with siRNA completely reversed Wnt3a-stimulated Mepe expression. The Mepe mRNA expression level was increased by overexpression of β-catenin and Lef-1, even in the absence of Wnt3a. Highly conserved Lef-1 response elements were identified in the mouse Mepe promoter. The direct binding of Lef-1 to these elements is critical for Mepe expression, indicating that Mepe is a direct target of canonical Wnt signaling. Meanwhile, we also found that Wnt3a treatment strongly stimulated Bmp-2 expression, and that the subsequent increase in Bmp-2 protein was determined in Wnt3a-treated conditioned medium (CM). Treatment of MC3T3-E1 cells with CM stimulated phosphorylation of the Smad1/5 proteins and their downstream Dlx5 mRNA expression. The CM-mediated increases of phospho-Smad and Dlx5 expression were not blocked completely by a Wnt3a antagonist, Dkk-1, but were almost completely suppressed by the addition of a Bmp-2 antagonist, Noggin. Collectively, Wnt3a stimulates Mepe transcription directly by a canonical Wnt signaling pathway through β-catenin and Lef-1 and indirectly through the activation of a Bmp-2 autocrine loop.

  17. AFM as an analysis tool for high-capacity sulfur cathodes for Li–S batteries

    PubMed Central

    Sörgel, Seniz; Costa, Rémi; Carlé, Linus; Galm, Ines; Cañas, Natalia; Pascucci, Brigitta; Friedrich, K Andreas

    2013-01-01

    Summary In this work, material-sensitive atomic force microscopy (AFM) techniques were used to analyse the cathodes of lithium–sulfur batteries. A comparison of their nanoscale electrical, electrochemical, and morphological properties was performed with samples prepared by either suspension-spraying or doctor-blade coating with different binders. Morphological studies of the cathodes before and after the electrochemical tests were performed by using AFM and scanning electron microscopy (SEM). The cathodes that contained polyvinylidene fluoride (PVDF) and were prepared by spray-coating exhibited a superior stability of the morphology and the electric network associated with the capacity and cycling stability of these batteries. A reduction of the conductive area determined by conductive AFM was found to correlate to the battery capacity loss for all cathodes. X-ray diffraction (XRD) measurements of Li2S exposed to ambient air showed that insulating Li2S hydrolyses to insulating LiOH. This validates the significance of electrical ex-situ AFM analysis after cycling. Conductive tapping mode AFM indicated the existence of large carbon-coated sulfur particles. Based on the analytical findings, the first results of an optimized cathode showed a much improved discharge capacity of 800 mA·g(sulfur)−1 after 43 cycles. PMID:24205455

  18. Mutant AFM 2 of Alcaligenes faecalis for phenol biodegradation using He-Ne laser irradiation.

    PubMed

    Jiang, Yan; Wen, Jianping; Caiyin, Qinggele; Lin, Liangcai; Hu, Zongding

    2006-11-01

    He-Ne laser technology was utilized in this study to investigate the response of Alcaligenes faecalis to laser stimulation. The irradiation experiments were conducted by the adjustment of the output power from 5 to 25 mW and the exposure time from 5 to 25 min. The results showed that the survival rate changed regularly with the variety of irradiation dose, and high positive mutation frequency was determined by both the energy density and the output power. The mutant strain AFM 2 was obtained. Phenol biodegradation assay demonstrated that AFM 2 possessed a more prominent phenol-degrading potential than its parent strain, which presumably attributed to the improvements of phenol hydroxylase and catechol 1,2-dioxygenase activities. The phenol of 2000 mgl(-1) was completely degraded by AFM 2 within 85.5h at 30 degrees C. In addition, the cell growth and phenol degradation kinetics of the mutant strain AFM 2 and its parent strain in batch cultures were also investigated at the wide initial phenol concentration ranging from 0 to 2000 mgl(-1) by Haldane model. The results of these experiments further demonstrated that the mutant strain AFM 2 possessed a higher capacity to resist phenol.

  19. Accelerated design and quality control of impact modifiers for plastics through atomic force microscopy (AFM) analysis

    NASA Astrophysics Data System (ADS)

    Moeller, Gunter

    2011-03-01

    Standard polymer resins are often too brittle or do not meet other mechanical property requirements for typical polymer applications. To achieve desired properties it is common to disperse so called ``impact modifiers'', which are spherical latex particles with diameters of much less than one micrometer, into the pure resin. Understanding and control of the entire process from latex particle formation to subsequent dispersion into polymer resins are necessary to accelerate the development of new materials that meet specific application requirements. In this work AFM imaging and nanoindentation techniques in combination with AFM-based spectroscopic techniques were applied to assess latex formation and dispersion. The size and size distribution of the latex particles can be measured based on AFM amplitude modulation images. AFM phase images provide information about the chemical homogeneity of individual particles. Nanoindentation may be used to estimate their elastic and viscoelastic properties. Proprietary creep and nanoscale Dynamic Mechanical Analysis (DMA) tests that we have developed were used to measure these mechanical properties. The small size of dispersed latex inclusions requires local mechanical and spectroscopic analysis techniques with high lateral and spatial resolution. We applied the CRAVE AFM method, developed at NIST, to perform mechanical analysis of individual latex inclusions and compared results with those obtained using nanoscale DMA. NanoIR, developed by Anasys Inc., and principal component confocal Raman were used for spectroscopic analysis and results from both techniques compared.

  20. Enabling accurate gate profile control with inline 3D-AFM

    NASA Astrophysics Data System (ADS)

    Bao, Tianming; Lopez, Andrew; Dawson, Dean

    2009-05-01

    The logic and memory semiconductor device technology strives to follow the aggressive ITRS roadmap. The ITRS calls for increased 3D metrology to meet the demand for tighter process control at 45nm and 32nm nodes. In particular, gate engineering has advanced to a level where conventional metrology by CD-SEM and optical scatterometry (OCD) faces fundamental limitations without involvement of 3D atomic force microscope (3D-AFM or CD-AFM). This paper reports recent progress in 3D-AFM to address the metrology need to control gate dimension in MOSFET transistor formation. 3D-AFM metrology measures the gate electrode at post-etch with the lowest measurement uncertainty for critical gate geometry, including linewidth, sidewall profile, sidewall angle (SWA), line width roughness (LWR), and line edge roughness (LER). 3D-AFM enables accurate gate profile control in three types of metrology applications: reference metrology to validate CD-SEM and OCD, inline depth or 3D monitoring, or replacing TEM for 3D characterization for engineering analysis.

  1. Quantitating membrane bleb stiffness using AFM force spectroscopy and an optical sideview setup.

    PubMed

    Gonnermann, Carina; Huang, Chaolie; Becker, Sarah F; Stamov, Dimitar R; Wedlich, Doris; Kashef, Jubin; Franz, Clemens M

    2015-03-01

    AFM-based force spectroscopy in combination with optical microscopy is a powerful tool for investigating cell mechanics and adhesion on the single cell level. However, standard setups featuring an AFM mounted on an inverted light microscope only provide a bottom view of cell and AFM cantilever but cannot visualize vertical cell shape changes, for instance occurring during motile membrane blebbing. Here, we have integrated a mirror-based sideview system to monitor cell shape changes resulting from motile bleb behavior of Xenopus cranial neural crest (CNC) cells during AFM elasticity and adhesion measurements. Using the sideview setup, we quantitatively investigate mechanical changes associated with bleb formation and compared cell elasticity values recorded during membrane bleb and non-bleb events. Bleb protrusions displayed significantly lower stiffness compared to the non-blebbing membrane in the same cell. Bleb stiffness values were comparable to values obtained from blebbistatin-treated cells, consistent with the absence of a functional actomyosin network in bleb protrusions. Furthermore, we show that membrane blebs forming within the cell-cell contact zone have a detrimental effect on cell-cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell-cell detachment or prevent adhesion reinforcement. Incorporating a sideview setup into an AFM platform therefore provides a new tool to correlate changes in cell morphology with results from force spectroscopy experiments.

  2. Combined force spectroscopy, AFM and calorimetric studies to reveal the nanostructural organization of biomimetic membranes.

    PubMed

    Suárez-Germà, C; Morros, A; Montero, M T; Hernández-Borrell, J; Domènech, Ò

    2014-10-01

    In this work we studied a binary lipid matrix of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), a composition that mimics the inner membrane of Escherichia coli. More specifically, liposomes with varying fractions of POPG were analysed by differential scanning calorimetry (DSC) and a binary phase diagram of the system was created. Additionally, we performed atomic force microscopy (AFM) imaging of supported lipid bilayers (SLBs) of similar compositions at different temperatures, in order to create a pseudo-binary phase diagram specific to this membrane model. AFM study of SLBs is of particular interest, as it is conceived as the most adequate technique not only for studying lipid bilayer systems but also for imaging and even nanomanipulating inserted membrane proteins. The construction of the above-mentioned phase diagram enabled us to grasp better the thermodynamics of the thermal lipid transition from a gel-like POPE:POPG phase system to a more fluid phase system. Finally, AFM force spectroscopy (FS) was used to determine the nanomechanics of these two lipid phases at 27°C and at different POPG fractions. The resulting data correlated with the specific composition of each phase was calculated from the AFM phase diagram obtained. All the experiments were done in the presence of 10 mM of Ca(2+), as this ion is commonly used when performing AFM with negatively charged phospholipids.

  3. Noise in NC-AFM measurements with significant tip–sample interaction

    PubMed Central

    Lübbe, Jannis; Temmen, Matthias

    2016-01-01

    The frequency shift noise in non-contact atomic force microscopy (NC-AFM) imaging and spectroscopy consists of thermal noise and detection system noise with an additional contribution from amplitude noise if there are significant tip–sample interactions. The total noise power spectral density D Δ f(f m) is, however, not just the sum of these noise contributions. Instead its magnitude and spectral characteristics are determined by the strongly non-linear tip–sample interaction, by the coupling between the amplitude and tip–sample distance control loops of the NC-AFM system as well as by the characteristics of the phase locked loop (PLL) detector used for frequency demodulation. Here, we measure D Δ f(f m) for various NC-AFM parameter settings representing realistic measurement conditions and compare experimental data to simulations based on a model of the NC-AFM system that includes the tip–sample interaction. The good agreement between predicted and measured noise spectra confirms that the model covers the relevant noise contributions and interactions. Results yield a general understanding of noise generation and propagation in the NC-AFM and provide a quantitative prediction of noise for given experimental parameters. We derive strategies for noise-optimised imaging and spectroscopy and outline a full optimisation procedure for the instrumentation and control loops. PMID:28144538

  4. Morphology of Vapor-Deposited Ice at Low Temperatures by Atomic Force Microscopy (AFM)

    NASA Astrophysics Data System (ADS)

    Fain, , Jr.; Donev, J. M. K.; Tait, B. R. Long, Jr.; Yu, Q.

    2002-03-01

    The morphology of multilayer films of ice on various substrates is measured by AFM as a function of vapor-deposition and annealing temperatures below 150K. The films are deposited in-situ in UHV from an effusive doser at 67 degrees from the surface normal. For depositions near 100K on clean Au(111), previous measurements by Donev et al. using needle-sensor AFM indicate that 3-D clustering starts near 120K for initially flat thin films of amorphous solid water (ASW). For depositions below 85K on clean Au(111), preliminary measurements using non-contact AFM (nc-AFM) indicate that clustering does not occur during annealing until bulk diffusion becomes operative at T>140K. Deposition at glancing angle at the lower temperatures is known to increase porosity and is also expected to decrease the number of crystalline nuclei in the ASW. For depositions near 100K on mica that had been annealed in UHV, preliminary measurements using ncAFM show clustering near 120K. Supported by U. W. Nanotechnology Fellowship (J.M.K.D.), Mary Gates Fellowship (B. R. L.), and M. J. Murdock Charitable Trust.

  5. Effect of AFM probe geometry on visco-hyperelastic characterization of soft materials

    NASA Astrophysics Data System (ADS)

    Boccaccio, Antonio; Lamberti, Luciano; Papi, Massimiliano; De Spirito, Marco; Pappalettere, Carmine

    2015-08-01

    Atomic force microscopy (AFM) nanoindentation is very suited for nano- and microscale mechanical characterization of soft materials. Although the structural response of polymeric networks that form soft matter depends on viscous effects caused by the relative slippage of polymeric chains, the usual assumption made in the AFM-based characterization is that the specimen behaves as a purely elastic material and viscous forces are negligible. However, for each geometric configuration of the AFM tip, there will be a limit indentation rate above which viscous effects must be taken into account to correctly determine mechanical properties. A parametric finite element study conducted on 12 geometric configurations of a blunt cone AFM tip (overall, the study included about 200 finite element analyses) allowed us to determine the limit indentation rate for each configuration. The selected tip dimensions cover commercially available products and account for changes in tip geometry caused by serial measurements. Nanoindentation rates cover typical experimental conditions set in AFM bio-measurements on soft matter. Viscous effects appear to be more significant in the case of sharper tips. This implies that, if quantitative data on sample viscosity are not available, using a rounded indenter and carrying out experiments below the limit indentation rate will allow errors in the determination of mechanical properties to be minimized.

  6. Fractal analysis of AFM images of the surface of Bowman's membrane of the human cornea.

    PubMed

    Ţălu, Ştefan; Stach, Sebastian; Sueiras, Vivian; Ziebarth, Noël Marysa

    2015-04-01

    The objective of this study is to further investigate the ultrastructural details of the surface of Bowman's membrane of the human cornea, using atomic force microscopy (AFM) images. One representative image acquired of Bowman's membrane of a human cornea was investigated. The three-dimensional (3-D) surface of the sample was imaged using AFM in contact mode, while the sample was completely submerged in optisol solution. Height and deflection images were acquired at multiple scan lengths using the MFP-3D AFM system software (Asylum Research, Santa Barbara, CA), based in IGOR Pro (WaveMetrics, Lake Oswego, OR). A novel approach, based on computational algorithms for fractal analysis of surfaces applied for AFM data, was utilized to analyze the surface structure. The surfaces revealed a fractal structure at the nanometer scale. The fractal dimension, D, provided quantitative values that characterize the scale properties of surface geometry. Detailed characterization of the surface topography was obtained using statistical parameters, in accordance with ISO 25178-2: 2012. Results obtained by fractal analysis confirm the relationship between the value of the fractal dimension and the statistical surface roughness parameters. The surface structure of Bowman's membrane of the human cornea is complex. The analyzed AFM images confirm a fractal nature of the surface, which is not taken into account by classical surface statistical parameters. Surface fractal dimension could be useful in ophthalmology to quantify corneal architectural changes associated with different disease states to further our understanding of disease evolution.

  7. In Drosophila melanogaster the COM Locus Directs the Somatic Silencing of Two Retrotransposons through both Piwi-Dependent and -Independent Pathways

    PubMed Central

    Meignin, Carine; Coiffet, Michael; Vaury, Chantal

    2008-01-01

    Background In the Drosophila germ line, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous transposable elements. This RNA silencing involves small RNAs of 26-30 nucleotides that are mainly produced from the antisense strand and function through the Piwi protein. Piwi belongs to the subclass of the Argonaute family of RNA interference effector proteins, which are expressed in the germline and in surrounding somatic tissues of the reproductive apparatus. In addition to this germ-line expression, Piwi has also been implicated in diverse functions in somatic cells. Principal Findings Here, we show that two LTR retrotransposons from Drosophila melanogaster, ZAM and Idefix, are silenced by an RNA silencing pathway that has characteristics of the rasiRNA pathway and that specifically recognizes and destroys the sense-strand RNAs of the retrotransposons. This silencing depends on Piwi in the follicle cells surrounding the oocyte. Interestingly, this silencing is active in all the somatic tissues examined from embryos to adult flies. In these somatic cells, while the silencing still involves the strict recognition of sense-strand transcripts, it displays the marked difference of being independent of the Piwi protein. Finally, we present evidence that in all the tissues examined, the repression is controlled by the heterochromatic COM locus. Conclusion Our data shed further light on the silencing mechanism that acts to target Drosophila LTR retrotransposons in somatic cells throughout fly development. They demonstrate that different RNA silencing pathways are involved in ovarian versus other somatic tissues, since Piwi is necessary for silencing in the former tissues but is dispensable in the latter. They further demonstrate that these pathways are controlled by the heterochromatic COM locus which ensures the overall protection of Drosophila against the detrimental effects of random retrotransposon mobilization. PMID

  8. The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding

    NASA Astrophysics Data System (ADS)

    Hughes, Megan L.; Dougan, Lorna

    2016-07-01

    One of the most exciting developments in the field of biological physics in recent years is the ability to manipulate single molecules and probe their properties and function. Since its emergence over two decades ago, single molecule force spectroscopy has become a powerful tool to explore the response of biological molecules, including proteins, DNA, RNA and their complexes, to the application of an applied force. The force versus extension response of molecules can provide valuable insight into its mechanical stability, as well as details of the underlying energy landscape. In this review we will introduce the technique of single molecule force spectroscopy using the atomic force microscope (AFM), with particular focus on its application to study proteins. We will review the models which have been developed and employed to extract information from single molecule force spectroscopy experiments. Finally, we will end with a discussion of future directions in this field.

  9. Oyster viperin retains direct antiviral activity and its transcription occurs via a signalling pathway involving a heat-stable haemolymph protein.

    PubMed

    Green, Timothy J; Speck, Peter; Geng, Lu; Raftos, David; Beard, Michael R; Helbig, Karla J

    2015-12-01

    Little is known about the response of non-model invertebrates, such as oysters, to virus infection. The vertebrate innate immune system detects virus-derived nucleic acids to trigger the type I IFN pathway, leading to the transcription of hundreds of IFN-stimulated genes (ISGs) that exert antiviral functions. Invertebrates were thought to lack the IFN pathway based on the absence of IFN or ISGs encoded in model invertebrate genomes. However, the oyster genome encodes many ISGs, including the well-described antiviral protein viperin. In this study, we characterized oyster viperin and showed that it localizes to caveolin-1 and inhibits dengue virus replication in a heterologous model. In a second set of experiments, we have provided evidence that the haemolymph from poly(I : C)-injected oysters contains a heat-stable, protease-susceptible factor that induces haemocyte transcription of viperin mRNA in conjunction with upregulation of IFN regulatory factor. Collectively, these results support the concept that oysters have antiviral systems that are homologous to the vertebrate IFN pathway.

  10. AFM study of the thermotropic behaviour of supported DPPC bilayers with and without the model peptide WALP23.

    PubMed

    Yarrow, F; Kuipers, B W M

    2011-01-01

    Temperature-controlled Atomic Force Microscopy (TC-AFM) in Contact Mode is used here to directly image the mechanisms by which melting and crystallization of supported, hydrated DPPC bilayers proceed in the presence and absence of the model peptide WALP23. Melting from the gel L(β)' to the liquid-crystalline L(α) phase starts at pre-existing line-type packing defects (grain boundaries) in absence of the peptide. The exact transition temperature is shown to be influenced by the magnitude of the force exerted by the AFM probe on the bilayer, but is higher than the main transition temperature of non-supported DPPC vesicles in all cases due to bilayer-substrate interactions. Cooling of the fluid L(α) bilayer shows the formation of the line-type defects at the borders between different gel-phase regions that originate from different nuclei. The number of these defects depends directly on the rate of cooling through the transition, as predicted by classical nucleation theory. The presence of the transmembrane, synthetic model peptide WALP23 is known to give rise to heterogeneity in the bilayer as microdomains with a striped appearance are formed in the DPPC bilayer. This striated phase consists of alternating lines of lipids and peptide. It is shown here that melting starts with the peptide-associated lipids in the domains, whose melting temperature is lowered by 0.8-2.0°C compared to the remaining, peptide-free parts of the bilayer. The stabilization of the fluid phase is ascribed to adaptations of the lipids to the shorter peptide. The lipids not associated with the peptide melt at the same temperature as those in the pure DPPC supported bilayer.

  11. Effect of enamel morphology on nanoscale adhesion forces of streptococcal bacteria : An AFM study.

    PubMed

    Wang, Chuanyong; Zhao, Yongqi; Zheng, Sainan; Xue, Jing; Zhou, Jinglin; Tang, Yi; Jiang, Li; Li, Wei

    2015-01-01

    We explore the influence of enamel surface morphology on nanoscale bacterial adhesion forces. Three dimensional morphology characteristics of enamel slices, which were treated with phosphoric acid (for 0 s, 5 s, 10 s, 20 s, and 30 s), were acquired. Adhesion forces of three initial colonizers (Streptococcus oralis, Streptococcus sanguinis, and Streptococcus mitis) and two cariogenic bacterial strains (Streptococcus mutans and Streptococcus sobrinus) with etched enamel surfaces were determined. Comparison of the forces was made by using bacterial probe method under atomic force microscope (AFM) in adhesion buffer. The results showed that enamel morphology was significantly altered by etching treatment. The roughness, peak-to-valley height, and valley-to-valley width of the depth profile, surface area, and volume increased linearly with acid exposure time, and reached the maximum at 30s, respectively. The adhesion forces of different strains increased accordingly with etching time. Adhesion forces of S. oralis, S. mitis, S. mutans, and S. sobrinus reached the maximum values of 0.81 nN, 0.84 nN, 0.73 nN, and 0.64 nN with enamel treated for 20s, respectively, whereas that of S. sanguinis at 10s (1.28nN), and dropped on coarser enamel surfaces. In conclusion, enamel micro-scale morphology may significantly alter the direct adhesion forces of bacteria. And there may be a threshold roughness for bacterial adhesion on enamel surface.

  12. Recombinant albumin adsorption on mica studied by AFM and streaming potential measurements.

    PubMed

    Kujda, Marta; Adamczyk, Zbigniew; Morga, Maria; Sofińska, Kamila

    2015-03-01

    Recombinant human serum albumin (rHSA) in monomeric state is widely used in pharmaceutical industry as a drug excipient and for preparing coatings for medical devices. In this work the adsorption process of rHSA on model mica surface at pH 3.5 was studied using the atomic force microscopy (AFM) and in situ streaming potential measurements. The kinetics of albumin adsorption was determined by a direct enumeration of single molecules over various substrate areas. These results were consistent with streaming potential measurements carried out for the parallel-plate channel flow and with theoretical predictions derived from the random sequential adsorption (RSA) model. Desorption kinetics of albumin under flow conditions was also evaluated via the streaming potential measurements. In this way, the amount of irreversibly bound albumin was quantitatively evaluated to be 0.64 and 1.2 mg m(-2) for ionic strength of 0.01 and 0.15 M, respectively. This agrees with previous results obtained for HSA and theoretical calculations derived from the RSA model. Additionally, it was demonstrated that there existed a fraction of reversibly bound albumin that can be fully eluted within a few hours. The binding energy of these fraction of molecules was -18 kT that is consistent with the electrostatic controlled adsorption mechanism of albumin at this pH. It was concluded that the rHSA monolayers of well-defined coverage can find applications for quantitatively analyzing ligand binding and for performing efficient biomaterials and immunological tests.

  13. Lipid asymmetry in DLPC/DSPC supported lipid bilayers, a combined AFM and fluorescence microscopy study

    SciTech Connect

    Lin, W; Blanchette, C D; Ratto, T V; Longo, M L

    2005-06-20

    A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy (AFM) and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald Ripening, the smaller domains in one leaflet erode away while the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have nearly identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have direct biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.

  14. DNA-duplex linker for AFM-SELEX of DNA aptamer against human serum albumin.

    PubMed

    Takenaka, Musashi; Okumura, Yuzo; Amino, Tomokazu; Miyachi, Yusuke; Ogino, Chiaki; Kondo, Akihiko

    2017-02-15

    DNA-duplex interactions in thymines and adenins are used as a linker for the novel methodology of Atomic Force Microscope-Systematic Evolution of Ligands by EXpotential enrichment (AFM-SELEX). This study used the hydrogen bonds in 10 mer of both thymines (T10) and adenines (A10). Initially, the interactive force in T10-A10 was measured by AFM, which returned an average interactive force of approximately 350pN. Based on this result, DNA aptamers against human serum albumin could be selected in the 4th round, and 15 different clones could be sequenced. The lowest dissociation constant of the selected aptamer was identified via surface plasmon resonance, and it proved to be identical to that of the commercial aptamer. Therefore, specific hydrogen bonds in DNA can be useful linkers for AFM-SELEX.

  15. Imaging and force measurement of LDL and HDL by AFM in air and liquid

    PubMed Central

    Gan, Chaoye; Ao, Meiying; Liu, Zhanghua; Chen, Yong

    2015-01-01

    The size and biomechanical properties of lipoproteins are tightly correlated with their structures/functions. While atomic force microscopy (AFM) has been used to image lipoproteins the force measurement of these nano-sized particles is missing. We detected that the sizes of LDL and HDL in liquid are close to the commonly known values. The Young’s modulus of LDL or HDL is ∼0.4 GPa which is similar to that of some viral capsids or nanovesicles but greatly larger than that of various liposomes. The adhesive force of LDL or HDL is small (∼200 pN). The comparison of AFM detection in air and liquid was also performed which is currently lacking. Our data may provide useful information for better understanding and AFM detection of lipoproteins. PMID:25893163

  16. AFM characterization of nonwoven material functionalized by ZnO sputter coating

    SciTech Connect

    Deng Bingyao; Yan Xiong; Wei Qufu Gao Weidong

    2007-10-15

    Sputter coatings provide new approaches to the surface functionalization of textile materials. In this study, polyethylene terephthalate (PET) nonwoven material was used as a substrate for creating functional nanostructures on the fiber surfaces. A magnetron sputter coating was used to deposit functional zinc oxide (ZnO) nanostructures onto the nonwoven substrate. The evolution of the surface morphology of the fibers in the nonwoven web was examined using atomic force microscopy (AFM). The AFM observations revealed a significant difference in the morphology of the fibers before and after the sputter coating. The AFM images also indicated the effect of the sputtering conditions on the surface morphology of the fibers. The increase in the sputtering time led to the growth of the ZnO grains on the fiber surfaces. The higher pressure in the sputtering chamber could cause the formation of larger grains on the fiber surfaces. The higher power used also generated larger grains on the fiber surfaces.

  17. AFM study of forces between silica, silicon nitride and polyurethane pads.

    PubMed

    Sokolov, Igor; Ong, Quy K; Shodiev, Hasan; Chechik, Nina; James, David; Oliver, Mike

    2006-08-15

    Interaction of silica and silicon nitride with polyurethane surfaces is rather poorly studied despite being of great interest for modern semiconductor industry, e.g., for chemical-mechanical planarization (CMP) processes. Here we show the results from the application of the atomic force microscopy (AFM) technique to study the forces between silica or silicon nitride (AFM tips) and polyurethane surfaces in aqueous solutions of different acidity. The polyurethane surface potentials are derived from the measured AFM data. The obtained potentials are in rather good agreement with measurements of zeta-potentials using the streaming-potentials method. Another important parameter, adhesion, is also measured. While the surface potentials of silica are well known, there are ambiguous results on the potentials of silicon nitride that is naturally oxidized. Deriving the surface potential of the naturally oxidized silicon nitride from our measurements, we show that it is not oxidized to silica despite some earlier published expectations.

  18. Imaging and force measurement of LDL and HDL by AFM in air and liquid.

    PubMed

    Gan, Chaoye; Ao, Meiying; Liu, Zhanghua; Chen, Yong

    2015-01-01

    The size and biomechanical properties of lipoproteins are tightly correlated with their structures/functions. While atomic force microscopy (AFM) has been used to image lipoproteins the force measurement of these nano-sized particles is missing. We detected that the sizes of LDL and HDL in liquid are close to the commonly known values. The Young's modulus of LDL or HDL is ∼0.4 GPa which is similar to that of some viral capsids or nanovesicles but greatly larger than that of various liposomes. The adhesive force of LDL or HDL is small (∼200 pN). The comparison of AFM detection in air and liquid was also performed which is currently lacking. Our data may provide useful information for better understanding and AFM detection of lipoproteins.

  19. The AFM Observation of Single Polyethylene Molecules in Coiled State on Mica

    NASA Astrophysics Data System (ADS)

    Prokhorov, V. V.; Yaminsky, I. V.

    2003-12-01

    Single polyethylene molecules and their small aggregates have been deposited on mica from diluted solutions at elevated temperatures and visualized by AFM in coiled and crystalline states. Coils have two-dimensional conformations with both highly tangled sites and locally extended segments with a length much exceeding the persistent length in a solution. The length measurements of coils reveal a wide distribution with the length of a maximum much smaller than the length of fully stretched molecules, moreover the long coils have been observed indicating the existence of linear multimolecular aggregates. Two models have been considered for the explanation of the observed deficit in the coils length, correspondingly the model implying the substantial smoothing of a winding chain trajectory due to the lack of the AFM resolution and the model of locally extended surface conformations with the long intramolecular folds. The roots of the apparent negative AFM height contrast of coils have been discussed.

  20. Custom AFM for X-ray beamlines: in situ biological investigations under physiological conditions

    PubMed Central

    Gumí-Audenis, B.; Carlà, F.; Vitorino, M. V.; Panzarella, A.; Porcar, L.; Boilot, M.; Guerber, S.; Bernard, P.; Rodrigues, M. S.; Sanz, F.; Giannotti, M. I.; Costa, L.

    2015-01-01

    A fast atomic force microscope (AFM) has been developed that can be installed as a sample holder for grazing-incidence X-ray experiments at solid/gas or solid/liquid interfaces. It allows a wide range of possible investigations, including soft and biological samples under physiological conditions (hydrated specimens). The structural information obtained using the X-rays is combined with the data gathered with the AFM (morphology and mechanical properties), providing a unique characterization of the specimen and its dynamics in situ during an experiment. In this work, lipid monolayers and bilayers in air or liquid environment have been investigated by means of AFM, both with imaging and force spectroscopy, and X-ray reflectivity. In addition, this combination allows the radiation damage induced by the beam on the sample to be studied, as has been observed on DOPC and DPPC supported lipid bilayers under physiological conditions. PMID:26524300

  1. Multiparametric high-resolution imaging of native proteins by force-distance curve-based AFM.

    PubMed

    Pfreundschuh, Moritz; Martinez-Martin, David; Mulvihill, Estefania; Wegmann, Susanne; Muller, Daniel J

    2014-05-01

    A current challenge in the life sciences is to understand how the properties of individual molecular machines adjust in order to meet the functional requirements of the cell. Recent developments in force-distance (FD) curve-based atomic force microscopy (FD-based AFM) enable researchers to combine sub-nanometer imaging with quantitative mapping of physical, chemical and biological properties. Here we present a protocol to apply FD-based AFM to the multiparametric imaging of native proteins under physiological conditions. We describe procedures for experimental FD-based AFM setup, high-resolution imaging of proteins in the native unperturbed state with simultaneous quantitative mapping of multiple parameters, and data interpretation and analysis. The protocol, which can be completed in 1-3 d, enables researchers to image proteins and protein complexes in the native unperturbed state and to simultaneously map their biophysical and biochemical properties at sub-nanometer resolution.

  2. Direct and indirect pharmacological modulation of CCL2/CCR2 pathway results in attenuation of neuropathic pain - In vivo and in vitro evidence.

    PubMed

    Piotrowska, Anna; Kwiatkowski, Klaudia; Rojewska, Ewelina; Slusarczyk, Joanna; Makuch, Wioletta; Basta-Kaim, Agnieszka; Przewlocka, Barbara; Mika, Joanna

    2016-08-15

    The repeated administration of microglial inhibitor (minocycline) and CCR2 antagonist (RS504393) attenuated the neuropathic pain symptoms in rats following chronic constriction injury of the sciatic nerve, which was associated with decreased spinal microglia activation and the protein level of CCL2 and CCR2. Furthermore, in microglia primary cell cultures minocycline downregulated both CCL2 and CCR2 protein levels after lipopolysaccharide-stimulation. Additionally, in astroglia primary cell cultures minocycline decreased the expression of CCL2, but not CCR2. Our results provide new evidence that modulation of CCL2/CCR2 pathway by microglial inhibitor as well as CCR2 antagonist is effective for neuropathic pain development in rats.

  3. An Evaluation of the Impacts of AF-M315E Propulsion Systems for Varied Mission Applications

    NASA Technical Reports Server (NTRS)

    Deans, Matthew C.; Oleson, Steven R.; Fittje, James; Colozza, Anthony; Packard, Tom; Gyekenyesi, John; McLean, Christopher H.; Spores, Ronald A.

    2015-01-01

    The purpose of the AF-M315E COMPASS study is to identify near-term (3-5 years) and long term (5 years +) opportunities for infusion, specifically the thruster and associated component technologies being developed as part of the GPIM project. Develop design reference missions which show the advantages of the AF-M315E green propulsion system. Utilize a combination of past COMPASS designs and selected new designs to demonstrate AF-M315E advantages. Use the COMPASS process to show the puts and takes of using AF-M315E at the integrated system level.

  4. Studying post-etching silicon crystal defects on 300mm wafer by automatic defect review AFM

    NASA Astrophysics Data System (ADS)

    Zandiatashbar, Ardavan; Taylor, Patrick A.; Kim, Byong; Yoo, Young-kook; Lee, Keibock; Jo, Ahjin; Lee, Ju Suk; Cho, Sang-Joon; Park, Sang-il

    2016-03-01

    Single crystal silicon wafers are the fundamental elements of semiconductor manufacturing industry. The wafers produced by Czochralski (CZ) process are very high quality single crystalline materials with known defects that are formed during the crystal growth or modified by further processing. While defects can be unfavorable for yield for some manufactured electrical devices, a group of defects like oxide precipitates can have both positive and negative impacts on the final device. The spatial distribution of these defects may be found by scattering techniques. However, due to limitations of scattering (i.e. light wavelength), many crystal defects are either poorly classified or not detected. Therefore a high throughput and accurate characterization of their shape and dimension is essential for reviewing the defects and proper classification. While scanning electron microscopy (SEM) can provide high resolution twodimensional images, atomic force microscopy (AFM) is essential for obtaining three-dimensional information of the defects of interest (DOI) as it is known to provide the highest vertical resolution among all techniques [1]. However AFM's low throughput, limited tip life, and laborious efforts for locating the DOI have been the limitations of this technique for defect review for 300 mm wafers. To address these limitations of AFM, automatic defect review AFM has been introduced recently [2], and is utilized in this work for studying DOI on 300 mm silicon wafer. In this work, we carefully etched a 300 mm silicon wafer with a gaseous acid in a reducing atmosphere at a temperature and for a sufficient duration to decorate and grow the crystal defects to a size capable of being detected as light scattering defects [3]. The etched defects form a shallow structure and their distribution and relative size are inspected by laser light scattering (LLS). However, several groups of defects couldn't be properly sized by the LLS due to the very shallow depth and low

  5. FRAME (Force Review Automation Environment): MATLAB-based AFM data processor.

    PubMed

    Partola, Kostyantyn R; Lykotrafitis, George

    2016-05-03

    Data processing of force-displacement curves generated by atomic force microscopes (AFMs) for elastic moduli and unbinding event measurements is very time consuming and susceptible to user error or bias. There is an evident need for consistent, dependable, and easy-to-use AFM data processing software. We have developed an open-source software application, the force review automation environment (or FRAME), that provides users with an intuitive graphical user interface, automating data processing, and tools for expediting manual processing. We did not observe a significant difference between manually processed and automatically processed results from the same data sets.

  6. Delivery of germinal granules and localized RNAs via the messenger transport organizer pathway to the vegetal cortex of Xenopus oocytes occurs through directional expansion of the mitochondrial cloud.

    PubMed

    Wilk, Katarzyna; Bilinski, Szczepan; Dougherty, Matthew T; Kloc, Malgorzata

    2005-01-01

    During Xenopus oogenesis, the message transport organizer (METRO) pathway delivers germinal granules and localized RNAs to the vegetal cortex of the oocyte via the mitochondrial cloud (Balbiani body). According to the traditional model, the mitochondrial cloud is thought to break up at the onset of vitellogenesis and the germinal granules and METRO-localized RNAs are transported within the mitochondrial cloud fragments to the vegetal cortex of the oocyte. We used light and electron microscopy in situ hybridization and three-dimensional reconstruction to show that germinal granules and METRO-localized RNAs are delivered to the oocyte cortex before the onset of mitochondrial cloud fragmentation and that the delivery involves accumulation of localized RNAs and aggregation of germinal granules at the vegetal tip of the mitochondrial cloud and subsequent internal expansion of the mitochondrial cloud between its animal (nuclear) and vegetal tips, which drives the germinal granules and METRO-localized RNAs toward the vegetal cortex. Thus the fragmentation of the cloud that occurs later in oogenesis is irrelevant to the movement of METRO-localized RNAs and germinal granules. On the basis of these findings, we propose here a revised model of germinal granule and localized RNAs delivery to the oocyte vegetal cortex via the METRO pathway.

  7. Absorption Spectroscopy and Imaging from the Visible through Mid-IR with 20 nm Resolution Using AFM probes

    NASA Astrophysics Data System (ADS)

    Centrone, Andrea

    2015-03-01

    Correlated nanoscale composition and optical property maps are important to engineer nanomaterials in applications ranging from photovoltaics to sensing and therapeutics. Wavelengths (λs) from the visible to near-IR probe electronic transitions in materials, providing information regarding band gap and defects while light in mid-IR probes vibrational transitions and provide chemical composition. However, light diffraction limits the lateral resolution of conventional micro-spectroscopic techniques to approximately λ/2, which is insufficient to image nanomaterials. Additionally, the λ-dependent resolution impedes direct comparison of spectral maps from different spectral ranges. Photo Thermal Induced Resonance (PTIR) is a novel technique that circumvents light diffraction by employing an AFM tip as a local detector for measuring light absorption with λ-independent nanoscale resolution. Our PTIR setup combines an AFM microscope with three lasers providing λ-tunability from 500 nm to 16000 nm continuously. The AFM tip transduces locally the sample thermal expansion induced by light absorption into large cantilever oscillations. Local absorption spectra (electronic or vibrational) and maps are obtained recording the amplitude of the tip deflection as a function of λ and position, respectively. The working principles of the PTIR technique will be described first, and nano-patterned polymer samples will be used to evaluate its lateral resolution, sensitivity and linearity. Results show that the PTIR signal intensity is proportional to the local absorbed energy suggesting applicability of this technique for quantitative chemical analysis at nanoscale, at least for thin (less than 1000 nm thick) samples. Additionally, a λ-independent resolution as high as 20 nm is demonstrated across the whole spectral range. In the second part of the talk, PTIR will be applied to image the dark plasmonic resonance of gold Asymmetric Split Ring Resonators (A-SRRs) in the mid

  8. Investigation of growth rate dispersion in lactose crystallisation by AFM

    NASA Astrophysics Data System (ADS)

    Dincer, T. D.; Ogden, M. I.; Parkinson, G. M.

    2014-09-01

    α-Lactose monohydrate crystals have been reported to exhibit growth rate dispersion (GRD). Variation in surface dislocations has been suggested as the cause of GRD, but this has not been further investigated to date. In this study, growth rate dispersion and the change in morphology were investigated in situ and via bottle roller experiments. The surfaces of the (0 1 0) faces of crystals were examined with Atomic Force Microscopy. Smaller, slow growing crystals tend to have smaller (0 1 0) faces with narrow bases and displayed a single double spiral in the centre of the crystal with 2 nm high steps. Additional double spirals in other crystals resulted in faster growth rates. Large, fast growing crystals were observed to have larger (0 1 0) faces with fast growth in both the a and b directions (giving a broader crystal base) with macro steps parallel to the (c direction). The number and location of spirals or existence of macro steps appears to influence the crystal morphology, growth rates and growth rate dispersion in lactose crystals.

  9. The HD-GYP domain of RpfG mediates a direct linkage between the Rpf quorum-sensing pathway and a subset of diguanylate cyclase proteins in the phytopathogen Xanthomonas axonopodis pv citri.

    PubMed

    Andrade, Maxuel O; Alegria, Marcos Castanheira; Guzzo, Cristiane R; Docena, Cassia; Rosa, Maria Claudia Pareda; Ramos, Carlos H I; Farah, Chuck S

    2006-10-01

    Bacteria use extracellular levels of small diffusible autoinducers to estimate local cell-density (quorum-sensing) and to regulate complex physiological processes. The quorum-sensing signal transduction pathway of Xanthomonas spp. phytopathogens has special features that distinguish it from that of other pathogens. This pathway consists of RpfF, necessary for the production of the unique autoinducer 'diffusible signalling factor' (DSF), and RpfC and RpfG, a two-component system necessary for the DSF-dependent production of extracellular pathogenicity factors and cellular dispersion. Yeast two-hybrid and direct in vitro assays were used to identify interactions involving the Rpf group of proteins. We show that RpfC, a protein consisting of N-terminal transmembrane, histidine kinase, response-regulator and C-terminal histidine phosphotransfer domains interacts with both RpfG, a protein consisting of an N-terminal response regulator domain and a C-terminal HD-GYP domain, and with RpfF. We also show that RpfC interacts with the only known homologue of 'conditioned medium factor', which is involved in quorum-sensing in Dictyostelium discoideum under conditions of nutritional stress. Furthermore, RpfCG is shown to interact with a second two-component system made up of NtrB and NtrC homologues. Finally we show that the recently characterized HD-GYP phosphodiesterase domain of RpfG interacts directly with diguanylate cyclase GGDEF domain-containing proteins coded by the Xanthomonas axonopodis pv. citri genome, which in other bacteria produce cyclic diGMP, an important second messenger involved in the regulation of complex bacterial processes including biofilm production, virulence and motility. These results demonstrate a direct physical linkage between quorum-sensing and cyclic diGMP signalling pathways in bacteria.

  10. MicroRNA-29c-5p suppresses gallbladder carcinoma progression by directly targeting CPEB4 and inhibiting the MAPK pathway

    PubMed Central

    Shu, Yi-Jun; Bao, Run-Fa; Jiang, Lin; Wang, Zheng; Wang, Xu-An; Zhang, Fei; Liang, Han-Bin; Li, Huai-Feng; Ye, Yuan-Yuan; Xiang, Shan-Shan; Weng, Hao; Wu, Xiang-Song; Li, Mao-Lan; Hu, Yun-Ping; Lu, Wei; Zhang, Yi-Jian; Zhu, Jian; Dong, Ping; Liu, Ying-Bin

    2017-01-01

    Gallbladder cancer (GBC) is a leading cause of cancer-related deaths worldwide, and its prognosis remains poor, with a 5-year survival rate of ~5%. Given the crucial role of microRNAs (miRNAs) in cancer metastasis, we aimed to analyze the expression and function of the metastasis-associated miRNA miR-29c-5p in GBC.We validated that expression of miR-29c-5p was significantly downregulated in GBC and was closely associated with lymph node metastasis, overall survival and disease-free survival in 40 GBC patients who were followed clinically. Ectopic overexpression of miR-29c-5p dramatically repressed proliferation, metastasis, and colony formation and induced apoptosis in vitro, and it suppressed tumorigenicity in vivo through the MAPK pathway. Cytoplasmic polyadenylation element binding protein 4 (CPEB4) was identified as a critical effector target of miR-29c-5p. Enforced expression of miR-29c-5p significantly inhibited the expression of CPEB4, and restoration of CPEB4 expression reversed the inhibitory effects of miR-29c-5p on GBC cell proliferation and metastasis. Transforming growth factor-β (TGF-β) upregulated CPEB4 by downregulating miR-29c-5p, leading to MAPK pathway activation. In conclusion, the TGF-β/miR-29c-5p/CPEB4 axis has a pivotal role in the pathogenesis and poor prognosis of GBC, suggesting that miR-29c-5p is a tumor-suppressive miRNA that may serve as potential prognostic biomarker or therapeutic target for GBC. PMID:28060377

  11. Herpes simplex virus 1 tegument protein US11 downmodulates the RLR signaling pathway via direct interaction with RIG-I and MDA-5.

    PubMed

    Xing, Junji; Wang, Shuai; Lin, Rongtuan; Mossman, Karen L; Zheng, Chunfu

    2012-04-01

    The interferon (IFN)-mediated antiviral response is a major defense of the host immune system. In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Herpes simplex virus 1 (HSV-1) is a large DNA virus containing more than 80 genes, many of which encode proteins that are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrate that the US11 protein, an RNA binding tegument protein of HSV-1, is a novel antagonist of the beta IFN (IFN-β) pathway. US11 significantly inhibited Sendai virus (SeV)-induced IFN-β production, and its double-stranded RNA (dsRNA) binding domain was indispensable for this inhibition activity. Additionally, wild-type HSV-1 coinfection showed stronger inhibition than US11 mutant HSV-1 in SeV-induced IFN-β production. Coimmunoprecipitation analysis demonstrated that the US11 protein in HSV-1-infected cells interacts with endogenous RIG-I and MDA-5 through its C-terminal RNA-binding domain, which was RNA independent. Expression of US11 in both transfected and HSV-1-infected cells interferes with the interaction between MAVS and RIG-I or MDA-5. Finally, US11 dampens SeV-mediated IRF3 activation. Taken together, the combined data indicate that HSV-1 US11 binds to RIG-I and MDA-5 and inhibits their downstream signaling pathway, preventing the production of IFN-β, which may contribute to the pathogenesis of HSV-1 infection.

  12. Investigation of Molecular Interactions between AFM-Tip and Thiol Films

    NASA Astrophysics Data System (ADS)

    Touhami, Ahmed; Moore, Justin; Lee, T. Randall

    Among various self-assembly processes, the formation of a self-assembled monolayer (SAM) is one of the most elegant ways for making an organic film with specific surface properties. Recently, much effort has been devoted in using AFM-based single-molecule force spectroscopy (SMFS) to understanding the formation of alkanethiol SAMs on gold surfaces. Investigating the factors that affect the AFM tip-SAMs interactions is necessary to clarify the controversial results of these studies. Here, we investigated the interactions between bare AFM-tips and several SAMs thiols-gold surfaces under controlled humidity conditions. Our results demonstrate that the Tip-SAM interactions can be used to precisely determine the length of the thiol chains, the adhesion force between thiols head groups and the AFM tip, and the strength of the thiol-gold contact. Our findings on the dynamics and the structure of the SAMs of alkanethiols on gold are useful for detail understanding of the thermodynamics, kinetics and mechanisms of SAM technology assembly. NSF.

  13. AFM tip characterization by using FFT filtered images of step structures.

    PubMed

    Yan, Yongda; Xue, Bo; Hu, Zhenjiang; Zhao, Xuesen

    2016-01-01

    The measurement resolution of an atomic force microscope (AFM) is largely dependent on the radius of the tip. Meanwhile, when using AFM to study nanoscale surface properties, the value of the tip radius is needed in calculations. As such, estimation of the tip radius is important for analyzing results taken using an AFM. In this study, a geometrical model created by scanning a step structure with an AFM tip was developed. The tip was assumed to have a hemispherical cone shape. Profiles simulated by tips with different scanning radii were calculated by fast Fourier transform (FFT). By analyzing the influence of tip radius variation on the spectra of simulated profiles, it was found that low-frequency harmonics were more susceptible, and that the relationship between the tip radius and the low-frequency harmonic amplitude of the step structure varied monotonically. Based on this regularity, we developed a new method to characterize the radius of the hemispherical tip. The tip radii estimated with this approach were comparable to the results obtained using scanning electron microscope imaging and blind reconstruction methods.

  14. Formation of hierarchical molecular assemblies from poly(oxypropylene)-segmented amido acids under AFM tapping.

    PubMed

    Lin, Jiang-Jen; Tsai, Wei-Cheng; Wang, Chi-He

    2007-04-10

    Molecular self-aligning of amphiphilic molecules into bundles with a constant width of 7-13 nm was observed under tapping-mode atomic force microscopy (TM-AFM). The requisite amphiphile, a poly(oxypropylene)-trimellitic amido acid sodium salt, is constituted of a symmetric amido acid structure with potential noncovalent forces of ionic charges, hydrogen bonds, pi-pi aromatic stacking, and hydrophobic interactions for intermolecular interaction. The amphiphiles are able to self-align into orderly hierarchical assemblies after simply being dissolved in water and dried under spin-coated evaporation. Under the TM-AFM tapping process, the bundles increased their length from an initial 20 to 600 nm. A sequential TM-AFM scanning and interval heating process was designed to probe the morphological transformations from the molecular bundles to lengthy strips (nearly micrometer scale) and to columns (with 5-7 nm spacing between the parallel strips). The formation of hierarchical arrays via molecular stretching, aligning, and connecting to each other was simultaneously observed and accelerated under the TM-AFM vibration energy. The molecular self-alignment caused by vibrations is envisioned to be a potential methodology for manipulating molecules into assembled templates, sensors, and optoelectronic devices.

  15. Single-Molecule Studies of Integrins by AFM-Based Force Spectroscopy on Living Cells

    NASA Astrophysics Data System (ADS)

    Eibl, Robert H.

    The characterization of cell adhesion between two living cells at the single-molecule level, i.e., between one adhesion receptor and its counter-receptor, appears to be an experimental challenge. Atomic force microscopy (AFM) can be used in its force spectroscopy mode to determine unbinding forces of a single pair of adhesion receptors, even with a living cell as a probe. This chapter provides an overview of AFM force measurements of the integrin family of cell adhesion receptors and their ligands. A focus is given to major integrins expressed on leukocytes, such as lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4). These receptors are crucial for leukocyte trafficking in health and disease. LFA-1 and VLA-1 can be activated within the bloodstream from a low-affinity to a high-affinity receptor by chemokines in order to adhere strongly to the vessel wall before the receptor-bearing leukocytes extravasate. The experimental considerations needed to provide near-physiological conditions for a living cell and to be able to measure adequate forces at the single-molecule level are discussed in detail. AFM technology has been developed into a modern and extremely sensitive tool in biomedical research. It appears now that AFM force spectroscopy could enter, within a few years, medical applications in diagnosis and therapy of cancer and autoimmune diseases.

  16. Combined AFM nano-machining and reactive ion etching to fabricate high aspect ratio structures.

    PubMed

    Peng, Ping; Shi, Tielin; Liao, Guanglan; Tang, Zirong

    2010-11-01

    In this paper, a new combined method of sub-micron high aspect ratio structure fabrication is developed which can be used for production of nano imprint template. The process includes atomic force microscope (AFM) scratch nano-machining and reactive ion etching (RIE) fabrication. First, 40 nm aluminum film was deposited on the silicon substrate by magnetron sputtering, and then sub-micron grooves were fabricated on the aluminum film by nano scratch using AFM diamond tip. As aluminum film is a good mask for etching silicon, high aspect ratio structures were finally fabricated by RIE process. The fabricated structures were studied by SEM, which shows that the grooves are about 400 nm in width and 5 microm in depth. To obtain sub-micron scale groove structures on the aluminum film, experiments of nanomachining on aluminum films under various machining conditions were conducted. The depths of the grooves fabricated using different scratch loads were also studied by the AFM. The result shows that the material properties of the film/substrate are elastic-plastic following nearly a bilinear law with isotropic strain hardening. Combined AFM nanomachining and RIE process provides a relative lower cost nano fabrication technique than traditional e-beam lithography, and it has a good prospect in nano imprint template fabrication.

  17. Fracture Mechanics Testing of Titanium 6AL-4V in AF-M315E

    NASA Technical Reports Server (NTRS)

    Sampson, J. W.; Martinez, J.; McLean, C.

    2016-01-01

    The Green Propellant Infusion Mission (GPIM) will demonstrate the performance of AF-M315E monopropellant on orbit. Flight certification requires a safe-life analysis of the titanium alloy fuel tank to ensure inherent processing flaws will not cause failure during the design life of the tank. Material property inputs for this analysis require testing to determine the stress intensity factor for environment-assisted cracking (KEAC) of Ti 6Al-4V in combination with the AF-M315E monopropellant. Testing of single-edge notched, or SE(B), specimens representing the bulk tank membrane and weld material were performed in accordance with ASTM E1681. Specimens with fatigue pre-cracks were loaded into test fixtures so that the crack tips were exposed to AF-M315E at 50 C for a duration of 1,000 hours. Specimens that did not fail during exposure were opened to inspect the crack surfaces for evidence of crack growth. The threshold stress intensity value, KEAC, is the highest applied stress intensity that produced neither a failure of the specimen during the exposure nor showed evidence of crack growth. The threshold stress intensity factor for environment-assisted cracking of the Ti 6Al-4V forged tank material was found to be at least 22 ksivin and at least 31 ksivin for the weld material when exposed to AF-M315E monopropellant.

  18. [AFM-based technologies as the way towards the reverse Avogadro number].

    PubMed

    Pleshakova, T O; Shumov, I D; Ivanov, Yu D; Malsagova, K A; Kaysheva, A L; Archakov, A I

    2015-01-01

    Achievement of the concentration detection limit for proteins at the level of the reverse Avogadro number determines the modern development of proteomics. In this review, the possibility of approximating the reverse Avogadro number by using nanotechnological methods (AFM-based fishing with mechanical and electrical stimulation, nanowire detectors, and other methods) are discussed. The ability of AFM to detect, count, visualize and characterize physico-chemical properties of proteins at concentrations up to 10(-17)-10(-18) M is demonstrated. The combination of AFM-fishing with mass-spectrometry allows the identification of proteins not only in pure solutions, but also in multi-component biological fluids (serum). The possibilities to improve the biospecific fishing efficiency by use of SOMAmers in both AFM and nanowire systems are discussed. The paper also provides criteria for evaluation of the sensitivity of fishing-based detection systems. The fishing efficiency depending on the detection system parameters is estimated. The practical implementation of protein fishing depending on the ratio of the sample solution volume and the surface of the detection system is discussed. The advantages and disadvantages of today's promising nanotechnological protein detection methods implemented on the basis of these schemes.

  19. First-principles AFM image simulation with frozen density embedding theory

    NASA Astrophysics Data System (ADS)

    Sakai, Yuki; Lee, Alex J.; Chelikowsky, James R.

    We present efficient first-principles method of non-contact atomic force microscopy (nc-AFM). Ordinary nc-AFM simulations based on density functional theory (DFT) require exhaustive computational cost because it involves thousands of total energy calculations. Regarding the sample as a fixed external potential can reduce the computational cost, and we adopt frozen density embedding theory (FDET) for this purpose. Simulated nc-AFM images with FDET using a carbon monoxide tip well reproduces the full DFT images of benzene, pentacene, and graphene, although optimized tip-sample distances and interaction energies in FDET are underestimated and overestimated, respectively. The FDET-based simulation method is promising for AFM image simulation of surfaces and two-dimensional materials. This work was supported by U.S. DOE under Grant No. DE-FG02-06ER46286 and Award No. DE-SC0008877, and by Welch Foundation under Grant F-1837. Computational resources are provided by NERSC and TACC.

  20. High-speed AFM for 1x node metrology and inspection: Does it damage the features?

    NASA Astrophysics Data System (ADS)

    Sadeghian, Hamed; van den Dool, Teun C.; Uziel, Yoram; Bar Or, Ron

    2015-03-01

    This paper aims at unraveling the mystery of damage in high speed AFMs for 1X node and below. With the device dimensions moving towards the 1X node and below, the semiconductor industry is rapidly approaching the point where existing metrology, inspection and review tools face huge challenges in terms of resolution, the ability to resolve 3D, and throughput. In this paper, we critically asses the important issue of damage in high speed AFM for metrology and inspection of semiconductor wafers. The issues of damage in four major scanning modes (contact mode, tapping mode, non-contact mode, and peak force tapping mode) are described to show which modes are suitable for which applications and which conditions are damaging. The effects of all important scanning parameters on resulting damage are taken into account for materials such as silicon, photoresists and low K materials. Finally, we recommend appropriate scanning parameters and conditions for several use cases (FinFET, patterned photoresist, HAR structures) that avoid exceeding a critical contact stress such that sample damage is minimized. In conclusion, we show using our theoretical analysis that selecting parameters that exceed the target contact stress, indeed leads to significant damage. This method provides AFM users for metrology with a better understanding of contact stresses and enables selection of AFM cantilevers and experimental parameters that prevent sample damage.

  1. Implications of the contact radius to line step (CRLS) ratio in AFM for nanotribology measurements.

    PubMed

    Helt, James M; Batteas, James D

    2006-07-04

    Investigating the mechanisms of defect generation and growth at surfaces on the nanometer scale typically requires high-resolution tools such as the atomic force microscope (AFM). To accurately assess the kinetics and activation parameters of defect production over a wide range of loads (F(z)), the AFM data should be properly conditioned. Generally, AFM wear trials are performed over an area defined by the length of the slow (L(sscan)) and fast scan axes. The ratio of L(sscan) to image resolution (res, lines per image) becomes an important experimental parameter in AFM wear trials because it defines the magnitude of the line step (LS = L(sscan)/res), the distance the AFM tip steps along the slow scan axis. Comparing the contact radius (a) to the line step (LS) indicates that the overlap of successive scans will result unless the contact radius-line step ratio (CRLS) is < or =(1)/(2). If this relationship is not considered, then the scan history (e.g., contact frequency) associated with a single scan is not equivalent at different loads owing to the scaling of contact radius with load (a proportional variant F(z)(1/3)). Here, we present a model in conjunction with empirical wear tests on muscovite mica to evaluate the effects of scan overlap on surface wear. Using the Hertz contact mechanics definition of a, the CRLS model shows that scan overlap pervades AFM wear trials even under low loads. Such findings indicate that simply counting the number of scans (N(scans)) in an experiment underestimates the full history conveyed to the surface by the tip and translates into an error in the actual extent to which a region on the surface is contacted. Utilizing the CRLS method described here provides an approach to account for image scan history accurately and to predict the extent of surface wear. This general model also has implications for any AFM measurement where one wishes to correlate scan-dependent history to image properties as well as feature resolution in scanned

  2. Versatile method for AFM-tip functionalization with biomolecules: fishing a ligand by means of an in situ click reaction.

    PubMed

    Kumar, Rakesh; Ramakrishna, Shivaprakash N; Naik, Vikrant V; Chu, Zonglin; Drew, Michael E; Spencer, Nicholas D; Yamakoshi, Yoko

    2015-04-21

    A facile and universal method for the functionalization of an AFM tip has been developed for chemical force spectroscopy (CFS) studies of intermolecular interactions of biomolecules. A click reaction between tripod-acetylene and an azide-linker-ligand molecule was successfully carried out on the AFM tip surface and used for the CFS study of ligand-receptor interactions.

  3. Identification of new autoantibody specificities directed at proteins involved in the transforming growth factor β pathway in patients with systemic sclerosis

    PubMed Central

    2011-01-01

    Introduction Antinuclear antibodies (ANAs), usually detected by indirect immunofluorescence on HEp-2 cells, are identified in 90% of patients with systemic sclerosis (SSc). Thus, approximately 10% of SSc patients have no routinely detectable autoantibodies, and for 20% to 40% of those with detectable ANAs, the ANAs do not have identified specificity (unidentified ANAs). In this work, we aimed to identify new target autoantigens in SSc patients. Methods Using a proteomic approach combining two-dimensional electrophoresis and immunoblotting with HEp-2 cell total and enriched nuclear protein extracts as sources of autoantigens, we systematically analysed autoantibodies in SSc patients. Sera from 45 SSc patients were tested in 15 pools from groups of three patients with the same phenotype. A sera pool from 12 healthy individuals was used as a control. Proteins of interest were identified by mass spectrometry and analysed using Pathway Studio software. Results We identified 974 and 832 protein spots in HEp-2 cell total and enriched nuclear protein extracts, respectively. Interestingly, α-enolase was recognised by immunoglobulin G (IgG) from all pools of patients in both extracts. Fourteen and four proteins were recognised by IgG from at least 75% of the 15 pools in total and enriched nuclear protein extracts, respectively, whereas 15 protein spots were specifically recognised by IgG from at least four of the ten pools from patients with unidentified ANAs. The IgG intensity for a number of antigens was higher in sera from patients than in sera from healthy controls. These antigens included triosephosphate isomerase, superoxide dismutase mitochondrial precursor, heterogeneous nuclear ribonucleoprotein L and lamin A/C. In addition, peroxiredoxin 2, cofilin 1 and calreticulin were specifically recognised by sera from phenotypic subsets of patients with unidentified ANAs. Interestingly, several identified target antigens were involved in the transforming growth factor

  4. Deficit, but Not Nondeficit, Schizophrenia Is Characterized by Mucosa-Associated Activation of the Tryptophan Catabolite (TRYCAT) Pathway with Highly Specific Increases in IgA Responses Directed to Picolinic, Xanthurenic, and Quinolinic Acid.

    PubMed

    Kanchanatawan, Buranee; Sirivichayakul, Sunee; Ruxrungtham, Kiat; Carvalho, André F; Geffard, Michel; Ormstad, Heidi; Anderson, George; Maes, Michael

    2017-02-08

    Evidence suggests that activation of the tryptophan catabolite (TRYCAT) pathway is involved in the pathophysiology of schizophrenia. However, no previous study examined whether TRYCAT pathway activation is associated with deficit schizophrenia. We measured IgA responses to TRYCATs, namely quinolinic acid, picolinic acid, kynurenic acid, xanthurenic acid, and anthranilic acid and 3-OH-kynurenine, in 40 healthy controls and in schizophrenic patients with (n = 40) and without (n = 40) deficit, defined according to the Schedule for the Deficit Syndrome (SDS). Primary deficit schizophrenia is accompanied by an activated TRYCAT pathway as compared to controls and nondeficit schizophrenia. Participants with deficit schizophrenia show increased IgA responses to xanthurenic acid, picolinic acid, and quinolinic acid and relatively lowered IgA responses to kynurenic and anthranilic acids, as compared to patients with nondeficit schizophrenia. Both schizophrenia subgroups show increased IgA responses to 3-OH-kynurenine as compared to controls. The IgA responses to noxious TRYCATs, namely xanthurenic acid, picolinic acid, quinolinic acid, and 3-OH-kynurenine, but not protective TRYCATS, namely anthranilic acid and kunyrenic acid, are significantly higher in deficit schizophrenia than in controls. The negative symptoms of schizophrenia are significantly and positively associated with increased IgA responses directed against picolinic acid and inversely with anthranilic acid, whereas no significant associations between positive symptoms and IgA responses to TRYCATs were found. In conclusion, primary deficit schizophrenia is characterized by TRYCAT pathway activation and differs from nondeficit schizophrenia by a highly specific TRYCAT pattern suggesting increased excitotoxicity, cytotoxicity, and neurotoxicity, as well as inflammation and oxidative stress. The specific alterations in IgA responses to TRYCATs provide further insight for the biological delineation of deficit

  5. Down-regulation of microRNA-9 leads to activation of IL-6/Jak/STAT3 pathway through directly targeting IL-6 in HeLa cell.

    PubMed

    Zhang, Jiangbo; Jia, Junqiao; Zhao, Lijun; Li, Xiaojun; Xie, Qing; Chen, Xiangmei; Wang, Jianliu; Lu, Fengmin

    2016-05-01

    MicroRNA-9 (miR-9) presents to exert distinct and even opposite functions in different kinds of tumors through targeting different cellular genes. However, its role in cervical adenocarcinoma remains uncertain. Here, we report that miR-9 is down-regulated in cervical adenocarcinoma due to its frequent promoter-hypermethylation and exerts its tumor suppressor role through inhibiting several novel target genes, including interleukin-6 (IL-6). The promoters of miR-9 precursors (mir-9-1, -2, and -3) were hypermethylated in cervical adenocarcinoma tissues. Demethylation treatment of HeLa dramatically increased the expression of mature miR-9. Both in vitro and in vivo functional experiments confirmed that miR-9 can inhibit the proliferation, migration, and malignant transformation abilities of HeLa cells. Bioinformatics methods and array-based RNA expression profiles were used to screen the downstream target genes of miR-9. Dual-luciferase reporting assay, real-time qPCR, and ELISA or Western blot confirmed four genes (CKAP2, HSPC159, IL-6, and TC10) to be novel direct target genes of miR-9. Pathway annotation analysis of the differently expressed genes (DEGs) induced by ectopic miR-9 expression revealed the enrichment in Jak/STAT3 pathway, which is one of the downstream pathways of IL-6. Ectopic expression of miR-9 in HeLa inhibited Jak/STAT3 signaling activity. Moreover, such effect could be partially reversed by the addition of exogenous IL-6. In conclusion, our results here present a tumor suppressor potential of miR-9 in cervical adenocarcinoma for the first time and suggest that miR-9 could repress tumorigenesis through inhibiting the activity of IL-6/Jak/STAT3 pathway.

  6. Reducing time to angiography and hospital stay for patients with high-risk non-ST-elevation acute coronary syndrome: retrospective analysis of a paramedic-activated direct access pathway

    PubMed Central

    Koganti, S; Patel, N; Seraphim, A; Kotecha, T; Whitbread, M; Rakhit, R D

    2016-01-01

    Objective To assess whether a novel ‘direct access pathway’ (DAP) for the management of high-risk non-ST-elevation acute coronary syndromes (NSTEACS) is safe, results in ‘shorter time to intervention and shorter admission times’. This pathway was developed locally to enable London Ambulance Service to rapidly transfer suspected high-risk NSTEACS from the community to our regional heart attack centre for consideration of early angiography. Methods This is a retrospective case–control analysis of 289 patients comparing patients with high-risk NSTEACS admitted via DAP with age-matched controls from the standard pan-London high-risk ACS pathway (PLP) and the conventional pathway (CP). The primary end point of the study was time from admission to coronary angiography/intervention. Secondary end point was total length of hospital stay. Results Over a period of 43 months, 101 patients were admitted by DAP, 109 matched patients by PLP and 79 matched patients through CP. Median times from admission to coronary angiography for DAP, PLP and CP were 2.8 (1.5–9), 16.6 (6–50) and 60 (33–116) hours, respectively (p<0.001). Median length of hospital stay for DAP and PLP was similar at 3.0 (2.0–5.0) days in comparison to 5 (3–7) days for CP (p<0.001). Conclusions DAP resulted in a significant reduction in time to angiography for patients with high-risk NSTEACS when compared to existing pathways. PMID:27324709

  7. Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides.

    PubMed

    Oehlenschlæger, Christian Berg; Løvgreen, Monika Nøhr; Reinauer, Eva; Lehtinen, Emilia; Pind, Marie-Louise Lindberg; Harris, Pernille; Martinussen, Jan; Willemoës, Martin

    2015-05-15

    Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.

  8. Direct endocardial recording from an accessory atrioventricular pathway: localization of the site of block, effect of antiarrhythmic drugs, and attempt at nonsurgical ablation.

    PubMed

    Jackman, W M; Friday, K J; Scherlag, B J; Dehning, M M; Schechter, E; Reynolds, D W; Olson, E G; Berbari, E J; Harrison, L A; Lazzara, R

    1983-11-01

    We recorded a discrete 0.95 mV potential consistent with accessory atrioventricular pathway (AP) activation during serial electrophysiologic studies in a patient with Ebstein's anomaly and Wolff-Parkinson-White syndrome. Bipolar pacing from the catheter electrode in which the AP potential was recorded resulted in a stimulus-ventricle interval identical to the AP-ventricle interval during antegrade conduction, and a stimulus-atrium interval identical to the AP-atrium interval during retrograde conduction. With the patient in the drug-free state, antegrade AP block during atrial pacing and retrograde AP block during ventricular pacing occurred distal to the AP potential (AP-ventricle junction and AP-atrium junction, respectively), supporting the "impedance mismatch" hypothesis. Procainamide and disopyramide each lengthened the antegrade AP effective refractory period by affecting the AP-ventricle junction (possibly by decreasing the current generated by the AP). Both drugs also lengthened the retrograde AP effective refractory period but produced a greater effect on the ventricle-AP junction than on the AP-atrium junction, suggesting marginal geometry of the former. R wave synchronous shocks of 160 and 320 W-sec delivered between the catheter electrode recording the largest unipolar AP potential and a skin electrode produced transient, complete, antegrade block over the AP, suggesting the feasibility of this new nonsurgical technique for AP ablation.

  9. The Brisbane Longitudinal Twin Study: Pathways to Cannabis Use, Abuse, and Dependence project-current status, preliminary results, and future directions.

    PubMed

    Gillespie, Nathan A; Henders, Anjali K; Davenport, Tracy A; Hermens, Daniel F; Wright, Margie J; Martin, Nicholas G; Hickie, Ian B

    2013-02-01

    We describe the data being collected from the Brisbane Longitudinal Twin Study in Australia as part of the US National Institute on Drug Abuse (NIDA)-funded project, Pathways to Cannabis Use, Abuse and Dependence. The history, recruitment, assessment, and retention of twin families in this project are described in detail, along with preliminary findings and plans for future research. The goal of this NIDA project is to make a significant contribution to the discovery of quantitative trait loci influencing cannabis use disorders. Although the focus is cannabis use, abuse, and dependence in young adults, measures of comorbid illicit drug use disorders are also being collected. In addition, a variety of internalizing and externalizing disorders are being assessed, funded by support from the Australian National Health and Medical Research Council. Because these same twins have participated in numerous twin studies since 1992, future plans will include linking different phenotypes to investigate relationships between drug use, psychiatric disorders, and psychological phenotypes within cross-sectional and longitudinal or developmental frameworks.

  10. Damage to the glycoshield activates PMT-directed O-mannosylation via the Msb2-Cek1 pathway in Candida albicans.

    PubMed

    Cantero, Pilar D; Ernst, Joachim F

    2011-05-01

    Protein-O-mannosyltransferases (Pmt) transfer mannosyl residues to secretory proteins. Five isoforms of Pmt proteins in the human fungal pathogen Candida albicans have distinct functions in growth, morphogenesis and antifungal resistance. We found that PMT genes encoding the major isoforms Pmt1, Pmt2, Pmt4 are regulated differently in response to impaired glycostructures. While the PMT1 transcript level increased in cell wall mutants and under inhibition of N-glycosylation by tunicamycin, PMT2 and PMT4 transcripts were upregulated only by inhibition of Pmt1 activity. Reporter fusions revealed specific promoter sequences to be required for PMT1 repression in undamaged cells, which was de-repressed by tunicamycin. Constitutive PMT1 de-repression was observed in mutants lacking the Cek1 MAP kinase and its upstream sensor Msb2. In contrast, in msb2 and cek1 mutants, upregulation of PMT2/PMT4 by Pmt1 inhibition did not occur and basal expression of both transcripts were decreased. We identified Ace2 as a novel transcription factor, which upregulates PMT basal expression and induction in response to glycostructure damage. Mutants lacking Msb2, Cek1 and Ace2 were supersensitive to glycosylation and cell wall inhibitors. We propose that a Msb2, Cek1 and Ace2 signalling pathway addresses PMT genes as downstream targets and that different modes of regulation have evolved for PMT1 and PMT2/PMT4 genes.

  11. High throughput nanofabrication of silicon nanowire and carbon nanotube tips on AFM probes by stencil-deposited catalysts.

    PubMed

    Engstrom, Daniel S; Savu, Veronica; Zhu, Xueni; Bu, Ian Y Y; Milne, William I; Brugger, Juergen; Boggild, Peter

    2011-04-13

    A new and versatile technique for the wafer scale nanofabrication of silicon nanowire (SiNW) and multiwalled carbon nanotube (MWNT) tips on atomic force microscope (AFM) probes is presented. Catalyst material for the SiNW and MWNT growth was deposited on prefabricated AFM probes using aligned wafer scale nanostencil lithography. Individual vertical SiNWs were grown epitaxially by a catalytic vapor-liquid-solid (VLS) process and MWNTs were grown by a plasma-enhanced chemical vapor (PECVD) process on the AFM probes. The AFM probes were tested for imaging micrometers-deep trenches, where they demonstrated a significantly better performance than commercial high aspect ratio tips. Our method demonstrates a reliable and cost-efficient route toward wafer scale manufacturing of SiNW and MWNT AFM probes.

  12. Two-Dimensional Measurement of n+-p Asymmetrical Junctions in Multicrystalline Silicon Solar Cells Using AFM-Based Electrical Techniques with Nanometer Resolution: Preprint

    SciTech Connect

    Jiang, C. S.; Moutinho, H. R.; Li, J. V.; Al-Jassim, M. M.; Heath, J. T.

    2011-07-01

    Lateral inhomogeneities of modern solar cells demand direct electrical imaging with nanometer resolution. We show that atomic force microscopy (AFM)-based electrical techniques provide unique junction characterizations, giving a two-dimensional determination of junction locations. Two AFM-based techniques, scanning capacitance microscopy/spectroscopy (SCM/SCS) and scanning Kelvin probe force microscopy (SKPFM), were significantly improved and applied to the junction characterizations of multicrystalline silicon (mc-Si) cells. The SCS spectra were taken pixel by pixel by precisely controlling the tip positions in the junction area. The spectra reveal distinctive features that depend closely on the position relative to the electrical junction, which allows us to indentify the electrical junction location. In addition, SKPFM directly probes the built-in potential over the junction area modified by the surface band bending, which allows us to deduce the metallurgical junction location by identifying a peak of the electric field. Our results demonstrate resolutions of 10-40 nm, depending on the techniques (SCS or SKPFM). These direct electrical measurements with nanometer resolution and intrinsic two-dimensional capability are well suited for investigating the junction distribution of solar cells with lateral inhomogeneities.

  13. Analysis the effect of different geometries of AFM's cantilever on the dynamic behavior and the critical forces of three-dimensional manipulation.

    PubMed

    Korayem, Moharam Habibnejad; Saraie, Maniya B; Saraee, Mahdieh B

    2017-01-13

    An important challenge when using an atomic force microscope (AFM) is to be able to control the force exerted by the AFM for performing various tasks. Nevertheless, the exerted force is proportional to the deflection of the AFM cantilever, which itself is affected by a cantilever's stiffness coefficient. Many papers have been published so far on the methods of obtaining the stiffness coefficients of AFM cantilevers in 2D; however, a comprehensive model is yet to be presented on 3D cantilever motion. The discrepancies between the equations of the 2D and 3D analysis are due to the number and direction of forces and moments that are applied to a cantilever. Moreover, in the 3D analysis, contrary to the 2D analysis, due to the interaction between the forces and moments applied on a cantilever, its stiffness values cannot be separately expressed for each direction; and instead, a stiffness matrix should be used to correctly derive the relevant equations. In this paper, 3D stiffness coefficient matrices have been obtained for three common cantilever geometries including the rectangular, V-shape and dagger-shape cantilevers. The obtained equations are validated by two methods. In the first approach, the Finite Element Method is combined with the cantilever deflection values computed by using the obtained stiffness matrices. In the second approach, by reducing the problem's parameters, the forces applied on a cantilever along different directions are compared with each other in 2D and 3D cases. Then the 3D manipulation of a stiff nanoparticle is modeled and simulated by using the stiffness matrices obtained for the three cantilever geometries. The obtained results indicate that during the manipulation process, the dagger-shaped and rectangular cantilevers exert the maximum and minimum amounts of forces on the stiff nanoparticle, respectively. Also, by examining the effects of different probe tip geometries, it is realized that a probe tip of cylindrical geometry exerts the

  14. Multi-drug loaded micelles delivering chemotherapy and targeted therapies directed against HSP90 and the PI3K/AKT/mTOR pathway in prostate cancer

    PubMed Central

    Le, Bao; Powers, Ginny L.; Tam, Yu Tong; Schumacher, Nicholas; Malinowski, Rita L.; Steinke, Laura; Kwon, Glen; Marker, Paul C.

    2017-01-01

    Background Advanced prostate cancers that are resistant to all current therapies create a need for new therapeutic strategies. One recent innovative approach to cancer therapy is the simultaneous use of multiple FDA-approved drugs to target multiple pathways. A challenge for this approach is caused by the different solubility requirements of each individual drug, resulting in the need for a drug vehicle that is non-toxic and capable of carrying multiple water-insoluble antitumor drugs. Micelles have recently been shown to be new candidate drug solubilizers for anti cancer therapy. Methods This study set out to examine the potential use of multi-drug loaded micelles for prostate cancer treatment in preclinical models including cell line and mouse models for prostate cancers with Pten deletions. Specifically antimitotic agent docetaxel, mTOR inhibitor rapamycin, and HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin were incorporated into the micelle system (DR17) and tested for antitumor efficacy. Results In vitro growth inhibition of prostate cancer cells was greater when all three drugs were used in combination compared to each individual drug, and packaging the drugs into micelles enhanced the cytotoxic effects. At the molecular level DR17 targeted simultaneously several molecular signaling axes important in prostate cancer including androgen receptor, mTOR, and PI3K/AKT. In a mouse genetic model of prostate cancer, DR17 treatment decreased prostate weight, which was achieved by both increasing caspase-dependent cell death and decreasing cell proliferation. Similar effects were also observed when DR17 was administered to nude mice bearing prostate cancer cells xenografts. Conclusion These results suggest that combining these three cancer drugs in multi-drug loaded micelles may be a promising strategy for prostate cancer therapy. PMID:28350865

  15. 13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum.

    PubMed

    Kleijn, Roelco J; van Winden, Wouter A; Ras, Cor; van Gulik, Walter M; Schipper, Dick; Heijnen, Joseph J

    2006-07-01

    In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.

  16. Genistein directly induces cardiac CFTR chloride current by a tyrosine kinase-independent and protein kinase A-independent pathway in guinea pig ventricular myocytes.

    PubMed

    Chiang, C E; Chen, S A; Chang, M S; Lin, C I; Luk, H N

    1997-06-09

    With one-suction electrode voltage-clamp technique, we demonstrated that genistein, a tyrosine kinase (TK) inhibitor, could directly activate cystic fibrosis transmembrane regulator (CFTR) chloride current in guinea pig ventricular myocytes. The activation showed concentration-dependent effect with the estimated IC50 of 39.7 microM. Tyrphostin 51, another TK inhibitor, had no effect, suggesting that genistein's effect might be unrelated to TK inhibition. After the chloride current had been activated by the maximally elevated intracellular cAMP content by saturating concentration of isoproterenol, forskolin and IBMX, genistein could further enhance the current. Pre-treatment with saturating concentration of a specific protein kinase A (PKA) inhibitor, H-89, or other protein kinase inhibitors H-8 and H-9 in the perfusate or intracellularly could not prevent the activation of the current by genistein, suggesting a PKA-independent activity. Furthermore, saturating concentration of calyculin A, a specific inhibitor of phosphotase 1 and 2A, in the perfusate or intracellularly could not block genistein's action. It is possible that genistein opens the channels directly or inhibits the dephosphorylation process of CFTR, which is not sensitive calyculin A.

  17. Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways.

    PubMed

    Sears, Catherine R; Turchi, John J

    2012-07-13

    The treatment for advanced stage non-small cell lung cancer (NSCLC) often includes platinum-based chemotherapy and IR. Cisplatin and IR combination therapy display schedule and dose-dependent synergy, the mechanism of which is not completely understood. In a series of in vitro and cell culture assays in a NSCLC model, we investigated both the downstream and direct treatment and damage effects of cisplatin on NHEJ catalyzed repair of a DNA DSB. The results demonstrate that extracts prepared from cisplatin-treated cells are fully capable of NHEJ catalyzed repair of a DSB using a non-cisplatin-damaged DNA substrate in vitro. Similarly, using two different host cell reactivation assays, treatment of cells prior to transfection of a linear, undamaged reporter plasmid revealed no reduction in NHEJ compared with untreated cells. In contrast, transfection of a linear GFP-reporter plasmid containing site-specific, cisplatin lesions 6-bp from the termini revealed a significant impairment in DSB repair of the cisplatin-damaged DNA substrates in the absence of cellular treatment with cisplatin. Together, these data demonstrate that impaired NHEJ in combined cisplatin-IR treated cells is likely the result of a direct effect of cisplatin-DNA lesions near a DSB and that the indirect cellular effects of cisplatin treatment are not significant contributors to the synergistic cytotoxicity observed with combination cisplatin-IR treatment.

  18. Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation.

    PubMed

    Andrushchenko, V; Leonenko, Z; Cramb, D; van de Sande, H; Wieser, H

    The interaction of natural calf thymus DNA with Cr(3+) ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr(3+) ion binding mainly to N(7) (G) and to phosphate groups was demonstrated. Psi-type VCD spectra resembling electronic CD (ECD) spectra, which appear during psi-type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large-scale helicity. Atomic force microscopy confirmed DNA condensation by Cr(3+) ions and the formation of tight DNA particles responsible for the psi-type VCD spectra. Upon increasing the Cr(3+) ion concentration the shape of the condensates changed from loose flower-like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr(3+) ions studied. The secondary structure of DNA remained in a B-form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles.

  19. Direct crosstalk between cancer and osteoblast lineage cells fuels metastatic growth in bone via auto-amplification of IL-6 and RANKL signaling pathways.

    PubMed

    Zheng, Yu; Chow, Shu-Oi; Boernert, Katja; Basel, Dennis; Mikuscheva, Anastasia; Kim, Sarah; Fong-Yee, Colette; Trivedi, Trupti; Buttgereit, Frank; Sutherland, Robert L; Dunstan, Colin R; Zhou, Hong; Seibel, Markus J

    2014-09-01

    The bone microenvironment and its modification by cancer and host cell interactions is a key driver of skeletal metastatic growth. Interleukin-6 (IL-6) stimulates receptor activator of NF-κB ligand (RANKL) expression in bone cells, and serum IL-6 levels are associated with poor clinical outcomes in cancer patients. We investigated the effects of RANKL on cancer cells and the role of tumor-derived IL-6 within the bone microenvironment. Using human breast cancer cell lines to induce tumors in the bone of immune-deficient mice, we first determined whether RANKL released by cells of the osteoblast lineage directly promotes IL-6 expression by cancer cells in vitro and in vivo. We then disrupted of IL-6 signaling in vivo either via knockdown of IL-6 in tumor cells or through treatment with specific anti-human or anti-mouse IL-6 receptor antibodies to investigate the tumor effect. Finally, we tested the effect of RANK knockdown in cancer cells on cancer growth. We demonstrate that osteoblast lineage-derived RANKL upregulates secretion of IL-6 by breast cancers in vivo and in vitro. IL-6, in turn, induces expression of RANK by cancer cells, which sensitizes the tumor to RANKL and significantly enhances cancer IL-6 release. Disruption in vivo of this auto-amplifying crosstalk by knockdown of IL-6 or RANK in cancer cells, or via treatment with anti-IL-6 receptor antibodies, significantly reduces tumor growth in bone but not in soft tissues. RANKL and IL-6 mediate direct paracrine-autocrine signaling between cells of the osteoblast lineage and cancer cells, significantly enhancing the growth of metastatic breast cancers within bone.

  20. Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells

    PubMed Central

    Acerbi, Irene; Luque, Tomás; Giménez, Alícia; Puig, Marta; Reguart, Noemi; Farré, Ramon; Navajas, Daniel; Alcaraz, Jordi

    2012-01-01

    Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ∼1 µm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis. PMID:22384196

  1. Aflatoxin M1 Concentration in Various Dairy Products: Evidence for Biologically Reduced Amount of AFM1 in Yoghurt

    PubMed Central

    RAHIMIRAD, Amir; MAALEKINEJAD, Hassan; OSTADI, Araz; YEGANEH, Samal; FAHIMI, Samira

    2014-01-01

    Abstract Background Aflatoxin M1 (AFM1), a carcinogenic substance is found in milk and dairy products. The effect of season and type of dairy products on AFMi level in northern Iran was investigated in this study. Methods Three hundred samples (each season 75 samples) including raw and pasteurized milk, yoghurt, cheese, and cream samples were collected from three distinct milk producing farms. The samples were subjected to chemical and solid phase extractions and were analyzed by using HPLC technique. Recovery percentages, limit of detection and limit of quantification values were determined. Results Seventy percent and 98% were the minimum and maximum recoveries for cheese and raw milk, respectively and 0.021 and 0.063 ppb were the limit of detection and limit of quantification values for AFM1. We found that in autumn and winter the highest level (0.121 ppb) of AFM1 in cheese and cream samples and failed to detect any AFM1 in spring samples. Interestingly, our data showed that the yoghurt samples had the lowest level of AFM1 in all seasons. Conclusion There are significant differences between the AFM1 levels in dairy products in various seasons and also various types of products, suggesting spring and summer yoghurt samples as the safest products from AFM1 level point of view. PMID:25927044

  2. Study of relaxation and transport processes by means of AFM based dielectric spectroscopy

    SciTech Connect

    Miccio, Luis A.

    2014-05-15

    Since its birth a few years ago, dielectric spectroscopy studies based on atomic force microscopy (AFM) have gained a growing interest. Not only the frequency and temperature ranges have become broader since then but also the kind of processes that can be studied by means of this approach. In this work we analyze the most adequate experimental setup for the study of several dielectric processes with a spatial resolution of a few nanometers by using force mode AFM based dielectric spectroscopy. Proof of concept experiments were performed on PS/PVAc blends and PMMA homopolymer films, for temperatures ranging from 300 to 400 K. Charge transport processes were also studied by this approach. The obtained results were analyzed in terms of cantilever stray contribution, film thickness and relaxation strength. We found that the method sensitivity is strongly coupled with the film thickness and the relaxation strength, and that it is possible to control it by using an adequate experimental setup.

  3. Confocal Raman spectroscopy and AFM for evaluation of sidewalls in type II superlattice FPAs

    NASA Astrophysics Data System (ADS)

    Rotter, T. J.; Busani, T.; Rathi, P.; Jaeckel, F.; Reyes, P. A.; Malloy, K. J.; Ukhanov, A. A.; Plis, E.; Krishna, S.; Jaime-Vasquez, M.; Baril, N. F.; Benson, J. D.; Tenne, D. A.

    2015-06-01

    We propose to utilize confocal Raman spectroscopy combined with high resolution atomic force microscopy (AFM) for nondestructive characterisation of the sidewalls of etched and passivated small pixel (24 μm×24 μm) focal plane arrays (FPA) fabricated using LW/LWIR InAs/GaSb type-II strained layer superlattice (T2SL) detector material. Special high aspect ratio Si and GaAs AFM probes, with tip length of 13 μm and tip aperture less than 7°, allow characterisation of the sidewall morphology. Confocal microscopy enables imaging of the sidewall profile through optical sectioning. Raman spectra measured on etched T2SL FPA single pixels enable us to quantify the non-uniformity of the mesa delineation process.

  4. Atom probe, AFM, and STM studies on vacuum-fired stainless steels.

    PubMed

    Stupnik, A; Frank, P; Leisch, M

    2009-04-01

    The surface morphology of grades 304L and 316LN stainless steels, after low-temperature bake-out process and vacuum annealing, has been studied by atomic force microscopy (AFM) and scanning tunnelling microscopy (STM). The local elemental composition on the surface before and after thermal treatment has been investigated by atom probe (AP) depth profiling measurements. After vacuum annealing, AFM and STM show significant changes in the surface structure and topology. Recrystallization and surface reconstruction is less pronounced on the 316LN stainless steel. AP depth profiling analyses result in noticeable nickel enrichment on the surface of grade 304L samples. Since hydrogen recombination is almost controlled by surface structure and composition, a strong influence on the outgassing behaviour by the particular surface microstructure can be deduced.

  5. Development of a novel nanoindentation technique by utilizing a dual-probe AFM system

    PubMed Central

    Sahin, Ferat; Yablon, Dalia

    2015-01-01

    Summary A novel instrumentation approach to nanoindentation is described that exhibits improved resolution and depth sensing. The approach is based on a multi-probe scanning probe microscopy (SPM) tool that utilizes tuning-fork based probes for both indentation and depth sensing. Unlike nanoindentation experiments performed with conventional AFM systems using beam-bounce technology, this technique incorporates a second probe system with an ultra-high resolution for depth sensing. The additional second probe measures only the vertical movement of the straight indenter attached to a tuning-fork probe with a high spring constant and it can also be used for AFM scanning to obtain an accurate profiling. Nanoindentation results are demonstrated on silicon, fused silica, and Corning Eagle Glass. The results show that this new approach is viable in terms of accurately characterizing mechanical properties of materials through nanoindentation with high accuracy, and it opens doors to many other exciting applications in the field of nanomechanical characterization. PMID:26665072

  6. Nano-Electrochemistry and Nano-Electrografting with an Original Combined AFM-SECM

    PubMed Central

    Ghorbal, Achraf; Grisotto, Federico; Charlier, Julienne; Palacin, Serge; Goyer, Cédric; Demaille, Christophe; Ben Brahim, Ammar

    2013-01-01

    This study demonstrates the advantages of the combination between atomic force microscopy and scanning electrochemical microscopy. The combined technique can perform nano-electrochemical measurements onto agarose surface and nano-electrografting of non-conducting polymers onto conducting surfaces. This work was achieved by manufacturing an original Atomic Force Microscopy-Scanning ElectroChemical Microscopy (AFM-SECM) electrode. The capabilities of the AFM-SECM-electrode were tested with the nano-electrografting of vinylic monomers initiated by aryl diazonium salts. Nano-electrochemical and technical processes were thoroughly described, so as to allow experiments reproducing. A plausible explanation of chemical and electrochemical mechanisms, leading to the nano-grafting process, was reported. This combined technique represents the first step towards improved nano-processes for the nano-electrografting.

  7. Exploring electron transport through organic monolayers using conductive tip AFM techniques

    NASA Astrophysics Data System (ADS)

    Scaini, Denis; Castronovo, Matteo; Dell'Angela, Martina; Hudej, Robert; Casalis, Loredana; Scoles, Giacinto

    2006-03-01

    We follow an alternative approach to the study of Metal-molecule-Metal junctions that uses a combination of two atomic force microscopy (AFM) techniques. We use Nanografting to build a nanopatch of the molecules of interest and a second made of a reference molecule into a hosting self assembled monolayer (SAM) typically made of alkanethiols. After the tip is changed to a conductive one CT-AFM is used to characterized the whole system recording, at the same time, the system topography. Some of the advantages of this approach are the possibility to build and study a wide range of different M-m-M junctions and the in-situ control of the quality of the monolayers and patches. Results will be presented on saturated and unsaturated thiols self-assembled and nanografted on Au(111) surfaces. The results will be compared with those obtained by Liang and Scoles at Princeton using similar techniques.

  8. BOREAS AFM-5 Level-2 Upper Air Network Standard Pressure Level Data

    NASA Technical Reports Server (NTRS)

    Barr, Alan; Hrynkiw, Charmaine; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Smith, David E. (Technical Monitor)

    2000-01-01

    The BOREAS AFM-5 team collected and processed data from the numerous radiosonde flights during the project. The goals of the AFM-05 team were to provide large-scale definition of the atmosphere by supplementing the existing AES aerological network, both temporally and spatially. This data set includes basic upper-air parameters interpolated at 0.5 kiloPascal increments of atmospheric pressure from data collected from the network of upper-air stations during the 1993, 1994, and 1996 field campaigns over the entire study region. The data are contained in tabular ASCII files. The data files are available on a CD-ROM (see document number 20010000884) or from the Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC).

  9. Characterization of the polycaprolactone melt crystallization: complementary optical microscopy, DSC, and AFM studies.

    PubMed

    Speranza, V; Sorrentino, A; De Santis, F; Pantani, R

    2014-01-01

    The first stages of the crystallization of polycaprolactone (PCL) were studied using several techniques. The crystallization exotherms measured by differential scanning calorimetry (DSC) were analyzed and compared with results obtained by polarized optical microscopy (POM), rheology, and atomic force microscope (AFM). The experimental results suggest a strong influence of the observation scale. In particular, the AFM, even if limited on time scale, appears to be the most sensitive technique to detect the first stages of crystallization. On the contrary, at least in the case analysed in this work, rheology appears to be the least sensitive technique. DSC and POM provide closer results. This suggests that the definition of induction time in the polymer crystallization is a vague concept that, in any case, requires the definition of the technique used for its characterization.

  10. Dynamics of a disturbed sessile drop measured by atomic force microscopy (AFM).

    PubMed

    McGuiggan, Patricia M; Grave, Daniel A; Wallace, Jay S; Cheng, Shengfeng; Prosperetti, Andrea; Robbins, Mark O

    2011-10-04

    A new method for studying the dynamics of a sessile drop by atomic force microscopy (AFM) is demonstrated. A hydrophobic microsphere (radius, r ∼ 20-30 μm) is brought into contact with a small sessile water drop resting on a polytetrafluoroethylene (PTFE) surface. When the microsphere touches the liquid surface, the meniscus rises onto it because of capillary forces. Although the microsphere volume is 6 orders of magnitude smaller than the drop, it excites the normal resonance modes of the liquid interface. The sphere is pinned at the interface, whose small (<100 nm) oscillations are readily measured with AFM. Resonance oscillation frequencies were measured for drop volumes between 5 and 200 μL. The results for the two lowest normal modes are quantitatively consistent with continuum calculations for the natural frequency of hemispherical drops with no adjustable parameters. The method may enable sensitive measurements of volume, surface tension, and viscosity of small drops.

  11. Molecular Dynamic Simulations of Interaction of an AFM Probe with the Surface of an SCN Sample

    NASA Technical Reports Server (NTRS)

    Bune, Adris; Kaukler, William; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    Molecular dynamic (MD) simulations is conducted in order to estimate forces of probe-substrate interaction in the Atomic Force Microscope (AFM). First a review of available molecular dynamic techniques is given. Implementation of MD simulation is based on an object-oriented code developed at the University of Delft. Modeling of the sample material - succinonitrile (SCN) - is based on the Lennard-Jones potentials. For the polystyrene probe an atomic interaction potential is used. Due to object-oriented structure of the code modification of an atomic interaction potential is straight forward. Calculation of melting temperature is used for validation of the code and of the interaction potentials. Various fitting parameters of the probe-substrate interaction potentials are considered, as potentials fitted to certain properties and temperature ranges may not be reliable for the others. This research provides theoretical foundation for an interpretation of actual measurements of an interaction forces using AFM.

  12. Accurate and precise calibration of AFM cantilever spring constants using laser Doppler vibrometry.

    PubMed

    Gates, Richard S; Pratt, Jon R

    2012-09-21

    Accurate cantilever spring constants are important in atomic force microscopy both in control of sensitive imaging and to provide correct nanomechanical property measurements. Conventional atomic force microscope (AFM) spring constant calibration techniques are usually performed in an AFM. They rely on significant handling and often require touching the cantilever probe tip to a surface to calibrate the optical lever sensitivity of the configuration. This can damage the tip. The thermal calibration technique developed for laser Doppler vibrometry (LDV) can be used to calibrate cantilevers without handling or touching the tip to a surface. Both flexural and torsional spring constants can be measured. Using both Euler-Bernoulli modeling and an SI traceable electrostatic force balance technique as a comparison we demonstrate that the LDV thermal technique is capable of providing rapid calibrations with a combination of ease, accuracy and precision beyond anything previously available.

  13. AFM fishing nanotechnology is the way to reverse the Avogadro number in proteomics.

    PubMed

    Archakov, Alexander Ivanovich; Ivanov, Yurii Dmitrievich; Lisitsa, Andrey Valerevich; Zgoda, Victor Gavrilovich

    2007-01-01

    Future development of proteomics may be hindered by limitations in the concentration sensitivity of widespread technological approaches. The concentration sensitivity limit (CSL) of currently used approaches, like 2-DE/LC separation coupled with MS detection, etc., varies from 10(-9) to 10(-12) M. Therefore, proteomic technologies enable detection of up to 20% of the protein species present in the plasma. New technologies, like atomic force microscopy (AFM molecular detector), enable the counting of single molecules, whereas biospecific fishing can be used to capture these molecules from the biomaterial. At the same time, fishing also has thermodynamic limitations due to the reversibility of the binding. In cases where the fishing becomes irreversible, its combination with an AFM detector enables the registration of single protein molecules, and that opens up a way to lower the CSL down to the reverse Avogadro number.

  14. Mechanism of high-resolution STM, AFM and IETS-STM imaging with functionalized tips

    NASA Astrophysics Data System (ADS)

    Temirov, R.; Hapala, P.; Tautz, F. S.; Jelinek, P.

    2015-03-01

    High-resolution AFM and STM with functionalized tips is well established, but a detailed understanding of the image mechanism is still missing. Moreover, recently this family of imaging techniques has been complemented by a method based on inelastic electron tunneling spectroscopy. Here we present a comprehensive mechanical and transport simulation model that explains essentially all image features in functionalized tip STM, AFM and IETS-STM. Important aspects of the mechanism are: (i) Images are dominantly determined by Pauli repulsion, (ii) in STM and IETS STM this force signal is transduced into an elastic or inelastic conductance signal, (iii) probe particle relaxation leads to image sharpening, (iv) the apparent imaging of hydrogen bonds can be explained by a relaxation effect, and (v) electrostatic forces may also influence the image contrast.

  15. Searching events in AFM force-extension curves: A wavelet approach.

    PubMed

    Benítez, R; Bolós, V J

    2017-01-01

    An algorithm, based on the wavelet scalogram energy, for automatically detecting events in force-extension AFM force spectroscopy experiments is introduced. The events to be detected are characterized by a discontinuity in the signal. It is shown how the wavelet scalogram energy has different decay rates at different points depending on the degree of regularity of the signal, showing faster decay rates at regular points and slower rates at singular points (jumps). It is shown that these differences produce peaks in the scalogram energy plot at the event points. Finally, the algorithm is illustrated in a tether analysis experiment by using it for the detection of events in the AFM force-extension curves susceptible to being considered tethers. Microsc. Res. Tech. 80:153-159, 2017. © 2016 Wiley Periodicals, Inc.

  16. U.S. Army Training and Testing Area Carrying Capacity (ATTACC) for Munitions (AFM)

    DTIC Science & Technology

    2006-11-01

    days. The RUSLE dataset delivered with the AFM model is defined below and in Appendix E. The Adapted RUSLE is defined as A=R*K* LS *C*P where: A...hr [hundreds of ft-tons]-1 in.-1) LS = slope length and steepness factor (dimensionless) C = cover and management factor (dimensionless) P...erosion index unit under standardized conditions. Higher K factor values indicate more easily erodible soils. • LS Factor. The rate of soil

  17. Study of mechanical behavior of AFM silicon tips under mechanical load

    NASA Astrophysics Data System (ADS)

    Kopycinska-Mueller, M.; Gluch, J.; Köhler, B.

    2016-11-01

    In this paper we address critical issues concerning calibration of AFM based methods used for nanoscale mechanical characterization of materials. It has been shown that calibration approaches based on macroscopic models for contact mechanics may yield excellent results in terms of the indentation modulus of the sample, but fail to provide a comprehensive and actual information concerning the tip-sample contact radius or the mechanical properties of the tip. Explanations for the severely reduced indentation modulus of the tip included the inadequacies of the models used for calculations of the tip-sample contact stiffness, discrepancies in the actual and ideal shape of the tip, presence of the amorphous silicon phase within the silicon tip, as well as negligence of the actual size of the stress field created in the tip during elastic interactions. To clarify these issues, we investigated the influence of the mechanical load applied to four AFM silicon tips on their crystalline state by exposing them to systematically increasing loads, evaluating the character of the tip-sample interactions via the load-unload stiffness curves, and assessing the state of the tips from HR-TEM images. The results presented in this paper were obtained in a series of relatively simple and basic atomic force acoustic microscopy (AFAM) experiments. The novel combination of TEM imaging of the AFM tips with the analysis of the load-unload stiffness curves gave us a detailed insight into their mechanical behavior under load conditions. We were able to identify the limits for the elastic interactions, as well as the hallmarks for phase transformation and dislocation formation and movement. The comparison of the physical dimensions of the AFM tips, geometry parameters determined from the values of the contact stiffness, and the information on the crystalline state of the tips allowed us a better understanding of the nanoscale contact.

  18. Up-regulated HMGB1 in EAM directly led to collagen deposition by a PKCβ/Erk1/2-dependent pathway: cardiac fibroblast/myofibroblast might be another source of HMGB1

    PubMed Central

    Su, Zhaoliang; Yin, Jingping; Wang, Ting; Sun, Yingkun; Ni, Ping; Ma, Rui; Zhu, Haitao; Zheng, Dong; Shen, Huiling; Xu, Wenlin; Xu, Huaxi

    2014-01-01

    High mobility group box 1 (HMGB1), an important inflammatory mediator, is actively secreted by immune cells and some non-immune cells or passively released by necrotic cells. HMGB1 has been implicated in many inflammatory diseases. Our previous published data demonstrated that HMGB1 was up-regulated in heart tissue or serum in experimental autoimmune myocarditis (EAM); HMGB1 blockade could ameliorate cardiac fibrosis at the last stage of EAM. And yet, until now, no data directly showed that HMGB1 was associated with cardiac fibrosis. Therefore, the aims of the present work were to assess whether (1) up-regulated HMGB1 could directly lead to cardiac fibrosis in EAM; (2) cardiac fibroblast/myofibroblasts could secrete HMGB1 as another source of high-level HMGB1 in EAM; and (3) HMGB1 blockade could effectively prevent cardiac fibrosis at the last stage of EAM. Our results clearly demonstrated that HMGB1 could directly lead to cardiac collagen deposition, which was associated with PKCβ/Erk1/2 signalling pathway; furthermore, cardiac fibroblast/myofibroblasts could actively secrete HMGB1 under external stress; and HMGB1 secreted by cardiac fibroblasts/myofibroblasts led to cardiac fibrosis via PKCβ activation by autocrine means; HMGB1 blockade could efficiently ameliorate cardiac fibrosis in EAM mice. PMID:24912759

  19. Enzymatic nanolithography of FRET peptide layer using V8 protease-immobilized AFM probe.

    PubMed

    Nakamura, Chikashi; Miyamoto, Chie; Obataya, Ikuo; Takeda, Seiji; Yabuta, Masayuki; Miyake, Jun

    2007-04-15

    In our study, a method based on Enzymatic nanolithography was successfully performed in a buffered solution using Staphylococcal serine V8 protease and AFM. To estimate the lithographing activity of the protease immobilized on the AFM tip to peptides immobilized on a substrate, we designed fluorescence resonance energy transfer (FRET) peptides as reporter peptides that showed enzymatic action specific to the V8 protease. When the protease digested the reporter peptide a quencher residue was released from the peptide and resulted in the appearance of fluorescence. In the designed 9-mer peptides, TAMRA functioned as a good quencher for FAM. When the fluorescence resonance energy transfer peptides immobilized on a glass substrate were hydrolyzed by V8 protease at the C-terminal of glutamic acid, fluorescence of a reporter dye was observed because of the release of a quencher from the substrate. After contacting and lateral scanning of the protease-immobilized AFM tip to the reporter peptide layer, a fluorescent area was observed by imaging using total internal refection fluorescence microscopy (TIRFM). The increment of fluorescence intensity of the digested peptide indicates the performance of lithography. Lithographing rates increased in inverse relation to scanning rates of the probe. The maximum limit of the scanning rate, i.e., that was too fast to permit cutting of the peptide on the substrate, and the lithographing performance are discussed in this study.

  20. Accurate, explicit formulae for higher harmonic force spectroscopy by frequency modulation-AFM.

    PubMed

    Kuchuk, Kfir; Sivan, Uri

    2015-01-01

    The nonlinear interaction between an AFM tip and a sample gives rise to oscillations of the cantilever at integral multiples (harmonics) of the fundamental resonance frequency. The higher order harmonics have long been recognized to hold invaluable information on short range interactions but their utilization has thus far been relatively limited due to theoretical and experimental complexities. In particular, existing approximations of the interaction force in terms of higher harmonic amplitudes generally require simultaneous measurements of multiple harmonics to achieve satisfactory accuracy. In the present letter we address the mathematical challenge and derive accurate, explicit formulae for both conservative and dissipative forces in terms of an arbitrary single harmonic. Additionally, we show that in frequency modulation-AFM (FM-AFM) each harmonic carries complete information on the force, obviating the need for multi-harmonic analysis. Finally, we show that higher harmonics may indeed be used to reconstruct short range forces more accurately than the fundamental harmonic when the oscillation amplitude is small compared with the interaction range.

  1. Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample

    NASA Astrophysics Data System (ADS)

    Guo, Shifeng; Zhu, Xiaoying; Jańczewski, Dominik; Lee, Serina Siew Chen; He, Tao; Teo, Serena Lay Ming; Vancso, G. Julius

    2016-09-01

    Protein charge at various pH and isoelectric point (pI) values is important in understanding protein function. However, often only trace amounts of unknown proteins are available and pI measurements cannot be obtained using conventional methods. Here, we show a method based on the atomic force microscope (AFM) to determine pI using minute quantities of proteins. The protein of interest is immobilized on AFM colloidal probes and the adhesion force of the protein is measured against a positively and a negatively charged substrate made by layer-by-layer deposition of polyelectrolytes. From the AFM force-distance curves, pI values with an estimated accuracy of ±0.25 were obtained for bovine serum albumin, myoglobin, fibrinogen and ribonuclease A over a range of 4.7-9.8. Using this method, we show that the pI of the ‘footprint’ of the temporary adhesive proteins secreted by the barnacle cyprid larvae of Amphibalanus amphitrite is in the range 9.6-9.7.

  2. Measuring protein isoelectric points by AFM-based force spectroscopy using trace amounts of sample.

    PubMed

    Guo, Shifeng; Zhu, Xiaoying; Jańczewski, Dominik; Lee, Serina Siew Chen; He, Tao; Teo, Serena Lay Ming; Vancso, G Julius

    2016-09-01

    Protein charge at various pH and isoelectric point (pI) values is important in understanding protein function. However, often only trace amounts of unknown proteins are available and pI measurements cannot be obtained using conventional methods. Here, we show a method based on the atomic force microscope (AFM) to determine pI using minute quantities of proteins. The protein of interest is immobilized on AFM colloidal probes and the adhesion force of the protein is measured against a positively and a negatively charged substrate made by layer-by-layer deposition of polyelectrolytes. From the AFM force-distance curves, pI values with an estimated accuracy of ±0.25 were obtained for bovine serum albumin, myoglobin, fibrinogen and ribonuclease A over a range of 4.7-9.8. Using this method, we show that the pI of the 'footprint' of the temporary adhesive proteins secreted by the barnacle cyprid larvae of Amphibalanus amphitrite is in the range 9.6-9.7.

  3. Single-cycle-PLL detection for real-time FM-AFM applications.

    PubMed

    Schlecker, Benedikt; Dukic, Maja; Erickson, Blake; Ortmanns, Maurits; Fantner, Georg; Anders, Jens

    2014-04-01

    In this paper we present a novel architecture for phase-locked loop (PLL) based high-speed demodulation of frequency-modulated (FM) atomic force microscopy (AFM) signals. In our approach, we use single-sideband (SSB) frequency upconversion to translate the AFM signal from the position sensitive detector to a fixed intermediate frequency (IF) of 10 MHz. In this way, we fully benefit from the excellent noise performance of PLL-based FM demodulators still avoiding the intrinsic bandwidth limitation of such systems. In addition, the upconversion to a fixed IF renders the PLL demodulator independent of the cantilever's resonance frequency, allowing the system to work with a large range of cantilever frequencies. To investigate if the additional noise introduced by the SSB upconverter degrades the system noise figure we present a model of the AM-to-FM noise conversion in PLLs incorporating a phase-frequency detector. Using this model, we can predict an upper corner frequency for the demodulation bandwidth above which the converted noise from the single-sideband upconverter becomes the dominant noise source and therefore begins to deteriorate the overall system performance. The approach is validated by both electrical and AFM measurements obtained with a PCB-based prototype implementing the proposed demodulator architecture.

  4. Bacterial adhesion to protein-coated surfaces: An AFM and QCM-D study

    NASA Astrophysics Data System (ADS)

    Strauss, Joshua; Liu, Yatao; Camesano, Terri A.

    2009-09-01

    Bacterial adhesion to biomaterials, mineral surfaces, or other industrial surfaces is strongly controlled by the way bacteria interact with protein layers or organic matter and other biomolecules that coat the materials. Despite this knowledge, many studies of bacterial adhesion are performed under clean conditions, instead of in the presence of proteins or organic molecules. We chose fetal bovine serum (FBS) as a model protein, and prepared FBS films on quartz crystals. The thickness of the FBS layer was characterized using atomic force microscopy (AFM) imaging under liquid and quartz crystal microbalance with dissipation (QCM-D). Next, we characterized how the model biomaterial surface would interact with the nocosomial pathogen Staphylococcus epidermidis. An AFM probe was coated with S. epidermidis cells and used to probe a gold slide that had been coated with FBS or another protein, fibronectin (FN). These experiments show that AFM and QCM-D can be used in complementary ways to study the complex interactions between bacteria, proteins, and surfaces.

  5. Topographical and electrical study of contact and intermittent contact mode InP AFM lithography

    NASA Astrophysics Data System (ADS)

    Tranvouez, E.; Budau, P.; Bremond, G.

    2006-01-01

    In order to fabricate nanoscale oxide patterns on an InP(001) surface, local anodization by atomic force microscopy (AFM) contact and intermittent contact modes has been performed. Contact mode results are similar to those obtained with the local anodization of silicon, and mainly limited by the effect of space charge that occurs during the oxide growth. The existence of this space charge associated with the poor dielectric quality of the obtained oxide has been verified by performing scanning capacitance microscopy (SCM) measurements. Results for oxidation using intermittent AFM contact mode associated with a modulated voltage are more specific. For a more than two decade variation of probe velocity (0.01-5 µm s-1), the AFM oxidation introduces no significant changes in the oxide pattern. Experiments on the influence of oxidation time give rise to two regimes. First, for times shorter than 100 ms, a high growth rate is found. Second, for oxidation times longer than 100 ms, we observe an oxide height saturation and a significant decrease of lateral growth rate. These results provide a way to easily control the oxide shape. The space charge neutralization in this mode has also been investigated by SCM. The interesting results for intermittent contact oxidation confirm the capability of this technique to modify a nanoscale InP surface.

  6. AFM and pulsed laser ablation methods for Cultural Heritage: application to archeometric analysis of stone artifacts

    NASA Astrophysics Data System (ADS)

    Barberio, M.; Veltri, S.; Stranges, F.; Bonanno, A.; Xu, F.; Antici, P.

    2015-09-01

    In this paper, we introduce the use of the atomic force microscope (AFM) and of the pulsed laser ablation as methods for morphological diagnostic with nanoscale precision of archeological artifacts and corrosive patina removal from stone artifacts. We test our methodology on stone artifacts extracted from the Church of Sotterra (located in Calabria, South Italy). The AFM microscopy was compared with different petrographic, chemical, optical and morphological analysis methods for identifying the textural characteristics, evaluating the state of preservation and formulating some hypotheses about the provenance and composition of the impurity patina located on the artifact surfaces. We demonstrate that with the nanometric precision obtained with AFM microscopy, it is possible to distinguish the different states of preservation, much better than using conventional petrographic methods. The surface's roughness is evaluated from very small artifact's fragments, reducing the coring at micrometric scale with a minimal damage to the artworks. After the diagnosis, we performed restoration tests using the pulsed laser ablation (PLA) method and compared it with the more common micro-sandblasting under dry conditions. We find that the PLA is highly effective for the removal of the surficial patina, with a control of a few hundreds of nanometers in the cleaning of surface, without introducing chemical or morphological damages to the artifacts. Moreover, PLA can be easily implemented in underwater conditions; this has the great advantage that stone and pottery artifacts for marine archeological sites do not need to be removed from the site.

  7. Fabrication of nanochannels with ladder nanostructure at the bottom using AFM nanoscratching method

    PubMed Central

    2014-01-01

    This letter presents a novel atomic force microscopy (AFM)-based nanomanufacturing method combining the tip scanning with the high-precision stage movement to fabricate nanochannels with ladder nanostructure at the bottom by continuous scanning with a fixed scan size. Different structures can be obtained according to the matching relation of the tip feeding velocity and the precision stage moving velocity. This relationship was first studied in detail to achieve nanochannels with different ladder nanostructures at the bottom. Machining experiments were then performed to fabricate nanochannels on an aluminum alloy surface to demonstrate the capability of this AFM-based fabrication method presented in this study. Results show that the feed value and the tip orientation in the removing action play important roles in this method which has a significant effect on the machined surfaces. Finally, the capacity of this method to fabricate a large-scale nanochannel was also demonstrated. This method has the potential to advance the existing AFM tip-based nanomanufacturing technique of the formation these complex structures by increasing the removal speed, simplifying the processing procedure and achieving the large-scale nanofabrication. PMID:24940171

  8. EEMD based pitch evaluation method for accurate grating measurement by AFM

    NASA Astrophysics Data System (ADS)

    Li, Changsheng; Yang, Shuming; Wang, Chenying; Jiang, Zhuangde

    2016-09-01

    The pitch measurement and AFM calibration precision are significantly influenced by the grating pitch evaluation method. This paper presents the ensemble empirical mode decomposition (EEMD) based pitch evaluation method to relieve the accuracy deterioration caused by high and low frequency components of scanning profile during pitch evaluation. The simulation analysis shows that the application of EEMD can improve the pitch accuracy of the FFT-FT algorithm. The pitch error is small when the iteration number of the FFT-FT algorithms was 8. The AFM measurement of the 500 nm-pitch one-dimensional grating shows that the EEMD based pitch evaluation method could improve the pitch precision, especially the grating line position precision, and greatly expand the applicability of the gravity center algorithm when particles and impression marks were distributed on the sample surface. The measurement indicates that the nonlinearity was stable, and the nonlinearity of x axis and forward scanning was much smaller than their counterpart. Finally, a detailed pitch measurement uncertainty evaluation model suitable for commercial AFMs was demonstrated and a pitch uncertainty in the sub-nanometer range was achieved. The pitch uncertainty was reduced about 10% by EEMD.

  9. On the determination of elastic moduli of cells by AFM based indentation

    PubMed Central

    Ding, Yue; Xu, Guang-Kui; Wang, Gang-Feng

    2017-01-01

    The atomic force microscopy (AFM) has been widely used to measure the mechanical properties of biological cells through indentations. In most of existing studies, the cell is supposed to be linear elastic within the small strain regime when analyzing the AFM indentation data. However, in experimental situations, the roles of large deformation and surface tension of cells should be taken into consideration. Here, we use the neo-Hookean model to describe the hyperelastic behavior of cells and investigate the influence of surface tension through finite element simulations. At large deformation, a correction factor, depending on the geometric ratio of indenter radius to cell radius, is introduced to modify the force-indent depth relation of classical Hertzian model. Moreover, when the indent depth is comparable with an intrinsic length defined as the ratio of surface tension to elastic modulus, the surface tension evidently affects the indentation response, indicating an overestimation of elastic modulus by the Hertzian model. The dimensionless-analysis-based theoretical predictions, which include both large deformation and surface tension, are in good agreement with our finite element simulation data. This study provides a novel method to more accurately measure the mechanical properties of biological cells and soft materials in AFM indentation experiments. PMID:28368053

  10. APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies

    NASA Astrophysics Data System (ADS)

    Shlyakhtenko, Luda S.; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S.; Lyubchenko, Yuri L.

    2015-10-01

    APOBEC3G (A3G) protein has antiviral activity against HIV and other pathogenic retroviruses. A3G has two domains: a catalytic C-terminal domain (CTD) that deaminates cytidine, and a N-terminal domain (NTD) that binds to ssDNA. Although abundant information exists about the biological activities of A3G protein, the interplay between sequence specific deaminase activity and A3G binding to ssDNA remains controversial. We used the topographic imaging and force spectroscopy modalities of Atomic Force Spectroscopy (AFM) to characterize the interaction of A3G protein with deaminase specific and nonspecific ssDNA substrates. AFM imaging demonstrated that A3G has elevated affinity for deaminase specific ssDNA than for nonspecific ssDNA. AFM force spectroscopy revealed two distinct binding modes by which A3G interacts with ssDNA. One mode requires sequence specificity, as demonstrated by stronger and more stable complexes with deaminase specific ssDNA than with nonspecific ssDNA. Overall these observations enforce prior studies suggesting that both domains of A3G contribute to the sequence specific binding of ssDNA.

  11. Investigation of surface changes of nanoparticles using TM-AFM phase imaging.

    PubMed

    Dong, Rong; Yu, Liya E

    2003-06-15

    Tapping-mode AFM (TM-AFM) phase imaging was utilized to characterize the surface changes of nanosize particles, in regard to the effects of different amounts of condensed water and organic coatings on particle surfaces. Model nanoparticles were continuously examined under various relative humidity (RH) levels by concurrently obtaining both topographic and phase images. The condensed water appeared to soften particle surfaces and to increase tip-sample attractive interaction over relatively stiff surfaces, which were shown with dark phase contrasts and negative phase shift values in phase images. Under high RH, a massive amount of water gave the particles a droplet-like surface, which reversed the original negative phase shifts to positive values with bright contrasts. Glutaric-acid coatings provided a compliant surface with high viscosity resulting in a dark phase contrast, whereas water droplets containing relatively low viscosity gave a bright phase contrast and positive phase shift. Overall, our results show that it is essential to describe the physical properties of a sample surface as solid, soft, or droplet-like material in order to derive a meaningful understanding of the surface changes of nanosize particles based on TM-AFM phase images. In contrast to other phase imaging studies, this work clearly correlates continuous surface changes with phase images, demonstrating a promising approach to characterize environmental nanoparticles.

  12. A novel dog-bone oscillating AFM probe with thermal actuation and piezoresistive detection.

    PubMed

    Xiong, Zhuang; Mairiaux, Estelle; Walter, Benjamin; Faucher, Marc; Buchaillot, Lionel; Legrand, Bernard

    2014-10-31

    In order to effectively increase the resonance frequency and the quality factor of atomic force microscope (AFM) probes, a novel oscillating probe based on a dog-bone shaped MEMS resonator was conceived, designed, fabricated and evaluated. The novel probe with 400 μm in length, 100 μm in width and 5 μm in thickness was enabled to feature MHz resonance frequencies with integrated thermal actuation and piezoresistive detection. Standard silicon micromachining was employed. Both electrical and optical measurements were carried out in air. The resonance frequency and the quality factor of the novel probe were measured to be 5.4 MHz and 4000 respectively, which are much higher than those (about several hundreds of kHz) of commonly used cantilever probes. The probe was mounted onto a commercial AFM set-up through a dedicated probe-holder and circuit board. Topographic images of patterned resist samples were obtained. It is expected that the resonance frequency and the measurement bandwidth of such probes will be further increased by a proper downscaling, thus leading to a significant increase in the scanning speed capability of AFM instruments.

  13. A Novel Dog-Bone Oscillating AFM Probe with Thermal Actuation and Piezoresistive Detection †

    PubMed Central

    Xiong, Zhuang; Mairiaux, Estelle; Walter, Benjamin; Faucher, Marc; Buchaillot, Lionel; Legrand, Bernard

    2014-01-01

    In order to effectively increase the resonance frequency and the quality factor of atomic force microscope (AFM) probes, a novel oscillating probe based on a dog-bone shaped MEMS resonator was conceived, designed, fabricated and evaluated. The novel probe with 400 μm in length, 100 μm in width and 5 μm in thickness was enabled to feature MHz resonance frequencies with integrated thermal actuation and piezoresistive detection. Standard silicon micromachining was employed. Both electrical and optical measurements were carried out in air. The resonance frequency and the quality factor of the novel probe were measured to be 5.4 MHz and 4000 respectively, which are much higher than those (about several hundreds of kHz) of commonly used cantilever probes. The probe was mounted onto a commercial AFM set-up through a dedicated probe-holder and circuit board. Topographic images of patterned resist samples were obtained. It is expected that the resonance frequency and the measurement bandwidth of such probes will be further increased by a proper downscaling, thus leading to a significant increase in the scanning speed capability of AFM instruments. PMID:25365463

  14. A study of water droplet between an AFM tip and a substrate using dissipative particle dynamics

    NASA Astrophysics Data System (ADS)

    Pal, Souvik; Lan, Chuanjin; Li, Zhen; Hirleman, E. Daniel; Ma, Yanbao

    2014-11-01

    Formation of a water droplet between a sharp AFM tip and a substrate due to capillary condensation affects the tip-substrate interaction. As a consequence, AFM measurements lose precision and often produce incorrect sample topology. Understanding the physics of liquid bridges is also important in the field of Dip-pen nanolithography (DPN). Significant research is being carried out to understand the mechanics of the formation of the liquid bridge and its dependence of surface properties, ambient conditions etc. The in-between length scale, i.e., mesoscale (~100 nm) associated with this phenomenon presents a steep challenge for experimental measurements. In addition, molecular dynamics (MD) can be computationally prohibitive to model the entire system, especially over microseconds to seconds. Theoretical analysis using Young Laplace equation has so far provided some qualitative insights only. We study this system using Dissipative Particle Dynamics (DPD) which is a simulation technique suitable for describing mesoscopic hydrodynamic behavior of fluids. In this work, we carry out simulations to improve understanding of the process of formation of the meniscus, the mechanics of manipulation and control of its shape, and better estimation of capillary forces. The knowledge gained through our study will help in correcting the AFM measurements affected by capillary condensation. Moreover, it will improve understanding of more accurate droplet manipulation in DPN.

  15. Fracture Growth Testing of Titanium 6AL-4V in AF-M315E

    NASA Technical Reports Server (NTRS)

    Sampson, Jeffrey W.; Martinez, Jonathan; McLean, Christopher

    2015-01-01

    The Green Propellant Infusion Mission (GPIM) will demonstrate the performance of AF-M315E monopropellant in orbit. Flight certification requires a safe-life analysis of the titanium alloy fuel tank to ensure inherent flaws will not cause failure during the design life. Material property inputs for this analysis require testing to determine the stress intensity factor for environmentally-assisted cracking (K (sub EAC)) of Ti 6Al-4V in combination with the AF-M315E monopropellant. Testing of single-edge notched specimens SE(B) representing the bulk tank membrane and weld material were performed in accordance with ASTM E1681. Specimens with fatigue pre-cracks were loaded into test fixtures so that the crack tips were exposed to the monopropellant at 50 degrees Centigrade for a duration of 1,000 hours. Specimens that did not fail during exposure were opened to inspect the crack surfaces for evidence of crack growth. The threshold stress intensity value, KEAC, is the highest applied stress intensity that produced neither a failure of the specimen during the exposure nor showed evidence of crack growth. The threshold stress intensity factor of the Ti 6Al-4V forged tank material when exposed to AF-M315E monopropellant was found to be at least 22.0 kilopounds per square inch. The stress intensity factor of the weld material was at least 31.3 kilopounds per square inch.

  16. Advantages and limitations of OM, SEM, TEM and AFM in the study of ancient decorated pottery

    NASA Astrophysics Data System (ADS)

    Arenas-Alatorre, J.; Silva-Velazquez, Y.; Alva Medina, A.; Rivera, M.

    2010-03-01

    This paper presents results from the study of two fragments of pre-Hispanic pottery, decorated with red pigment, using Optical Microscopy (OM), Scanning Electron Microscopy (SEM), High Resolution Transmission Electron Microscopy (HRTEM), Atomic Force Microscopy (AFM) and Magnetic Force Microscopy (MFM). Capabilities and limitations of these techniques in the analysis of archaeological material are highlighted with special emphasis on TEM, AFM and MFM due to their contribution in the study of the pigment layer at micro and nano scale. The analyzed samples come from the archaeological sites of El Tajin and Xochicalco, both in Mexico. Results of conventional TEM and HRTEM analysis of the red pigment showed nanometric Fe2O3 particles in both samples but different particle shape and size distributions: specimen from El Tajin presented irregular particles between 50-100 nm while that from Xochicalco exhibited semispherical shapes in the 3-25 nm range. AFM images showed the topography of the pigments, which are related to the texture of their surface and thus to the production process. Finally, MFM showed different contrast regions suggesting the presence of ferromagnetic elements forming clusters and domain orientations on the color layer.

  17. Robust deposition of lambda DNA on mica for imaging by AFM in air.

    PubMed

    Cervantes, Nancy Anabel Gerling; Gutiérrez-Medina, Braulio

    2014-01-01

    Long DNA molecules remain difficult to image by atomic force microscopy (AFM) because of their tendency to entanglement and spontaneous formation of networks. We present a comparison of two different DNA deposition methods operating at room temperature and humidity conditions, aimed at reproducible imaging of isolated and relaxed λ DNA conformations by AFM in air. We first demonstrate that a standard deposition procedure, consisting in adsorption of DNA in the presence of divalent cations followed by washing and air-drying steps, yields a coexistence of different types of λ DNA networks with a only a few isolated DNA chains. In contrast, deposition using a spin-coating-based technique results in reproducible coverage of a significant fraction of the substrate area by isolated and relaxed λ DNA molecules, with the added benefit of a reduction in the effect of a residual layer that normally embeds DNA strands and leads to an apparent DNA height closer to the expected value. Furthermore, we show that deposition by spin-coating is also well-suited to visualize DNA-protein complexes. These results indicate that spin-coating is a simple, powerful alternative for reproducible sample preparation for AFM imaging.

  18. Multilayer silicon rich oxy-nitride films characterization by SIMS, VASE and AFM

    NASA Astrophysics Data System (ADS)

    Barozzi, M.; Vanzetti, L.; Iacob, E.; Bersani, M.; Anderle, M.; Pucker, G.; Kompocholis, C.; Ghulinyan, M.; Bellutti, P.

    2008-03-01

    In this work secondary ion mass spectrometry (SIMS), variable angle spectroscopy ellipsometry (VASE) and atomic force microscopy (AFM) are used to investigate the structure, composition and morphology of multilayer SRON films. Three/four SRON sequential layers were deposited on silicon wafers by PECVD and silicon, nitrogen and oxygen content was varied by changing the N2O/SiH4 ratio. The total thickness of the resulting SRON stack is about 50nm. SIMS analyses of NCs+, OCs+, SiCs+, in MCs+ methodology are performed by a Cameca SC-ultra instrument. Depth profiles are obtained at 500eV of primary beam impact energy with sample rotation. An approximate method to obtain silicon concentration is used. Total layer thickness are obtained from both SIMS and VASE measurements. In addition, we compare the thickness of the single layers obtained from VASE with the SIMS depth profiles. A detailed analysis of films morphology is obtained by AFM. The SRON stack is sputtered by SIMS until a certain layer is exposed, which is then analyzed by AFM. The sputtered layers are then etched in HF solution to better resolve the exposed nano-crystals.

  19. On the determination of elastic moduli of cells by AFM based indentation.

    PubMed

    Ding, Yue; Xu, Guang-Kui; Wang, Gang-Feng

    2017-04-03

    The atomic force microscopy (AFM) has been widely used to measure the mechanical properties of biological cells through indentations. In most of existing studies, the cell is supposed to be linear elastic within the small strain regime when analyzing the AFM indentation data. However, in experimental situations, the roles of large deformation and surface tension of cells should be taken into consideration. Here, we use the neo-Hookean model to describe the hyperelastic behavior of cells and investigate the influence of surface tension through finite element simulations. At large deformation, a correction factor, depending on the geometric ratio of indenter radius to cell radius, is introduced to modify the force-indent depth relation of classical Hertzian model. Moreover, when the indent depth is comparable with an intrinsic length defined as the ratio of surface tension to elastic modulus, the surface tension evidently affects the indentation response, indicating an overestimation of elastic modulus by the Hertzian model. The dimensionless-analysis-based theoretical predictions, which include both large deformation and surface tension, are in good agreement with our finite element simulation data. This study provides a novel method to more accurately measure the mechanical properties of biological cells and soft materials in AFM indentation experiments.

  20. Non--Cubic Symmetry of the Electronic Response in AFM Late Transition--Metal Oxides.

    NASA Astrophysics Data System (ADS)

    Posternak, M.; Baldereschi, A.; Massidda, S.; Resta, R.

    1998-03-01

    The late transition--metal monoxides (MnO, FeO, CoO, NiO) have the rocksalt structure in their paramagnetic phase, while below the Neel temperature a weak structural distortion accompanies an AFM ordering of type II. Therefore, it is generally assumed that most nonmagnetic (i.e. spin--integrated) crystalline properties are essentially cubic: we give here convincing evidence of the contrary. We focus on the half--filled d shell oxide MnO as the most suitable case study, on which we perform accurate ab--initio, all--electron calculations, within different one--particle schemes. In order to study the symmetry lowering due to AFM ordering, we assume an ideal cubic geometry throughout. The calculated TO frequencies and Born effective charge tensor do not have cubic symmetry. The standard LSD severely exaggerates the deviations from cubic symmetry, confirming its unreliability for calculating properties of insulating AFM oxides, while a model self--energy correction scheme(S. Massidda et al.), Phys. Rev. B 55, 13494 (1997). reduces considerably the anisotropy. We also explain the origin and the magnitude of this effect in terms of the mixed charge--transfer/Mott--Hubbard character of MnO.

  1. Brain tumor classification using AFM in combination with data mining techniques.

    PubMed

    Huml, Marlene; Silye, René; Zauner, Gerald; Hutterer, Stephan; Schilcher, Kurt

    2013-01-01

    Although classification of astrocytic tumors is standardized by the WHO grading system, which is mainly based on microscopy-derived, histomorphological features, there is great interobserver variability. The main causes are thought to be the complexity of morphological details varying from tumor to tumor and from patient to patient, variations in the technical histopathological procedures like staining protocols, and finally the individual experience of the diagnosing pathologist. Thus, to raise astrocytoma grading to a more objective standard, this paper proposes a methodology based on atomic force microscopy (AFM) derived images made from histopathological samples in combination with data mining techniques. By comparing AFM images with corresponding light microscopy images of the same area, the progressive formation of cavities due to cell necrosis was identified as a typical morphological marker for a computer-assisted analysis. Using genetic programming as a tool for feature analysis, a best model was created that achieved 94.74% classification accuracy in distinguishing grade II tumors from grade IV ones. While utilizing modern image analysis techniques, AFM may become an important tool in astrocytic tumor diagnosis. By this way patients suffering from grade II tumors are identified unambiguously, having a less risk for malignant transformation. They would benefit from early adjuvant therapies.

  2. Complete active space second order perturbation theory (CASPT2) study of N(2D) + H2O reaction paths on D1 and D0 potential energy surfaces: Direct and roaming pathways

    NASA Astrophysics Data System (ADS)

    Isegawa, Miho; Liu, Fengyi; Maeda, Satoshi; Morokuma, Keiji

    2014-10-01

    We report reaction paths starting from N(2D) + H2O for doublet spin states, D0 and D1. The potential energy surfaces are explored in an automated fashion using the global reaction route mapping strategy. The critical points and reaction paths have been fully optimized at the complete active space second order perturbation theory level taking all valence electrons in the active space. In addition to direct dissociation pathways that would be dominant, three roaming processes, two roaming dissociation, and one roaming isomerization: (1) H2ON → H-O(H)N → H-HON → NO(2Π) + H2, (2) cis-HNOH → HNO-H → H-HNO → NO + H2, (3) H2NO → H-HNO → HNO-H → trans-HNOH, are confirmed on the D0 surface.

  3. Protocol for a multicentre study to assess feasibility, acceptability, effectiveness and direct costs of TRIumPH (Treatment and Recovery In PsycHosis): integrated care pathway for psychosis

    PubMed Central

    Rathod, Shanaya; Garner, Christie; Griffiths, Alison; Dimitrov, Borislav D; Newman-Taylor, Katherine; Woodfine, Chris; Hansen, Lars; Tabraham, Paul; Ward, Karen; Asher, Carolyn; Phiri, Peter; Naeem, Farooq; North, Pippa; Munshi, Tariq; Kingdon, David

    2016-01-01

    Introduction Duration of untreated psychosis (time between the onset of symptoms and start of treatment) is considered the strongest predictor of symptom severity and outcome. Integrated care pathways that prescribe timeframes around access and interventions can potentially improve quality of care. Methods and analysis A multicentre mixed methods study to assess feasibility, acceptability, effectiveness and analysis of direct costs of an integrated care pathway for psychosis. A pragmatic, non-randomised, controlled trial design is used to compare the impact of Treatment and Recovery In PsycHosis (TRIumPH; Intervention) by comparison between NHS organisations that adopt TRIumPH and those that continue with care as usual (Control). Quantitative and qualitative methods will be used. We will use routinely collected quantitative data and study-specific questionnaires and focus groups to compare service user outcomes, satisfaction and adherence to intervention between sites that adopt TRIumPH versus sites that continue with usual care pathways. Setting 4 UK Mental health organisations. Two will implement TRIumPH whereas two will continue care as usual. Participants Staff, carers, individuals accepted to early intervention in psychosis teams in participating organisations for the study period. Intervention TRIumPH—Integrated Care Pathway for psychosis that has a holistic approach and prescribes time frames against interventions; developed using intelligence from data; co-produced with patients, carers, clinicians and other stakeholders. Outcomes Feasibility will be assessed through adherence to the process measures. Satisfaction and acceptability will be assessed using questionnaires and focus groups. Effectiveness will be assessed through data collection and evaluation of patient outcomes, including clinical, functional and recovery outcomes, physical health, acute care use. Outcome measures will be assessed at baseline, 12 and 24 months to measure whether there is

  4. AFM/MFM hybrid nanocharacterization of martensitic transformation and degradation for Fe-Pd shape memory alloy

    NASA Astrophysics Data System (ADS)

    Suzuki, Takayuki; Nagatani, Kohei; Hirano, Kazumi; Teramoto, Tokuo; Taya, Minoru

    2003-07-01

    Martensitic transformation and degradation characteristics for Fe-Pd ferromagnetic shape memory alloy were investigated by the developed AFM (Atomic Force Microscope)/MFM (Magnetic Force Microscope) hybrid nano-characterization technique. In AFM martensitic transformation was detected by the changes of surface topography of martensite plates. In MFM martensitic transformation was detected by the changes of magnetic domain structures. This technique has an advantage that martensitic transformation characteristics such as martensitic transformation temperature and reverse transformation temperature can be measured at microscopic and nanoscopic small area. Degradation characteristics of martensitic transformation under cyclic loading were also detected by the changes of AFM and MFM images. In AFM images surface topography of martensite plates became flat and in MFM images the morphology of magnetic domain structures became unfocused under cyclic loading. Then it was found that the hybrid nano-characterization was very high sensitive technique to evaluate degradation for Fe-Pd ferromagnetic shape memory alloy.

  5. An NC-AFM and KPFM study of the adsorption of a triphenylene derivative on KBr(001)

    PubMed Central

    Hinaut, Antoine; Pujol, Adeline; Chaumeton, Florian; Martrou, David; Gourdon, André

    2012-01-01

    Summary The adsorption on KBr(001) of a specially designed molecule, consisting of a flat aromatic triphenylene core equipped with six flexible propyl chains ending with polar cyano groups, is investigated by using atomic force microscopy in the noncontact mode (NC-AFM) coupled to Kelvin probe force microscopy (KPFM) in ultrahigh vacuum at room temperature. Two types of monolayers are identified, one in which the molecules lie flat on the surface (MLh) and another in which they stand approximately upright (MLv). The Kelvin voltage on these two structures is negatively shifted relative to that of the clean KBr surface, revealing the presence of surface dipoles with a component pointing along the normal to the surface. These findings are interpreted with the help of numerical simulations. It is shown that the surface–molecule interaction is dominated by the electrostatic interaction of the cyano groups with the K+ ions of the substrate. The molecule is strongly adsorbed in the MLh structure with an adsorption energy of 1.8 eV. In the MLv layer, the molecules form π-stacked rows aligned along the polar directions of the KBr surface. In these rows, the molecules are less strongly bound to the substrate, but the structure is stabilized by the strong intermolecular interaction due to π-stacking. PMID:22496995

  6. Proliferation and Glia-Directed Differentiation of Neural Stem Cells in the Subventricular Zone of the Lateral Ventricle and the Migratory Pathway to the Lesions after Cortical Devascularization of Adult Rats

    PubMed Central

    Wan, Feng; Bai, Hua-Jing; Liu, Jun-Qi; Tian, Mo; Wang, Yong-Xue; Niu, Xin; Si, Yin-Chu

    2016-01-01

    We investigated the effects of cortical devascularization on the proliferation, differentiation, and migration of neural stem cells (NSCs) in the subventricular zone (SVZ) of the lateral ventricle of adult rats. 60 adult male Wistar rats were randomly divided into control group and devascularized group. At 15 and 30 days after cerebral cortices were devascularized, rats were euthanized and immunohistochemical analysis was performed. The number of PCNA-, Vimentin-, and GFAP-positive cells in the bilateral SVZ of the lateral wall and the superior wall of the lateral ventricles of 15- and 30-day devascularized groups increased significantly compared with the control group (P < 0.05 and P < 0.01). The area density of PCNA-, Vimentin-, and GFAP-positive cells in cortical lesions of 15- and 30-day devascularized groups increased significantly compared with the control group (P < 0.05 and P < 0.01). PCNA-, GFAP-, and Vimentin-positive cells in the SVZ migrated through the rostral migratory stream (RMS), and PCNA-, GFAP-, and Vimentin-positive cells from both the ipsilateral and contralateral dorsolateral SVZ (dl-SVZ) migrated into the corpus callosum (CC) and accumulated, forming a migratory pathway within the CC to the lesioned site. Our study suggested that cortical devascularization induced proliferation, glia-directed differentiation, and migration of NSCs from the SVZ through the RMS or directly to the corpus callosum and finally migrating radially to cortical lesions. This may play a significant role in neural repair. PMID:27294116

  7. Versatile method for AFM-tip functionalization with biomolecules: fishing a ligand by means of an in situ click reaction

    NASA Astrophysics Data System (ADS)

    Kumar, Rakesh; Ramakrishna, Shivaprakash N.; Naik, Vikrant V.; Chu, Zonglin; Drew, Michael E.; Spencer, Nicholas D.; Yamakoshi, Yoko

    2015-04-01

    A facile and universal method for the functionalization of an AFM tip has been developed for chemical force spectroscopy (CFS) studies of intermolecular interactions of biomolecules. A click reaction between tripod-acetylene and an azide-linker-ligand molecule was successfully carried out on the AFM tip surface and used for the CFS study of ligand-receptor interactions.A facile and universal method for the functionalization of an AFM tip has been developed for chemical force spectroscopy (CFS) studies of intermolecular interactions of biomolecules. A click reaction between tripod-acetylene and an azide-linker-ligand molecule was successfully carried out on the AFM tip surface and used for the CFS study of ligand-receptor interactions. Electronic supplementary information (ESI) available: Experimental details with synthesis and characterization of compounds. Procedures for modifications of Au surfaces and AFM tips. AFM images and full PM-IRRAS spectra of modified surfaces. Detailed procedure for QCM measurement. A table showing ligand-receptor interaction probability. NMR, IR and MS charts. See DOI: 10.1039/c5nr01495f

  8. Applications of AFM in semiconductor R&D and manufacturing at 45 nm technology node and beyond

    NASA Astrophysics Data System (ADS)

    Lee, Moon-Keun; Shin, Minjung; Bao, Tianming; Song, Chul-Gi; Dawson, Dean; Ihm, Dong-Chul; Ukraintsev, Vladimir

    2009-03-01

    Continuing demand for high performance microelectronic products propelled integrated circuit technology into 45 nm node and beyond. The shrinking device feature geometry created unprecedented challenges for dimension metrology in semiconductor manufacturing and research and development. Automated atomic force microscope (AFM) has been used to meet the challenge and characterize narrower lines, trenches and holes at 45nm technology node and beyond. AFM is indispensable metrology techniques capable of non-destructive full three-dimensional imaging, surface morphology characterization and accurate critical dimension (CD) measurements. While all available dimensional metrology techniques approach their limits, AFM continues to provide reliable information for development and control of processes in memory, logic, photomask, image sensor and data storage manufacturing. In this paper we review up-todate applications of automated AFM in every mentioned above semiconductor industry sector. To demonstrate benefits of AFM at 45 nm node and beyond we compare capability of automated AFM with established in-line and off-line metrologies like critical dimension scanning electron microscopy (CDSEM), optical scatterometry (OCD) and transmission electronic microscopy (TEM).

  9. In vivo characterization of protein uptake by yeast cell envelope: single cell AFM imaging and μ-tip-enhanced Raman scattering study.

    PubMed

    Naumenko, Denys; Snitka, Valentinas; Serviene, Elena; Bruzaite, Ingrida; Snopok, Boris

    2013-09-21

    Direct detection of biological transformations of single living cells in vivo has been performed by the advanced combination of local topographic imaging by Atomic Force Microscopy (AFM) and label-free sub-surface chemical characterization using new μ-Tip-Enhanced Raman Spectroscopy (μ-TERS). The enhancing mechanism for μ-TERS tips with micrometre range radius differs significantly to that of the conventional tapered structures terminated by a sharp apex and conditioned by the effects of propagating instead of localizing surface plasmon resonance phenomena. Sub-wavelength light confinement in the form of a nonradiative evanescent wave near the tip surface with penetration depth in the sub-micrometre range opens the way for monitoring of subsurface processes near or within the cell wall, inaccessible by other methods. The efficiency of the approach has been demonstrated by the analysis of the cell envelope of genetically modified (by glucose dehydrogenase (GDH) gene bearing Kluyveromyces lactis toxin signal sequence) yeast cells enriched by GDH protein. The presence of trans-membrane fragments in GDH together with the tendency to form active dimers and tetramers causes the accumulation of the proteins within the periplasmic space. These results demonstrate that the advanced combination of AFM imaging and subsurface chemical characterization by the novel μ-TERS technique provides a new analytical tool for the investigation of single living cells in vivo.

  10. 3-Dimensional atomic scale structure of the ionic liquid-graphite interface elucidated by AM-AFM and quantum chemical simulations.

    PubMed

    Page, Alister J; Elbourne, Aaron; Stefanovic, Ryan; Addicoat, Matthew A; Warr, Gregory G; Voïtchovsky, Kislon; Atkin, Rob

    2014-07-21

    In situ amplitude modulated atomic force microscopy (AM-AFM) and quantum chemical simulations are used to resolve the structure of the highly ordered pyrolytic graphite (HOPG)-bulk propylammonium nitrate (PAN) interface with resolution comparable with that achieved for frozen ionic liquid (IL) monolayers using STM. This is the first time that (a) molecular resolution images of bulk IL-solid interfaces have been achieved, (b) the lateral structure of the IL graphite interface has been imaged for any IL, (c) AM-AFM has elucidated molecular level structure immersed in a viscous liquid and (d) it has been demonstrated that the IL structure at solid surfaces is a consequence of both thermodynamic and kinetic effects. The lateral structure of the PAN-graphite interface is highly ordered and consists of remarkably well-defined domains of a rhomboidal superstructure composed of propylammonium cations preferentially aligned along two of the three directions in the underlying graphite lattice. The nanostructure is primarily determined by the cation. Van der Waals interactions between the propylammonium chains and the surface mean that the cation is enriched in the surface layer, and is much less mobile than the anion. The presence of a heterogeneous lateral structure at an ionic liquid-solid interface has wide ranging ramifications for ionic liquid applications, including lubrication, capacitive charge storage and electrodeposition.

  11. Studying Chemical Reactions, One Bond at a Time, with Single Molecule AFM Techniques

    NASA Astrophysics Data System (ADS)

    Fernandez, Julio M.

    2008-03-01

    atomic force microscopy (AFM) techniques, as shown here, can probe dynamic rearrangements within an enzyme's active site which cannot be resolved with any other current structural biological technique. Furthermore, our work at the single bond level directly demonstrates that thiol/disulfide exchange in proteins is a force-dependent chemical reaction. Our findings suggest that mechanical force plays a role in disulfide reduction in vivo, a property which has never been explored by traditional biochemistry. 1.-Wiita, A.P., Ainavarapu, S.R.K., Huang, H.H. and Julio M. Fernandez (2006) Force-dependent chemical kinetics of disulfide bond reduction observed with single molecule techniques. Proc Natl Acad Sci U S A. 103(19):7222-7 2.-Wiita, A.P., Perez-Jimenez, R., Walther, K.A., Gräter, F. Berne, B.J., Holmgren, A., Sanchez-Ruiz, J.M., and Fernandez, J.M. (2007) Probing the chemistry of thioredoxin catalysis with force. Nature, 450:124-7.

  12. Tolerogenic response: allorecognition pathways.

    PubMed

    Caballero, A; Fernandez, N; Lavado, R; Bravo, M J; Miranda, J M; Alonso, A

    2006-12-01

    The induction of immune tolerance in transplant recipients has been sought for many years but only a fundamental understanding of the immunological mechanisms underlying graft rejection will allow manipulation of the anti-graft immune response. In general, acute rejection is better understood and treated than chronic rejection, as they occur through partially different mechanisms. It is now generally accepted that recognition of same-species, non-self antigens (allorecognition) occurs through at least two different mechanisms, the direct and indirect pathways. In the direct pathway, donor MHC molecules on the surface of donor antigen-presenting cells (APCs) are recognised directly by the recipient's T cells. This mechanism is so immediate that it seems to be primarily involved in acute graft rejection. Since APCs of donor origin are depleted with time a second mechanism, the indirect pathway, takes over to cause chronic rejection, in which foreign MHC molecules are internalised, partially digested and presented as peptides to recipient T cells. Nonetheless, a number of studies are only fully understood when a third proposed allorecognition mechanism is taken into account. This is the semi-indirect pathway, as discussed in this short report.

  13. Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers EVIDENCE FOR A DIRECT PATHWAY BETWEEN THE 4′-AMINOPYRIMIDINE N1′ ATOMS

    SciTech Connect

    Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Mossad, Madouna; Tittmann, Kai; Furey, William; Jordan, Frank

    2010-11-03

    Kinetic, spectroscopic, and structural analysis tested the hypothesis that a chain of residues connecting the 4{prime}-aminopyrimidine N1{prime} atoms of thiamin diphosphates (ThDPs) in the two active centers of the Escherichia coli pyruvate dehydrogenase complex E1 component provides a signal transduction pathway. Substitution of the three acidic residues (Glu{sup 571}, Glu{sup 235}, and Glu{sup 237}) and Arg{sup 606} resulted in impaired binding of the second ThDP, once the first active center was filled, suggesting a pathway for communication between the two ThDPs. (1) Steady-state kinetic and fluorescence quenching studies revealed that upon E571A, E235A, E237A, and R606A substitutions, ThDP binding in the second active center was affected. (2) Analysis of the kinetics of thiazolium C2 hydrogen/deuterium exchange of enzyme-bound ThDP suggests half-of-the-sites reactivity for the E1 component, with fast (activated site) and slow exchanging sites (dormant site). The E235A and E571A variants gave no evidence for the slow exchanging site, indicating that only one of two active sites is filled with ThDP. (3) Titration of the E235A and E237A variants with methyl acetylphosphonate monitored by circular dichroism suggested that only half of the active sites were filled with a covalent predecarboxylation intermediate analog. (4) Crystal structures of E235A and E571A in complex with ThDP revealed the structural basis for the spectroscopic and kinetic observations and showed that either substitution affects cofactor binding, despite the fact that Glu{sup 235} makes no direct contact with the cofactor. The role of the conserved Glu{sup 571} residue in both catalysis and cofactor orientation is revealed by the combined results for the first time.

  14. Characterization of Pebax angioplasty balloon surfaces with AFM, SEM, TEM, and SAXS.

    PubMed

    Warner, Jacob A; Forsyth, Bruce; Zhou, Fang; Myers, Jason; Frethem, Chris; Haugstad, Greg

    2016-04-01

    In the medical device industry, angioplasty balloons have been widely used in the less invasive treatment of heart disease by expanding and relieving clogged structures in various arterial segments. However, new applications using thin coatings on the balloon surface have been explored to enhance therapeutic value in the delivery of pharmaceuticals (drug-elution) or control thermal energy output (RF ablation). In this study, angioplasty balloon materials comprised of poly(ether-block-amide) (Pebax) were investigated via atomic force microscopy (AFM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and small-angle X-ray scattering (SAXS) to characterize physical properties at the balloon surface that may affect coating adhesion. The soft segment of this Pebax 1074 material is polyethylene oxide (PEO) and the hard segment is nylon-12. The morphology of the hard segments of this block co-polymer are found via AFM stiffness measurements to be (40 ± 20) nm by (300 ± 150) nm and are oriented parallel to the surface of the balloon. SAXS measurements found the lamellar spacing to be (18.5 ± 0.5) nm, and demonstrate a preferential orientation in agreement with TEM and AFM measurements. Fixation of this balloon in resin, followed by cryo-sectioning is shown to provide a novel manner in which to investigate surface characteristics on the balloon such as material or coating thickness as well as uniformity in comparison to the bulk structure. These outputs were deemed critical to improve overall balloon processing such as molding and surface treatment options for robust designs toward better procedural outcomes targeting new therapeutic areas.

  15. Unspecific membrane protein-lipid recognition: combination of AFM imaging, force spectroscopy, DSC and FRET measurements.

    PubMed

    Borrell, Jordi H; Montero, M Teresa; Morros, Antoni; Domènech, Òscar

    2015-11-01

    In this work, we will describe in quantitative terms the unspecific recognition between lactose permease (LacY) of Escherichia coli, a polytopic model membrane protein, and one of the main components of the inner membrane of this bacterium. Supported lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol) in the presence of Ca(2+) display lateral phase segregation that can be distinguished by atomic force microscopy (AFM) as well as force spectroscopy. LacY shows preference for fluid (Lα) phases when it is reconstituted in POPE : POPG (3:1, mol/mol) proteoliposomes at a lipid-to-protein ratio of 40. When the lipid-to-protein ratio is decreased down to 0.5, two domains can be distinguished by AFM. While the upper domain is formed by self-segregated units of LacY, the lower domain is constituted only by phospholipids in gel (Lβ) phase. On the one hand, classical differential scanning calorimetry (DSC) measurements evidenced the segregation of a population of phospholipids and point to the existence of a boundary region at the lipid-protein interface. On the other hand, Förster Resonance Energy Transfer (FRET) measurements in solution evidenced that POPE is selectively recognized by LacY. A binary pseudophase diagram of POPE : POPG built from AFM observations enables to calculate the composition of the fluid phase where LacY is inserted. These results are consistent with a model where POPE constitutes the main component of the lipid-LacY interface segregated from the fluid bulk phase where POPG predominates.

  16. microRNA-130a Promotes Human Keratinocyte Viability and Migration and Inhibits Apoptosis Through Direct Regulation of STK40-Mediated NF-κB Pathway and Indirect Regulation of SOX9-Meditated JNK/MAPK Pathway: A Potential Role in Psoriasis.

    PubMed

    Xiong, Ying; Chen, Hongxiao; Liu, Liqian; Lu, Leihong; Wang, Zongshan; Tian, Fujun; Zhao, Yongliang

    2017-03-01

    Psoriasis is a chronic inflammatory skin disorder. The aim of this study was to determine a potential role of microRNA (miR)-130a in psoriasis, and underlying mechanism. Expression levels of miR-130a in psoriasis specimens and normal skin tissues were analyzed. MiR-130a mimic, inhibitor, miR-control, small interfering RNA (siRNA) specific serine/threonine kinase 40 (STK40), or sex-determining region Y chromosome-box 9 (SOX9) were transfected to human keratinocyte HaCaT cells, respectively. After transfection, the cell viability, apoptosis, and migration were determined. Luciferase reporter assay, quantitative reverse transcription-polymerase chain reaction, and western blot were performed to explore whether STK40 was a target of miR-130a. The effects of aberrant expressions of miR-130a, STK40, or SOX9 on key proteins of NF-κB and c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) pathway were assessed. The miR-130a levels were significantly higher in patients with psoriasis compared to the healthy controls (p < 0.01). Overexpressing miR-130a strikingly promoted HaCaT cell viability and migration and inhibited apoptosis (p < 0.01 or p < 0.05). We confirmed that STK40 was a direct target of miR-130a, and STK40 was involved in miR-130a-induced cell functions. Overexpressing miR-130a significantly upregulated NF-κB p65, SOX9, p-c-Jun, p-JNK, and p-p38MAPK proteins and silencing miR-130a downregulated them. In addition, silencing STK40 alleviated the effects of anti-miR-130a on SOX9 expression. Furthermore, silencing SOX9 also decreased levels of p-c-Jun, p-JNK, and p-p38MAPK proteins. MiR-130a regulates human keratinocyte HaCaT viability, migration and apoptosis might be by direct regulation of STK40-mediated NF-κB pathway and by indirect regulation of SOX9-mediated downstream JNK/MAPK signaling pathway.

  17. BOREAS AFM-3 NCAR Electra 1994 Aircraft Flux and Moving Window Data

    NASA Technical Reports Server (NTRS)

    Lenschow, Donald H.; Hall, Forrest G. (Editor); Knapp, David E. (Editor); Shanot, Al; Oncley, Steven P.; Cooper, Al; Smith, David E. (Technical Monitor)

    2000-01-01

    The BOREAS AFM-3 team used the NCAR Electra aircraft data to make measurements of the fluxes of momentum, sensible and latent heat, carbon dioxide, and ozone over the entire BOREAS region to tie together measurements made in both the SSA and the NSA in 1994. These data were also used to study the planetary boundary layer using both in situ and remote sensing measurements. This data set contains both the aircraft flux and the moving window data. These data are stored in tabular ASCII files. The data files are available on a CD-ROM (see document number 20010000884) or from the Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC).

  18. Lateral Tip Control Effects in CD-AFM Metrology: The Large Tip Limit.

    PubMed

    Dixson, Ronald G; Orji, Ndubuisi G; Goldband, Ryan S

    2016-01-25

    Sidewall sensing in critical dimension atomic force microscopes (CD-AFMs) usually involves continuous lateral dithering of the tip or the use of a control algorithm and fast response piezo actuator to position the tip in a manner that resembles touch-triggering of coordinate measuring machine (CMM) probes. All methods of tip position control, however, induce an effective tip width that may deviate from the actual geometrical tip width. Understanding the influence and dependence of the effective tip width on the dither settings and lateral stiffness of the tip can improve the measurement accuracy and uncertainty estimation for CD-AFM measurements. Since CD-AFM typically uses tips that range from 15 nm to 850 nm in geometrical width, the behavior of effective tip width throughout this range should be understood. The National Institute of Standards and Technology (NIST) has been investigating the dependence of effective tip width on the dither settings and lateral stiffness of the tip, as well as the possibility of material effects due to sample composition. For tip widths of 130 nm and lower, which also have lower lateral stiffness, the response of the effective tip width to lateral dither is greater than for larger tips. However, we have concluded that these effects will not generally result in a residual bias, provided that the tip calibration and sample measurement are performed under the same conditions. To validate that our prior conclusions about the dependence of effective tip width on lateral stiffness are valid for large CD-tips, we recently performed experiments using a very large non-CD tip with an etched plateau of approximately 2 μm width. The effective lateral stiffness of these tips is at least 20 times greater than typical CD-AFM tips, and these results supported our prior conclusions about the expected behavior for larger tips. The bottom-line importance of these latest observations is that we can now reasonably conclude that a dither slope of 3 nm