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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. Mapping site-specific endonuclease binding to DNA by direct imaging with AFM

    SciTech Connect

    Allison, D.P.; Thundat, T.; Doktycz, M.J.; Kerper, P.S.; Warmack, R.J.; Modrich, P.; Isfort, R.J.

    1995-12-31

    Physical mapping of DNA can be accomplished by direct AFM imaging of site specific proteins bound to DNA molecules. Using Gln-111, a mutant of EcoRI endonuclease with a specific affinity for EcoRI sites 1,000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 10{sup 4}, the authors demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS{sup +}) or two (pMP{sup 32}) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin gold prior to imaging. Image enhancement coupled with improvements in the preparation techniques for imaging large DNA molecules, such as lambda DNA (47 kb), has the potential to contribute to direct AFM restriction mapping of cosmid-sized genomic DNAs.

  3. Mapping site-specific endonuclease binding to DNA by direct imaging with atomic force microscopy (AFM)

    NASA Astrophysics Data System (ADS)

    Allison, David P.; Thundat, Thomas G.; Modrich, P.; Isfort, R. J.; Doktycz, Mitchel J.; Kerper, P. S.; Warmack, R. J.

    1995-04-01

    Physical mapping of DNA can be accomplished by direct AFM imaging of site specific proteins bound to DNA molecules. Using Gln-111, a mutant of EcoRI endonuclease with a specific affinity for EcoRI sites 1000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 104, we demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS+) or two (pMP32) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin gold prior to imaging. Image enhancement coupled with improvements in our preparation techniques for imaging large DNA molecules, such as lambda DNA (47 kb), has the potential to contribute to direct AFM restriction mapping of cosmid-sized genomic DNAs.

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

    DOE PAGES

    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 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

  5. 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.

  6. 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

  7. 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

  8. Direct observation of hydration of TiO 2 on Ti using electrochemical AFM: freely corroding versus potentiostatically held

    NASA Astrophysics Data System (ADS)

    Bearinger, Jane P.; Orme, Christine A.; Gilbert, Jeremy L.

    2001-10-01

    Hydration of titanium/titanium oxide surfaces under freely corroding and potentiostatically held conditions has been characterized using electrochemical atomic force microscopy (EC AFM). In contrast to conventional high vacuum techniques, AFM enables measurement of morphological surface structure in the in situ hydrated state. Electrochemical probes in the imaging environment further enable acquisition of electrical characteristics during AFM imaging. Experiments were performed on etched, electropolished commercially pure titanium. As noted by direct observation and corroborated by power spectral density (Fourier analysis) measurements, oxide domes cover the titanium surface and grow laterally during hydration. Applied potential altered the growth rate. Under open circuit potential conditions, growth proceeded approximately six times faster than under a -1 V applied voltage ( 1098±52 nm2/ min ± versus 184.84±19 nm2/min). Film growth increased electrical resistance and lowered interfacial capacitance based on step polarization impedance spectroscopy tests.

  9. 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).

  10. Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging

    SciTech Connect

    Carnally, Stewart M.; Dev, Harveer S.; Stewart, Andrew P.; Barrera, Nelson P.; Van Bemmelen, Miguel X.; Schild, Laurent; Henderson, Robert M.; Edwardson, J.Michael

    2008-08-08

    There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica. We detected a mixture of subunit monomers, dimers and trimers. In some cases, triple-subunit clusters were clearly visible, confirming the trimeric structure of the channel, and indicating that the trimer sometimes disaggregated after adhesion to the mica surface. This AFM-based technique will now enable us to determine the subunit arrangement within heteromeric ASICs.

  11. Direct and quantitative AFM measurements of the concentration and temperature dependence of the hydrophobic force law at nanoscopic contacts.

    PubMed

    Stock, Philipp; Utzig, Thomas; Valtiner, Markus

    2015-05-15

    By virtue of its importance for self-organization of biological matter the hydrophobic force law and the range of hydrophobic interactions (HI) have been debated extensively over the last 40 years. Here, we directly measure and quantify the hydrophobic force-distance law over large temperature and concentration ranges. In particular, we study the HI between molecularly smooth hydrophobic self-assembled monolayers, and similarly modified gold-coated AFM tips (radii∼8-50 nm). We present quantitative and direct evidence that the hydrophobic force is both long-ranged and exponential down to distances of about 1-2 nm. Therefore, we introduce a self-consistent radius-normalization for atomic force microscopy data. This approach allows quantitative data fitting of AFM-based experimental data to the recently proposed Hydra-model. With a statistical significance of r(2)⩾0.96 our fitting and data directly reveal an exponential HI decay length of 7.2±1.2 Å that is independent of the salt concentration up to 750 mM. As such, electrostatic screening does not have a significant influence on the HI in electrolyte concentrations ranging from 1 mM to 750 mM. In 1 M solutions the observed instability during approach shifts to longer distances, indicating ion correlation/adsorption effects at high salt concentrations. With increasing temperature the magnitude of HI decreases monotonically, while the range increases slightly. We compare our results to the large body of available literature, and shed new light into range and magnitude of hydrophobic interactions at very close distances and over wide temperature and concentration regimes. PMID:25678158

  12. Cytochrome c self-assembly on alkanethiol monolayer electrodes as characterized by AFM, IR, QCM, and direct electrochemistry.

    PubMed

    Nakano, Koji; Yoshitake, Tadateru; Yamashita, Yasunori; Bowden, Edmond F

    2007-05-22

    With the advantage of carbodiimide coupling chemistry, horse heart cytochrome c (cyt c) has been covalently immobilized onto self-assembled monolayers (SAMs) from 11-mercaptoundecanoic acid (MUDA) developed on single-crystal or polycrystalline gold substrate surfaces. The cyt c immobilized substrates thus prepared have been characterized by atomic force microscopy (AFM); we have succeeded in obtaining surface topographical images down to single-protein resolution. AFM imaging has also shown densely packed, uniform protein monolayer formation that is highly suggestive of self-assembly of cyt c molecules on MUDA SAMs. Covalent attachment of cyt c has been further evidenced by reflection-absorption FT-IR as well as microgravimetric analysis using a quartz crystal microbalance (QCM). In the latter, the specific MUDA and cyt c surface concentrations were determined to be 0.86 +/- 0.11 nmol cm-2 (n = 5) and 28 +/- 12 pmol cm-2 (n = 5), both of which agree fairly well with their theoretical counterparts. The obtained QCM chips having the cyt c/MUDA/Au interfacial structure were found to be capable of the direct electrochemistry of the surface-attached cyt c molecules. Cyclic voltammetric measurements on the chips gave particular redox waves showing characteristics of surface process. The electroactive protein surface concentration was determined to be 7.2 +/- 4.8 pmol cm-2 (n = 6); it was almost consistent with values found in literature, while it was limited to 26% in magnitude for the QCM data. This was deemed to have arisen from the orientation variation of the surface-confined cyt c molecules and is discussed briefly.

  13. Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach.

    PubMed

    Upadhye, Kalpesh V; Candiello, Joseph E; Davidson, Lance A; Lin, Hai

    2011-06-01

    Patch clamp is a powerful tool for studying the properties of ion-channels and cellular membrane. In recent years, planar patch clamp chips have been fabricated from various materials including glass, quartz, silicon, silicon nitride, polydimethyl-siloxane (PDMS), and silicon dioxide. Planar patch clamps have made automation of patch clamp recordings possible. However, most planar patch clamp chips have limitations when used in combination with other techniques. Furthermore, the fabrication methods used are often expensive and require specialized equipments. An improved design as well as fabrication and characterization of a silicon-based planar patch clamp chip are described in this report. Fabrication involves true batch fabrication processes that can be performed in most common microfabrication facilities using well established MEMS techniques. Our planar patch clamp chips can form giga-ohm seals with the cell plasma membrane with success rate comparable to existing patch clamp techniques. The chip permits whole-cell voltage clamp recordings on variety of cell types including Chinese Hamster Ovary (CHO) cells and pheochromocytoma (PC12) cells, for times longer than most available patch clamp chips. When combined with a custom microfluidics chamber, we demonstrate that it is possible to perfuse the extra-cellular as well as intra-cellular buffers. The chamber design allows integration of planar patch clamp with atomic force microscope (AFM). Using our planar patch clamp chip and microfluidics chamber, we have recorded whole-cell mechanosensitive (MS) currents produced by directly stimulating human keratinocyte (HaCaT) cells using an AFM cantilever. Our results reveal the spatial distribution of MS ion channels and temporal details of the responses from MS channels. The results show that planar patch clamp chips have great potential for multi-parametric high throughput studies of ion channel proteins. PMID:22174731

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

    PubMed

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

    2014-07-21

    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.

  15. 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.

  16. 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.

  17. Identifying individual chemical bonds in single-molecule chemical reaction products using nc-AFM

    NASA Astrophysics Data System (ADS)

    Wickenburg, Sebastian; de Oteyza, Dimas G.; Chen, Yen-Chia; Riss, Alexander; Tsai, Hsin-Zon; Pedramrazi, Zahra; Bradley, Aaron J.; Ugeda, Miguel M.; Gorman, Patrick; Etkin, Grisha; Mowbray, Duncan J.; Perez, Alejandro; Rubio, Angel; Crommie, Michael F.; Fischer, Felix R.

    2014-03-01

    Determining reaction pathways and products is an integral part of chemical synthesis. Ensemble measurements are commonly used, but identifying products of complex reactions at surfaces presents a significant challenge. Here we present a non-contact AFM (nc-AFM) study to directly address this issue. We followed the change of the chemical structures, from reactants to products of enediyne cyclization reactions on metal surfaces. Thermal annealing of enediynes induced a series of cyclization cascades leading to radical species and the formation of dimers. Atomically resolved nc-AFM images reveal the precise chemical structure and the formation of chemical bonds between single molecular units. With the support of DFT calculations, we identified the underlying chemical pathways and barriers, demonstrating the potential of this atomically resolved AFM technique to study unknown reaction products in surface chemistry at the single-molecule level.

  18. Defective sumoylation pathway directs congenital heart disease

    PubMed Central

    Wang, Jun; Chen, Li; Wen, Shu; Zhu, Huiping; Yu, Wei; Moskowitz, Ivan P.; Shaw, Gary M.; Finnell, Richard H.; Schwartz, Robert J

    2016-01-01

    Congenital heart defects (CHDs) are the most common of all birth defects, yet molecular mechanism(s) underlying highly prevalent atrial septal defects (ASDs) and ventricular septal defects (VSDs) have remained elusive. We demonstrate the indispensability of “balanced” post-translational SUMO conjugation-deconjugation pathway for normal cardiac development. Both hetero- and homo-zygous SUMO-1 knockout mice exhibited ASDs and VSDs with high mortality rates, which were rescued by cardiac re-expression of the SUMO-1 transgene. Since SUMO-1 was also involved in cleft lip/palate in human patients, the above findings provided a powerful rationale to question whether SUMO-1 was mutated in babies born with cleft palates and ASDs. Sequence analysis of DNA from newborn screening blood spots revealed a single 16 bp substitution in the SUMO-1 regulatory promoter of a patient displaying both oral-facial clefts and ASDs. Diminished sumoylation activity whether by genetics, environmental toxins and/or pharmaceuticals may significantly contribute to susceptibility to the induction of congenital heart disease worldwide. PMID:21563299

  19. The role of confinement and corona crystallinity on the bending modulus of copolymer micelles measured directly by AFM flexural tests.

    PubMed

    Jennings, L; Glazer, P; Laan, A C; de Kruijff, R M; Waton, G; Schosseler, F; Mendes, E

    2016-09-21

    We present an approach which makes it possible to directly determine the bending modulus of single elongated block copolymer micelles. This is done by forming arrays of suspended micelles onto microfabricated substrates and by performing three-point bending flexural tests, using an atomic force microscope, on their suspended portions. By coupling the direct atomic force microscopy measurements with differential scanning calorimetry data, we show that the presence of a crystalline corona strongly increases the modulus of the copolymer elongated micelles. This large increase suggests that crystallites in the corona are larger and more uniformly oriented due to confinement effects. Our findings together with this hypothesis open new interesting avenues for the preparation of core-templated polymer fibres with enhanced mechanical properties. PMID:27506248

  20. Qplus AFM driven nanostencil.

    PubMed

    Grévin, B; Fakir, M; Hayton, J; Brun, M; Demadrille, R; Faure-Vincent, J

    2011-06-01

    We describe the development of a novel setup, in which large stencils with suspended silicon nitride membranes are combined with atomic force microscopy (AFM) regulation by using tuning forks. This system offers the possibility to perform separate AFM and nanostencil operations, as well as combined modes when using stencil chips with integrated tips. The flexibility and performances are demonstrated through a series of examples, including wide AFM scans in closed loop mode, probe positioning repeatability of a few tens of nanometer, simultaneous evaporation of large (several hundred of micron square) and nanoscopic metals and fullerene patterns in static, multistep, and dynamic modes. This approach paves the way for further developments, as it fully combines the advantages of conventional stenciling with the ones of an AFM driven shadow mask. PMID:21721701

  1. AFM cantilever vibration detection with a transmitted electron beam

    NASA Astrophysics Data System (ADS)

    Woehl, Taylor; Wagner, Ryan; Keller, Robert; Killgore, Jason

    Cantilever oscillations for dynamic atomic force microscopy (AFM) are conventionally measured with an optical lever system. The speed of AFM cantilevers can be increased by decreasing the size of the cantilever; however, the fastest AFM cantilevers are currently nearing the smallest size that can be detected with the current optical lever approach. Here we demonstrate an electron detection scheme in an SEM for detecting AFM cantilever oscillations. An oscillating AFM tip is positioned perpendicular to the propagation direction of a stationary ~ 1 nm diameter electron probe, and the oscillatory change in electron scattering resulting from the changing thickness of the electron irradiated area of the AFM tip is detected with a transmitted electron detector positioned below the AFM tip. We perform frequency sweep and ring-down experiments to determine the first resonant frequency and Q factor of an AFM cantilever.

  2. 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.

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

    PubMed

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

    2016-04-01

    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. PMID:27046807

  4. Training directionally selective motion pathways can significantly improve reading efficiency

    NASA Astrophysics Data System (ADS)

    Lawton, Teri

    2004-06-01

    This study examined whether perceptual learning at early levels of visual processing would facilitate learning at higher levels of processing. This was examined by determining whether training the motion pathways by practicing leftright movement discrimination, as found previously, would improve the reading skills of inefficient readers significantly more than another computer game, a word discrimination game, or the reading program offered by the school. This controlled validation study found that practicing left-right movement discrimination 5-10 minutes twice a week (rapidly) for 15 weeks doubled reading fluency, and significantly improved all reading skills by more than one grade level, whereas inefficient readers in the control groups barely improved on these reading skills. In contrast to previous studies of perceptual learning, these experiments show that perceptual learning of direction discrimination significantly improved reading skills determined at higher levels of cognitive processing, thereby being generalized to a new task. The deficits in reading performance and attentional focus experienced by the person who struggles when reading are suggested to result from an information overload, resulting from timing deficits in the direction-selectivity network proposed by Russell De Valois et al. (2000), that following practice on direction discrimination goes away. This study found that practicing direction discrimination rapidly transitions the inefficient 7-year-old reader to an efficient reader.

  5. Identification of a direct GABAergic pallidocortical pathway in rodents

    PubMed Central

    Chen, Michael C.; Ferrari, Loris; Sacchet, Matthew D.; Foland-Ross, Lara C.; Qiu, Mei-Hong; Gotlib, Ian H.; Fuller, Patrick M.; Arrigoni, Elda; Lu, Jun

    2014-01-01

    The basal ganglia, interacting with the cortex, play a critical role in a range of behaviors. Output from the basal ganglia to the cortex is thought to relay through the thalamus, yet an intriguing alternative is that the basal ganglia may directly project to, and communicate with, the cortex. We explored an efferent projection from the globus pallidus externa (GPe), a key hub in the basal ganglia system, to the cortex of rats and mice. Anterograde and retrograde tracing revealed projections to the frontal premotor cortex, especially the deep projecting layers, originating from GPe neurons that receive axonal inputs from the dorsal striatum. Cre-dependent anterograde tracing in GPe Vgat-ires-cre mice confirmed that the pallidocortical projection is GABAergic, and in vitro optogenetic stimulation in the cortex of these projections produced a fast inhibitory postsynaptic current in targeted cells that was abolished by bicucculine. The pallidocortical projections targeted GABAergic interneurons and, to a lesser extent, pyramidal neurons. This GABAergic pallidocortical pathway directly links the basal ganglia and cortex and may play a key role in behavior and cognition in normal and disease states. PMID:25581560

  6. 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. PMID:26515095

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

    DOE PAGES

    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-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

  8. 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.

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

    PubMed Central

    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.

    2015-01-01

    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 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. PMID:26515095

  10. Current perspectives of the signaling pathways directing neural crest induction.

    PubMed

    Stuhlmiller, Timothy J; García-Castro, Martín I

    2012-11-01

    The neural crest is a migratory population of embryonic cells with a tremendous potential to differentiate and contribute to nearly every organ system in the adult body. Over the past two decades, an incredible amount of research has given us a reasonable understanding of how these cells are generated. Neural crest induction involves the combinatorial input of multiple signaling pathways and transcription factors, and is thought to occur in two phases from gastrulation to neurulation. In the first phase, FGF and Wnt signaling induce NC progenitors at the border of the neural plate, activating the expression of members of the Msx, Pax, and Zic families, among others. In the second phase, BMP, Wnt, and Notch signaling maintain these progenitors and bring about the expression of definitive NC markers including Snail2, FoxD3, and Sox9/10. In recent years, additional signaling molecules and modulators of these pathways have been uncovered, creating an increasingly complex regulatory network. In this work, we provide a comprehensive review of the major signaling pathways that participate in neural crest induction, with a focus on recent developments and current perspectives. We provide a simplified model of early neural crest development and stress similarities and differences between four major model organisms: Xenopus, chick, zebrafish, and mouse. PMID:22547091

  11. Current perspectives of the signaling pathways directing neural crest induction.

    PubMed

    Stuhlmiller, Timothy J; García-Castro, Martín I

    2012-11-01

    The neural crest is a migratory population of embryonic cells with a tremendous potential to differentiate and contribute to nearly every organ system in the adult body. Over the past two decades, an incredible amount of research has given us a reasonable understanding of how these cells are generated. Neural crest induction involves the combinatorial input of multiple signaling pathways and transcription factors, and is thought to occur in two phases from gastrulation to neurulation. In the first phase, FGF and Wnt signaling induce NC progenitors at the border of the neural plate, activating the expression of members of the Msx, Pax, and Zic families, among others. In the second phase, BMP, Wnt, and Notch signaling maintain these progenitors and bring about the expression of definitive NC markers including Snail2, FoxD3, and Sox9/10. In recent years, additional signaling molecules and modulators of these pathways have been uncovered, creating an increasingly complex regulatory network. In this work, we provide a comprehensive review of the major signaling pathways that participate in neural crest induction, with a focus on recent developments and current perspectives. We provide a simplified model of early neural crest development and stress similarities and differences between four major model organisms: Xenopus, chick, zebrafish, and mouse.

  12. Direct Observation of the Formation Pathway of [Mo132] Keplerates.

    PubMed

    Biswas, Subharanjan; Melgar, Dolores; Srimany, Amitava; Rodríguez-Fortea, Antonio; Pradeep, Thalappil; Bo, Carles; Poblet, Josep M; Roy, Soumyajit

    2016-09-01

    The formation pathway of a closed spherical cluster [Mo132], starting from a library of building blocks of molybdate anions, has been reported. Electrospray ionization mass spectrometry, Raman spectroscopy, and theoretical studies describe the formation of such a complex cluster from a reduced and acidified aqueous solution of molybdate. Understanding the emergence of such an enormous spherical model cluster may lead to the design of new clusters in the future. Formation of such a highly symmetric cluster is principally controlled by charge balance and the emergence of more symmetric structures at the expense of less symmetric ones.

  13. Direct Observation of the Formation Pathway of [Mo132] Keplerates.

    PubMed

    Biswas, Subharanjan; Melgar, Dolores; Srimany, Amitava; Rodríguez-Fortea, Antonio; Pradeep, Thalappil; Bo, Carles; Poblet, Josep M; Roy, Soumyajit

    2016-09-01

    The formation pathway of a closed spherical cluster [Mo132], starting from a library of building blocks of molybdate anions, has been reported. Electrospray ionization mass spectrometry, Raman spectroscopy, and theoretical studies describe the formation of such a complex cluster from a reduced and acidified aqueous solution of molybdate. Understanding the emergence of such an enormous spherical model cluster may lead to the design of new clusters in the future. Formation of such a highly symmetric cluster is principally controlled by charge balance and the emergence of more symmetric structures at the expense of less symmetric ones. PMID:27560767

  14. Heterogeneous Effects of Direct Hypoxia Pathway Activation in Kidney Cancer

    PubMed Central

    Salama, Rafik; Masson, Norma; Simpson, Peter; Sciesielski, Lina Katrin; Sun, Min; Tian, Ya-Min; Ratcliffe, Peter John; Mole, David Robert

    2015-01-01

    General activation of hypoxia-inducible factor (HIF) pathways is classically associated with adverse prognosis in cancer and has been proposed to contribute to oncogenic drive. In clear cell renal carcinoma (CCRC) HIF pathways are upregulated by inactivation of the von-Hippel-Lindau tumor suppressor. However HIF-1α and HIF-2α have contrasting effects on experimental tumor progression. To better understand this paradox we examined pan-genomic patterns of HIF DNA binding and associated gene expression in response to manipulation of HIF-1α and HIF-2α and related the findings to CCRC prognosis. Our findings reveal distinct pan-genomic organization of canonical and non-canonical HIF isoform-specific DNA binding at thousands of sites. Overall associations were observed between HIF-1α-specific binding, and genes associated with favorable prognosis and between HIF-2α-specific binding and adverse prognosis. However within each isoform-specific set, individual gene associations were heterogeneous in sign and magnitude, suggesting that activation of each HIF-α isoform contributes a highly complex mix of pro- and anti-tumorigenic effects. PMID:26262842

  15. Direct AFM force measurements between air bubbles in aqueous polydisperse sodium poly(styrene sulfonate) solutions: effect of collision speed, polyelectrolyte concentration and molar mass.

    PubMed

    Browne, Christine; Tabor, Rico F; Grieser, Franz; Dagastine, Raymond R

    2015-07-01

    Interactions between colliding air bubbles in aqueous solutions of polydisperse sodium poly(styrene sulfonate) (NaPSS) using direct force measurements were studied. The forces measured with deformable interfaces were shown to be more sensitive to the presence of the polyelectrolytes when compared to similar measurements using rigid interfaces. The experimental factors that were examined were NaPSS concentration, bubble collision velocity and polyelectrolyte molar mass. These measurements were then compared with an analytical model based on polyelectrolyte scaling theory in order to explain the effects of concentration and bubble deformation on the interaction between bubbles. Typically structural forces from the presence of monodisperse polyelectrolyte between interacting surfaces may be expected, however, it was found that the polydispersity in molar mass resulted in the structural forces to be smoothed and only a depletion interaction was able to be measured between interacting bubbles. It was found that an increase in number density of NaPSS molecules resulted in an increase in the magnitude of the depletion interaction. Conversely this interaction was overwhelmed by an increase in the fluid flow in the system at higher bubble collision velocities. Polymer molar mass dispersity plays a significant role in the interactions present between the bubbles and has implications that also affect the polyelectrolyte overlap concentration of the solution. Further understanding of these implications can be expected to play a role in the improvement in operations in such fields as water treatment and mineral processing where polyelectrolytes are used extensively.

  16. [Distinct roles of the direct and indirect pathways in the basal ganglia circuit mechanism].

    PubMed

    Morita, Makiko; Hikida, Takatoshi

    2015-11-01

    The basal ganglia are key neural substrates that control not only motor balance but also emotion, motivation, cognition, learning, and decision-making. Dysfunction of the basal ganglia leads to neurodegenerative diseases (e.g. Parkinson's disease and Huntington's disease) and psychiatric disorders (e.g. drug addiction, schizophrenia, and depression). In the basal ganglia circuit, there are two important pathways: the direct and indirect striatal pathways. Recently, new molecular techniques that activate or inactive selectively the direct or indirect pathway neurons have revealed the function of each pathway. Here we review the distinct roles of the direct and indirect striatal pathways in brain function and drug addiction. We have developed a reversible neurotransmission blocking technique, in which transmission of each pathway is selectively blocked by specific expression of transmission-blocking tetanus toxin, and revealed that the activation of D1 receptors in the direct pathway is critical for reward learning/cocaine addiction, and that the inactivation of D2 receptors is critical for aversive learning/learning flexibility. We propose a new circuit mechanism by which the dopaminergic input from the ventral tegmental area can switch the direct and indirect pathways in the nucleus accumbens. These basal ganglia circuit mechanisms will give us insights into the pathophysiology of mental diseases. PMID:26785520

  17. 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.

  18. 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…

  19. 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

  20. Liquid contact resonance AFM: analytical models, experiments, and limitations

    NASA Astrophysics Data System (ADS)

    Parlak, Zehra; Tu, Qing; Zauscher, Stefan

    2014-11-01

    Contact resonance AFM (CR-AFM) is a scanning probe microscopy technique that utilizes the contact resonances of the AFM cantilever for concurrent imaging of topography and surface stiffness. The technique has not been used in liquid until recently due to analytical and experimental difficulties, associated with viscous damping of cantilever vibrations and fluid loading effects. To address these difficulties, (i) an analytical approach for contact resonances in liquid is developed, and (ii) direct excitation of the contact resonances is demonstrated by actuating the cantilever directly in a magnetic field. By implementing the analytical approach and the direct actuation through magnetic particles, quantitative stiffness imaging on surfaces with a wide range of stiffness can be achieved in liquid with soft cantilevers and low contact forces.

  1. Competition between direct and indirect dissociation pathways in ultraviolet photodissociation of HNCO.

    PubMed

    Yu, Shengrui; Su, Shu; Dorenkamp, Yvonne; Wodtke, Alec M; Dai, Dongxu; Yuan, Kaijun; Yang, Xueming

    2013-11-21

    Photodissociation dynamics of HNCO at photolysis wavelengths between 200 and 240 nm have been studied using the H-atom Rydberg tagging time-of-flight technique. Product translational energy distributions and angular distributions have been determined. At low photon energy excitation, the product translational energy distribution is nearly statistical and the angular distribution is isotropic, which is consistent with an indirect dissociation mechanism, i.e., internal conversion from S1 to S0 surface and dissociation on S0 surface. As the photon energy increases, a direct dissociation pathway on S1 surface opens up. The product translational energy distribution appears to be quite nonstatistical and the product angular distribution is anisotropic. The fraction of direct dissociation pathway is determined to be 36 ± 5% at 202.67 nm photolysis. Vibrational structures are observed in both direct and indirect dissociation pathways, which can be assigned to the NCO bending mode excitation with some stretching excitation. PMID:23496141

  2. Direct interaction of Ste11 and Mkk1/2 through Nst1 integrates high-osmolarity glycerol and pheromone pathways to the cell wall integrity MAPK pathway.

    PubMed

    Leng, Gang; Song, Kiwon

    2016-01-01

    Coordination and cross talks of MAPK pathways are critical for signaling efficiency, but their mechanisms are not well understood. Slt2, the MAP kinase of cell wall integrity pathway (CWI), is activated by heat stress even in the absence of upstream components of this pathway, suggesting a supplementary input for Slt2 activation. Here, we identify a new interaction of Ste11 and Mkk1, mediated by Nst1 that connects the high-osmolarity glycerol and pheromone pathways directly to CWI pathway in response to heat and pheromone. We suggest that Ser(407) and Thr(411) are novel residues of Mkk1 activated by these MAPK pathways.

  3. Different Roles of Direct and Indirect Frontoparietal Pathways for Individual Working Memory Capacity.

    PubMed

    Ekman, Matthias; Fiebach, Christian J; Melzer, Corina; Tittgemeyer, Marc; Derrfuss, Jan

    2016-03-01

    The ability to temporarily store and manipulate information in working memory is a hallmark of human intelligence and differs considerably across individuals, but the structural brain correlates underlying these differences in working memory capacity (WMC) are only poorly understood. In two separate studies, diffusion MRI data and WMC scores were collected for 70 and 109 healthy individuals. Using a combination of probabilistic tractography and network analysis of the white matter tracts, we examined whether structural brain network properties were predictive of individual WMC. Converging evidence from both studies showed that lateral prefrontal cortex and posterior parietal cortex of high-capacity individuals are more densely connected compared with low-capacity individuals. Importantly, our network approach was further able to dissociate putative functional roles associated with two different pathways connecting frontal and parietal regions: a corticocortical pathway and a subcortical pathway. In Study 1, where participants were required to maintain and update working memory items, the connectivity of the direct and indirect pathway was predictive of WMC. In contrast, in Study 2, where participants were required to maintain working memory items without updating, only the connectivity of the direct pathway was predictive of individual WMC. Our results suggest an important dissociation in the circuitry connecting frontal and parietal regions, where direct frontoparietal connections might support storage and maintenance, whereas subcortically mediated connections support the flexible updating of working memory content.

  4. Modality-Based Organization of Ascending Somatosensory Axons in the Direct Dorsal Column Pathway

    PubMed Central

    Niu, Jingwen; Ding, Long; Li, Jian J.; Kim, Hyukmin; Liu, Jiakun; Li, Haipeng; Moberly, Andrew; Badea, Tudor C.; Duncan, Ian D.; Son, Young-Jin; Scherer, Steven S.

    2013-01-01

    The long-standing doctrine regarding the functional organization of the direct dorsal column (DDC) pathway is the “somatotopic map” model, which suggests that somatosensory afferents are primarily organized by receptive field instead of modality. Using modality-specific genetic tracing, here we show that ascending mechanosensory and proprioceptive axons, two main types of the DDC afferents, are largely segregated into a medial–lateral pattern in the mouse dorsal column and medulla. In addition, we found that this modality-based organization is likely to be conserved in other mammalian species, including human. Furthermore, we identified key morphological differences between these two types of afferents, which explains how modality segregation is formed and why a rough “somatotopic map” was previously detected. Collectively, our results establish a new functional organization model for the mammalian direct dorsal column pathway and provide insight into how somatotopic and modality-based organization coexist in the central somatosensory pathway. PMID:24198362

  5. Evidence for "direct" and "indirect" pathways through the song system basal ganglia.

    PubMed

    Farries, Michael A; Ding, Long; Perkel, David J

    2005-03-28

    Song learning in oscine birds relies on a circuit known as the "anterior forebrain pathway," which includes a specialized region of the avian basal ganglia. This region, area X, is embedded within a telencephalic structure considered homologous to the striatum, the input structure of the mammalian basal ganglia. Area X has many features in common with the mammalian striatum, yet has distinctive traits, including largely aspiny projection neurons that directly innervate the thalamus and a cell type that physiologically resembles neurons recorded in the mammalian globus pallidus. We have proposed that area X is a mixture of striatum and globus pallidus and has the same functional organization as circuits in the mammalian basal ganglia. Using electrophysiological and anatomical approaches, we found that area X contains a functional analog of the "direct" striatopallidothalamic pathway of mammals: axons of the striatal spiny neurons make close contacts on the somata and dendrites of pallidal cells. A subset of pallidal neurons project directly to the thalamus. Surprisingly, we found evidence that many pallidal cells may not project to the thalamus, but rather participate in a functional analog of the mammalian "indirect" pathway, which may oppose the effects of the direct pathway. Our results deepen our understanding of how information flows through area X and provide more support for the notion that song learning in oscines employs physiological mechanisms similar to basal ganglia-dependent forms of motor learning in mammals. PMID:15717304

  6. 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.

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

    PubMed

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

    2015-10-01

    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

  8. Turning over renal osteodystrophy dogma: direct actions of FGF23 on osteoblast β-catenin pathway.

    PubMed

    Schiavi, Susan C; Moysés, Rosa M A

    2016-07-01

    Although recognized as a major complication of chronic kidney disease (CKD), the pathophysiology of the CKD-related mineral and bone disorder (CKD-MBD) is not completely understood. Recently, the inhibition of Wnt/β-catenin pathway in osteocytes by sclerostin has been shown to play a role in CKD-MBD. The study by Carrilo-Lopez et al. confirms this inhibition in an experimental model of CKD. Moreover, they describe direct actions of FGF23-Klotho on osteoblasts, increasing the expression of DKK1, another Wnt/β-catenin pathway inhibitor. PMID:27312441

  9. 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.

  10. 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...

  11. Direct and indirect spino-cerebellar pathways: shared ideas but different functions in motor control

    PubMed Central

    Jiang, Juan; Azim, Eiman; Ekerot, Carl-Fredrik; Alstermark, Bror

    2015-01-01

    The impressive precision of mammalian limb movements relies on internal feedback pathways that convey information about ongoing motor output to cerebellar circuits. The spino-cerebellar tracts (SCT) in the cervical, thoracic and lumbar spinal cord have long been considered canonical neural substrates for the conveyance of internal feedback signals. Here we consider the distinct features of an indirect spino-cerebellar route, via the brainstem lateral reticular nucleus (LRN), and the implications of this pre-cerebellar “detour” for the execution and evolution of limb motor control. Both direct and indirect spino-cerebellar pathways signal spinal interneuronal activity to the cerebellum during movements, but evidence suggests that direct SCT neurons are mainly modulated by rhythmic activity, whereas the LRN also receives information from systems active during postural adjustment, reaching and grasping. Thus, while direct and indirect spino-cerebellar circuits can both be regarded as internal copy pathways, it seems likely that the direct system is principally dedicated to rhythmic motor acts like locomotion, while the indirect system also provides a means of pre-cerebellar integration relevant to the execution and coordination of dexterous limb movements. PMID:26217214

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

    PubMed

    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 14years. 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

  13. 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

  14. Evolution of branched regulatory genetic pathways: directional selection on pleiotropic loci accelerates developmental system drift.

    PubMed

    Johnson, Norman A; Porter, Adam H

    2007-01-01

    Developmental systems are regulated by a web of interacting loci. One common and useful approach in studying the evolution of development is to focus on classes of interacting elements within these systems. Here, we use individual-based simulations to study the evolution of traits controlled by branched developmental pathways involving three loci, where one locus regulates two different traits. We examined the system under a variety of selective regimes. In the case where one branch was under stabilizing selection and the other under directional selection, we observed "developmental system drift": the trait under stabilizing selection showed little phenotypic change even though the loci underlying that trait showed considerable evolutionary divergence. This occurs because the pleiotropic locus responds to directional selection and compensatory mutants are then favored in the pathway under stabilizing selection. Though developmental system drift may be caused by other mechanisms, it seems likely that it is accelerated by the same underlying genetic mechanism as that producing the Dobzhansky-Muller incompatibilities that lead to speciation in both linear and branched pathways. We also discuss predictions of our model for developmental system drift and how different selective regimes affect probabilities of speciation in the branched pathway system.

  15. 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).

  16. In situ TEM imaging of CaCO₃ nucleation reveals coexistence of direct and indirect pathways.

    PubMed

    Nielsen, Michael H; Aloni, Shaul; De Yoreo, James J

    2014-09-01

    Mechanisms of nucleation from electrolyte solutions have been debated for more than a century. Recent discoveries of amorphous precursors and evidence for cluster aggregation and liquid-liquid separation contradict common assumptions of classical nucleation theory. Using in situ transmission electron microscopy (TEM) to explore calcium carbonate (CaCO3) nucleation in a cell that enables reagent mixing, we demonstrate that multiple nucleation pathways are simultaneously operative, including formation both directly from solution and indirectly through transformation of amorphous and crystalline precursors. However, an amorphous-to-calcite transformation is not observed. The behavior of amorphous calcium carbonate upon dissolution suggests that it encompasses a spectrum of structures, including liquids and solids. These observations of competing direct and indirect pathways are consistent with classical predictions, whereas the behavior of amorphous particles hints at an underlying commonality among recently proposed precursor-based mechanisms. PMID:25190792

  17. 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.

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

    PubMed Central

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

    2015-01-01

    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 13C-phospho- and neutral lipid fatty acids, we traced in-situ flows of recently photoassimilated C of 13CO2-exposed wheat (Triticum aestivum) through arbuscular mycorrhiza (AM) into root- and hyphae-associated soil microbial communities. Intraradical hyphae of AM fungi were significantly 13C-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 13C-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. PMID:25382456

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. Classical dendritic cells mediate fibrosis directly via the retinoic acid pathway in severe eye allergy

    PubMed Central

    Ahadome, Sarah D.; Mathew, Rose; Reyes, Nancy J.; Mettu, Priyatham S.; Cousins, Scott W.; Calder, Virginia L.; Saban, Daniel R.

    2016-01-01

    Fibrosis is a shared end-stage pathway to lung, liver, and heart failure. In the ocular mucosa (conjunctiva), fibrosis leads to blindness in trachoma, pemphigoid, and allergy. The indirect fibrogenic role of DCs via T cell activation and inflammatory cell recruitment is well documented. However, here we demonstrate that DCs can directly induce fibrosis. In the mouse model of allergic eye disease (AED), classical CD11b+ DCs in the ocular mucosa showed increased activity of aldehyde dehydrogenase (ALDH), the enzyme required for retinoic acid synthesis. In vitro, CD11b+ DC–derived ALDH was associated with 9-cis-retinoic acid ligation to retinoid x receptor (RXR), which induced conjunctival fibroblast activation. In vivo, stimulating RXR led to rapid onset of ocular mucosal fibrosis, whereas inhibiting ALDH activity in DCs or selectively depleting DCs markedly reduced fibrosis. Collectively, these data reveal a profibrotic ALDH-dependent pathway by DCs and uncover a role for DC retinoid metabolism. PMID:27595139

  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. PMID:27507940

  6. 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.

  7. 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

  8. Visible light may directly induce nuclear DNA damage triggering the death pathway in RGC-5 cells

    PubMed Central

    Fan, Bin; Ma, Tong-Hui

    2011-01-01

    Purpose Visible light has been previously demonstrated to induce retinal ganglion cell (RGC)-5 cell death through the mitochondrial pathway. The present study was designed to determine whether visible light might also directly trigger the death pathway by damaging nuclear DNA. Methods RGC-5 cells were exposed to various intensities and durations of visible light exposure. Cell viability and death were monitored with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide staining. Nuclear DNA damage caused by light was determined with the plasmid assay, genome DNA assay, and in situ terminal deoxynucleotidyl transferase dUTP nick end labeling. The subsequent activation of nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) was measured with western blot, and PARP-1’s role in the death pathway was assessed by using specific inhibitors. Poly (ADP-ribose) glycohydrolase and apoptosis-inducing factor (AIF) inhibitors were used to show their influence on light-induced cell death. Calcium influx was examined with the fura-2 assay and calcium channel blocker. Results We found that visible light induced RGC-5 cell death in a time- and intensity-dependent manner. After the light intensity was increased to 2,600 lx, activation of the death pathway in RGC-5 cells was clearly observed by detecting double-strand DNA breaks and nuclear DNA damage in vitro. Nuclear enzyme PARP-1 was promptly activated after exposure to 2,600 lx of light for 2 days, and specific inhibitors of PARP-1 had significant neuroprotective effects. The poly(ADP-ribose) glycohydrolase inhibitor tannic acid and AIF inhibitor N-phenylmaleimide partially protected RGC-5 cells from light injury. A massive calcium influx was detected after 2 days of light exposure, and a calcium channel blocker partially protected cells against light injury. Conclusions These results suggest that visible light exposure may directly cause nuclear DNA damage, which consequently activates

  9. Antipsychotic pathway genes with expression altered in opposite direction by antipsychotics and amphetamine.

    PubMed

    Ko, Françoise; Tallerico, Teresa; Seeman, Philip

    2006-08-01

    To develop a new strategy for identifying possible psychotic- or antipsychotic-related pathway genes, rats were treated with clinical doses of haloperidol and clozapine for 4 days, and the altered expression of genes was compared with the genes altered in expression after amphetamine sensitization. The objective was to identify genes with expression altered in the same direction by haloperidol and clozapine but in the opposite direction in the amphetamine-sensitized rat striatum. These criteria were met by 21 genes, consisting of 15 genes upregulated by amphetamine, and 6 genes downregulated by amphetamine. Of the 21 genes, 15 are not presently identified, and only 3 genes (cathepsin K, GRK6, and a gene with accession number AI177589) are located in chromosome regions known to be associated with schizophrenia.

  10. 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. PMID:26769097

  11. MDI: integrity index of cytoskeletal fibers observed by AFM

    NASA Astrophysics Data System (ADS)

    Manghi, Massimo; Bruni, Luca; Croci, Simonetta

    2016-06-01

    The Modified Directional Index (MDI) is a form factor of the angular spectrum computed from the 2D Fourier transform of an image marking the prevalence of rectilinear features throughout the picture. We study some properties of the index and we apply it to AFM images of cell cytoskeleton regions featuring patterns of rectilinear nearly parallel actin filaments as in the case of microfilaments grouped in bundles. The analysis of AFM images through MDI calculation quantifies the fiber directionality changes which could be related to fiber damages. This parameter is applied to the images of Hs 578Bst cell line, non-tumoral and not immortalized human epithelial cell line, irradiated with X-rays at doses equivalent to typical radiotherapy treatment fractions. In the reported samples, we could conclude that the damages are mainly born to the membrane and not to the cytoskeleton. It could be interesting to test the parameter also using other kinds of chemical or physical agents.

  12. High-speed AFM for Studying Dynamic Biomolecular Processes

    NASA Astrophysics Data System (ADS)

    Ando, Toshio

    2008-03-01

    Biological molecules show their vital activities only in aqueous solutions. It had been one of dreams in biological sciences to directly observe biological macromolecules (protein, DNA) at work under a physiological condition because such observation is straightforward to understanding their dynamic behaviors and functional mechanisms. Optical microscopy has no sufficient spatial resolution and electron microscopy is not applicable to in-liquid samples. Atomic force microscopy (AFM) can visualize molecules in liquids at high resolution but its imaging rate was too low to capture dynamic biological processes. This slow imaging rate is because AFM employs mechanical probes (cantilevers) and mechanical scanners to detect the sample height at each pixel. It is quite difficult to quickly move a mechanical device of macroscopic size with sub-nanometer accuracy without producing unwanted vibrations. It is also difficult to maintain the delicate contact between a probe tip and fragile samples. Two key techniques are required to realize high-speed AFM for biological research; fast feedback control to maintain a weak tip-sample interaction force and a technique to suppress mechanical vibrations of the scanner. Various efforts have been carried out in the past decade to materialize high-speed AFM. The current high-speed AFM can capture images on video at 30-60 frames/s for a scan range of 250nm and 100 scan lines, without significantly disturbing week biomolecular interaction. Our recent studies demonstrated that this new microscope can reveal biomolecular processes such as myosin V walking along actin tracks and association/dissociation dynamics of chaperonin GroEL-GroES that occurs in a negatively cooperative manner. The capacity of nanometer-scale visualization of dynamic processes in liquids will innovate on biological research. In addition, it will open a new way to study dynamic chemical/physical processes of various phenomena that occur at the liquid-solid interfaces.

  13. ezAFM: A low cost Atomic Force Microscope(AFM)

    NASA Astrophysics Data System (ADS)

    Celik, Umit; Celik, Kubra; Aslan, Husnu; Kehribar, Ihsan; Dede, Munir; Ozgur Ozer, H.; Oral, Ahmet

    2012-02-01

    A low cost AFM, ezAFM is developed for educational purposes as well as research. Optical beam deflection method is used to measure the deflection of cantilever. ezAFM scanner is built using voice coil motors (VCM) with ˜50x50x6 μm scan area. The microscope uses alignment free cantilevers, which minimizes setup times. FPGA based AFM feedback Control electronics is developed. FPGA technology allows us to drive all peripherals in parallel. ezAFM Controller is connected to PC by USB 2.0 interface as well as Wi-Fi. We have achieved <5nm lateral and ˜0.01nm vertical resolution. ezAFM can image single atomic steps in HOPG and mica. An optical microscope with <3 μm resolution is also integrated into the system. ezAFM supports different AFM operation modes such as dynamic mode, contact mode, lateral force microscopy. Advanced modes like magnetic force microscopy and electric force microscopy will be implemented later on. The new ezAFM system provides, short learning times for student labs, quick setup and easy to transport for portable applications with the best price/performance ratio. The cost of the system starts from 15,000, with system performance comparable with the traditional AFM systems.

  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. PMID:26934298

  15. 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.

  16. Direct vs. indirect pathway for nitrobenzene reduction reaction on a Ni catalyst surface: a density functional study.

    PubMed

    Mahata, Arup; Rai, Rohit K; Choudhuri, Indrani; Singh, Sanjay K; Pathak, Biswarup

    2014-12-21

    Density functional theory (DFT) calculations are performed to understand and address the previous experimental results that showed the reduction of nitrobenzene to aniline prefers direct over indirect reaction pathways irrespective of the catalyst surface. Nitrobenzene to aniline conversion occurs via the hydroxyl amine intermediate (direct pathway) or via the azoxybenzene intermediate (indirect pathway). Through our computational study we calculated the spin polarized and dispersion corrected reaction energies and activation barriers corresponding to various reaction pathways for the reduction of nitrobenzene to aniline over a Ni catalyst surface. The adsorption behaviour of the substrate, nitrobenzene, on the catalyst surface was also considered and the energetically most preferable structural orientation was elucidated. Our study indicates that the parallel adsorption behaviour of the molecules over a catalyst surface is preferable over vertical adsorption behaviour. Based on the reaction energies and activation barrier of the various elementary steps involved in direct or indirect reaction pathways, we find that the direct reduction pathway of nitrobenzene over the Ni(111) catalyst surface is more favourable than the indirect reaction pathway. PMID:25367892

  17. Direct Photolysis Rates and Transformation Pathways of the Lampricides TFM and Niclosamide in Simulated Sunlight.

    PubMed

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

    2016-09-20

    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. PMID:27508405

  18. The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis.

    PubMed

    Ausin, Israel; Greenberg, Maxim V C; Li, Carey Fei; Jacobsen, Steven E

    2012-01-01

    Cytosine DNA methylation is an epigenetic mark frequently associated with silencing of genes and transposons. In Arabidopsis, the establishment of cytosine DNA methylation is performed by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). DRM2 is guided to target sequences by small interfering RNAs (siRNAs) in a pathway termed RNA-directed DNA methylation (RdDM). We performed a screen for mutants that affect the establishment of DNA methylation by investigating genes that contain predicted RNA-interacting domains. After transforming FWA into 429 T-DNA insertion lines, we assayed for mutants that exhibited a late-flowering phenotype due to hypomethylated, thus ectopically expressed, copies of FWA. A T-DNA insertion line within the coding region of the spliceosome gene SR45 (sr45-1) flowered late after FWA transformation. Additionally, sr45-1 mutants display defects in the maintenance of DNA methylation. DNA methylation establishment and maintenance defects present in sr45-1 mutants are enhanced in dcl3-1 mutant background, suggesting a synergistic cooperation between SR45 and DICER-LIKE3 (DCL3) in the RdDM pathway. PMID:22274613

  19. The splicing factor SR45 affects the RNA-directed DNA methylation pathway in Arabidopsis

    PubMed Central

    Ausin, Israel; Greenberg, Maxim V.C.; Li, Carey Fei; Jacobsen, Steven E.

    2012-01-01

    Cytosine DNA methylation is an epigenetic mark frequently associated with silencing of genes and transposons. In Arabidopsis, the establishment of cytosine DNA methylation is performed by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). DRM2 is guided to target sequences by small interfering RNAs (siRNAs) in a pathway termed RNA-directed DNA methylation (RdDM). We performed a screen for mutants that affect the establishment of DNA methylation by investigating genes that contain predicted RNA-interacting domains. After transforming FWA into 429 T-DNA insertion lines, we assayed for mutants that exhibited a late-flowering phenotype due to hypomethylated, thus ectopically expressed, copies of FWA. A T-DNA insertion line within the coding region of the spliceosome gene SR45 (sr45-1) flowered late after FWA transformation. Additionally, sr45-1 mutants display defects in the maintenance of DNA methylation. DNA methylation establishment and maintenance defects present in sr45-1 mutants are enhanced in dcl3-1 mutant background, suggesting a synergistic cooperation between SR45 and DICER-LIKE3 (DCL3) in the RdDM pathway. PMID:22274613

  20. 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

  1. Pathways: lessons learned and future directions for school-based interventions among American Indians

    PubMed Central

    Gittelsohn, Joel; Davis, Sally M.; Steckler, Allan; Ethelbah, Becky; Clay, Theresa; Metcalfe, Lauve; Rock, Bonnie Holy

    2016-01-01

    Background Pathways, a multicenter study to test the effect of a school-based program to prevent obesity in American Indian children, yielded many benefits and encountered many challenges. This paper explores what we have learned from this study and examines possible future directions. Methods Information presented in this paper is based on formative research, study results, and discussions with staff and investigators. Results Some of the lessons learned relate to having a strong relationship with the tribes, how best to engage the communities, the importance of formative research and achieving standardization in culturally diverse settings, how to incorporate cultural information into curricula, and the importance of family involvement. One of the strengths of the study was the collaborative process that teamed American Indian and non-American Indian investigators and staff. Researchers recognized that they must work in cooperation with research participants including their schools and communities to address challenges, to ensure accurate findings and analyses, and to share benefits. Conclusions The lessons learned from Pathways offer valuable insights for researchers into successful approaches to the challenges inherent in research in American Indian communities, particularly in schools, and how to maximize the benefits of such a study. © 2003 American Health Foundation and Elsevier Inc. All rights reserved. PMID:14636815

  2. 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.

  3. Activated pathways for the directed insertion of patterned nanoparticles into polymer membranes.

    PubMed

    Ting, Christina L; Frischknecht, Amalie L

    2013-10-28

    We combine the string method with self-consistent field theory to compute the most probable transition pathway, i.e. the minimum free energy path, for the insertion of Janus and protein-like nanoparticles into a polymer membrane bilayer. The method makes no assumptions in the reaction coordinate and overcomes the long timescales challenge associated with simulating rare events. Our study suggests that one approach to building functional polymer–nanoparticle composite membranes with oriented nanoparticles is through electrostatic interactions. In particular, hydrophobic Janus nanoparticles with an asymmetric charge distribution can be made to directionally insert into charged membranes. This process is kinetically driven, and involves overcoming a thermally surmountable activation barrier, which requires favorable interactions between the nanoparticle and the hydrophilic block of the membrane. In contrast, the insertion of protein-like nanoparticles with alternating hydrophilic–hydrophobic–hydrophilic domains into polymer membranes does not occur as a thermally activated event. PMID:26029770

  4. 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.

  5. 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.

  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. Insights into Epoxy Network Nanostructural Heterogeneity Using AFM-IR.

    PubMed

    Morsch, Suzanne; Liu, Yanwen; Lyon, Stuart B; Gibbon, Simon R

    2016-01-13

    The first direct observation of a chemically heterogeneous nanostructure within an epoxy resin is reported. Epoxy resins comprise the matrix component of many high performance composites, coatings and adhesives, yet the molecular network structure that underpins the performance of these industrially essential materials is not well understood. Internal nodular morphologies have repeatedly been reported for epoxy resins analyzed using SEM or AFM, yet the origin of these features remains a contentious subject, and epoxies are still commonly assumed to be chemically homogeneous. Uniquely, in this contribution we use the recently developed AFM-IR technique to eliminate previous differences in interpretation, and establish that nodule features correspond to heterogeneous network connectivity within an epoxy phenolic formulation.

  8. 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. PMID:25944927

  9. 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.

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

    PubMed Central

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

    2015-01-01

    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 33P. 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. PMID:25944927

  11. Towards Direct Synthesis of Alane: A Predicted Defect-Mediated Pathway Confirmed Experimentally.

    PubMed

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

    2016-09-01

    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 10(4)  bar expected from bulk thermodynamics.

  12. Towards Direct Synthesis of Alane: A Predicted Defect-Mediated Pathway Confirmed Experimentally.

    PubMed

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

    2016-09-01

    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 10(4)  bar expected from bulk thermodynamics. PMID:27535100

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

    DOE PAGES

    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 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

  14. Probing Ternary Solvent Effect in High V(oc) Polymer Solar Cells Using Advanced AFM Techniques.

    PubMed

    Li, Chao; Ding, Yi; Soliman, Mikhael; Lorenzo, Josie; Dhasmana, Nitesh; Chantharasupawong, Panit; Ievlev, Anton V; Gesquiere, Andre J; Tetard, Laurene; Thomas, Jayan

    2016-02-01

    This work describes a simple method to develop a high V(oc) 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 V(oc) 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. Finally, 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. PMID:26807919

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

    PubMed

    Soto, Cinque; Ofek, Gilad; Joyce, M Gordon; 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.

  16. 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.

  17. Carcinogen-specific mutations in preferred Ras-Raf pathway oncogenes directed by strand bias.

    PubMed

    Keller, Ross R; Gestl, Shelley A; Lu, Amy Q; Hoke, Alicia; Feith, David J; Gunther, Edward J

    2016-08-01

    Carcinogen exposures inscribe mutation patterns on cancer genomes and sometimes bias the acquisition of driver mutations toward preferred oncogenes, potentially dictating sensitivity to targeted agents. Whether and how carcinogen-specific mutation patterns direct activation of preferred oncogenes remains poorly understood. Here, mouse models of breast cancer were exploited to uncover a mechanistic link between strand-biased mutagenesis and oncogene preference. When chemical carcinogens were employed during Wnt1-initiated mammary tumorigenesis, exposure to either 7,12-dimethylbenz(a)anthracene (DMBA) or N-ethyl-N-nitrosourea (ENU) dramatically accelerated tumor onset. Mammary tumors that followed DMBA exposure nearly always activated the Ras pathway via somatic Hras(CAA61CTA) mutations. Surprisingly, mammary tumors that followed ENU exposure typically lacked Hras mutations, and instead activated the Ras pathway downstream via Braf(GTG636GAG) mutations. Hras(CAA61CTA) mutations involve an A-to-T change on the sense strand, whereas Braf(GTG636GAG) mutations involve an inverse T-to-A change, suggesting that strand-biased mutagenesis may determine oncogene preference. To examine this possibility further, we turned to an alternative Wnt-driven tumor model in which carcinogen exposures augment a latent mammary tumor predisposition in Apc(min) mice. DMBA and ENU each accelerated mammary tumor onset in Apc(min) mice by introducing somatic, "second-hit" Apc mutations. Consistent with our strand bias model, DMBA and ENU generated strikingly distinct Apc mutation patterns, including stringently strand-inverse mutation signatures at A:T sites. Crucially, these contrasting signatures precisely match those proposed to confer bias toward Hras(CAA61CTA) versus Braf(GTG636GAG) mutations in the original tumor sets. Our findings highlight a novel mechanism whereby exposure history acts through strand-biased mutagenesis to specify activation of preferred oncogenes. PMID:27207659

  18. 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

  19. Confocal Raman-AFM, A New Tool for Materials Research

    NASA Astrophysics Data System (ADS)

    Schmidt, Ute

    2005-03-01

    Characterization of heterogeneous systems, e.g. polymers, on the nanometer scale continues to grow in importance and to impact key applications in the field of materials science, nanotechnology and catalysis. The development of advanced polymeric materials for such applications requires detailed information about the physical and chemical properties of these materials on the nanometer scale. However, some details about the phase-separation process in polymers are difficult to study with conventional characterization techniques due to the inability of these methods to chemically differentiate materials with good spatial resolution, without damage, staining or preferential solvent washing. The CR-AFM is a breakthrough in microscopy. It combines three measuring techniques in one instrument: a high resolution confocal optical microscope, an extremely sensitive Raman spectroscopy system, and an Atomic Force Microscope. Using this instrument, the high spatial and topographical resolution obtained with an AFM can be directly linked to the chemical information gained by Confocal Raman spectroscopy. To demonstrate the capabilities of this unique combination of measuring techniques, polymer blend films, spin coated on glass substrates, have been characterized. AFM measurements reveal the structural and mechanical properties of the films, whereas Raman spectral images show the chemical composition of the blends.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

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

    PubMed

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

    2015-07-22

    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

  5. 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

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

    PubMed Central

    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.

    2015-01-01

    Summary 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 CAPRISA-donor CAP256; each antibody contained the protruding tyrosine-sulfated, anionic antigen-binding loop (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 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, an important vaccine insight. PMID:24590074

  7. 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.

  8. DNA Double Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities

    PubMed Central

    Shibata, Atsushi; Moiani, Davide; Arvai, Andrew S.; Perry, J. Jefferson P.; Harding, Shane M.; Genois, Marie-Michelle; Maity, Ranjan; van Rossum-Fikkert, Sari; Kertokalio, Aryandi; Romoli, Filippo; Ismail, Amani; Ismalaj, Ermal; Petricci, Elena; Matthew, J Neale; Bristow, Robert G; Masson, Jean-Yves; Wyman, Claire; Jeggo, Penny; Tainer, John A.

    2014-01-01

    SUMMARY MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection and signaling; yet, how its endo- and exonuclease activities regulate DSB repair by non-homologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors we examined repair pathway choice at DSBs generated in G2 following radiation exposure. Whilst endo- or exonuclease inhibition impairs radiation-induced RPA chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, whilst exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exo and EXO1/BLM bidirectional resection towards and away from the DNA end, which commits to HR. PMID:24316220

  9. A new device concept for directly modulating spinal cord pathways: initial in vivo experimental results.

    PubMed

    Flouty, Oliver; Oya, Hiroyuki; Kawasaki, Hiroto; Wilson, Saul; Reddy, Chandan G; Jeffery, Nicholas D; Brennan, Timothy J; Gibson-Corley, Katherine N; Utz, Marcel; Gillies, George T; Howard, Matthew A

    2012-12-01

    We describe a novel spinal cord (SC) stimulator that is designed to overcome a major shortcoming of existing stimulator devices: their restricted capacity to selectively activate targeted axons within the dorsal columns. This device overcomes that limitation by delivering electrical stimuli directly to the pial surface of the SC. Our goal in testing this device was to measure its ability to physiologically activate the SC and examine its capacity to modulate somatosensory evoked potentials (SSEPs) triggered by peripheral stimulation. In this acute study on adult sheep (n = 7), local field potentials were recorded from a grid placed in the subdural space of the right hemisphere during electrical stimulation of the left tibial nerve and the spinal cord. Large amplitude SSEPs (>200 µV) in response to SC stimulation were consistently obtained at stimulation strengths well below the thresholds inducing neural injury. Moreover, stimulation of the dorsal columns with signals employed routinely by devices in standard clinical use, e.g., 50 Hz, 0.2 ms pulse width, produced long-lasting changes (>4.5 h) in the SSEP patterns produced by subsequent tibial nerve stimulation. The results of these acute experiments demonstrate that this device can be safely secured to the SC surface and effectively activate somatosensory pathways. PMID:23151433

  10. Slug contributes to cancer progression by direct regulation of ERα signaling pathway.

    PubMed

    Li, Youqiang; Wu, Yanyuan; Abbatiello, Thomas C; Wu, Warren L; Kim, Ju Ri; Sarkissyan, Marianna; Sarkissyan, Suren; Chung, Seyung S; Elshimali, Yahya; Vadgama, Jaydutt V

    2015-04-01

    Hormone therapy targeting estrogen receptor α (ERα) is the most effective treatment for breast cancer. However, this treatment eventually fails as the tumor develops resistance. Although reduced expression of ER-α is a known contributing factor to endocrine resistance, the mechanism of ER-α downregulation in endocrine resistance is still not fully understood. The present study shows that Slug has an inverse relationship with ERα in breast and prostate cancer patient samples. Also the inhibition of Slug blocks mammary stem cell activity in primary mammary epithelial cells. We hypothesize that Slug may be a key transcription factor in the regulation of ERα expression. To understand the Slug-ERα signaling pathway, we employed resistant cell line MCF-TAMR (ERα relatively negative) derived from its parental MCF-7 (ERα positive) cell line and assessed changes in cell phenotype, activity and response to therapy. Conversely, we performed knockdown of Slug in the high-Slug expressing cell line MDA-MB-231 and assessed reversal of the mesenchymal phenotype. Microarray analysis showed that Slug is overexpressed in high grade breast and prostate cancer tissues. Additionally, Slug overexpression leads to drug resistance. Furthermore, we demonstrated that Slug binds directly to ERα promoter E-boxes and represses ERα expression. This resulted in decrease in epithelial-to-mesenchymal transition in cancer cells. These findings demonstrate that Slug, by regulation of ERα expression, contributes to tumor progression and could serve as an important target for cancer therapy.

  11. 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.

  12. 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.

  13. 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.…

  14. 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,…

  15. 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…

  16. Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

    PubMed Central

    Spence, Jennifer S.; Krause, Tyler B.; Mittler, Eva; Jangra, Rohit K.

    2016-01-01

    ABSTRACT Ebola virus (EBOV) makes extensive and intricate use of host factors in the cellular endosomal/lysosomal pathway to release its genome into the cytoplasm and initiate infection. Following viral internalization into endosomes, host cysteine proteases cleave the EBOV fusion glycoprotein (GP) to unmask the binding site for its intracellular receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). GP-NPC1 interaction is required for viral entry. Despite these and other recent discoveries, late events in EBOV entry following GP-NPC1 binding and culminating in GP-catalyzed fusion between viral and cellular lipid bilayers remain enigmatic. A mechanistic understanding of EBOV membrane fusion has been hampered by the failure of previous efforts to reconstitute fusion in vitro or at the cell surface. This report describes an assay to monitor initial steps directly in EBOV membrane fusion—triggering of GP and virus-cell lipid mixing—by single virions in live cells. Fusogenic triggering of GP occurs predominantly in Rab7-positive (Rab7+) endosomes, absolutely requires interaction between proteolytically primed GP and NPC1, and is blocked by key GP-specific neutralizing antibodies with therapeutic potential. Unexpectedly, cysteine protease inhibitors do not inhibit lipid mixing by virions bearing precleaved GP, even though they completely block cytoplasmic entry by these viruses, as shown previously. These results point to distinct cellular requirements for different steps in EBOV membrane fusion and suggest a model in which host cysteine proteases are dispensable for GP fusion triggering after NPC1 binding but are required for the formation of fusion pores that permit genome delivery. PMID:26861015

  17. An approach towards 3D sensitive AFM cantilevers

    NASA Astrophysics Data System (ADS)

    Koops, Richard; Fokkema, Vincent

    2014-04-01

    The atomic force microscope (AFM) tapping mode is a highly sensitive local probing technique that is very useful to study and measure surface properties down to the atomic scale. The tapping mode is mostly implemented using the resonance of the first bending mode of the cantilever and therefore provides sensitivity mainly along the direction of this oscillation. Driven by the semiconductor industry, there is an increasing need for accurate measurements of nanoscale structures for side wall characterization by AFM that requires additional sensitivity in the lateral direction. The conventional tapping mode has been augmented by various authors, for example by tilting the cantilever system (Cho et al 2011 Rev. Sci. Instrum. 82 023707) to access the sidewall or using a torsion mode (Dai et al 2011 Meas. Sci. Technol. 22 094009) of the cantilever to provide additional lateral sensitivity. These approaches however trade lateral sensitivity for vertical sensitivity or still lack sensitivity in the remaining lateral direction. We present an approach towards true 3D sensitivity for AFM cantilevers based on simultaneous excitation and optical detection of multiple cantilever resonance modes along three axes. Tuning the excitation of the cantilever to specific frequencies provides a mechanism to select only those cantilever modes that have the desired characteristics. Additionally, cantilever engineering has been used to design and create a substructure within the cantilever that has been optimized for specific resonance behavior around 4 MHz. In contrast to the conventional approach of using a piezo to actuate the cantilever modulation, we present results on photo-thermal excitation using an intensity modulated low-power laser source. By tightly focusing the excitation spot on the cantilever we were able to attain a deflection efficiency of 0.7 nm µW-1 for the first bending mode. The presented approach results in an efficient all optical excitation and deflection detection

  18. Insight into mechanics of AFM tip-based nanomachining: bending of cantilevers and machined grooves

    NASA Astrophysics Data System (ADS)

    Al-Musawi, R. S. J.; Brousseau, E. B.; Geng, Y.; Borodich, F. M.

    2016-09-01

    Atomic force microscope (AFM) tip-based nanomachining is currently the object of intense research investigations. Values of the load applied to the tip at the free end of the AFM cantilever probe used for nanomachining are always large enough to induce plastic deformation on the specimen surface contrary to the small load values used for the conventional contact mode AFM imaging. This study describes an important phenomenon specific for AFM nanomachining in the forward direction: under certain processing conditions, the deformed shape of the cantilever probe may change from a convex to a concave orientation. The phenomenon can principally change the depth and width of grooves machined, e.g. the grooves machined on a single crystal copper specimen may increase by 50% on average following such a change in the deformed shape of the cantilever. It is argued that this phenomenon can take place even when the AFM-based tool is operated in the so-called force-controlled mode. The study involves the refined theoretical analysis of cantilever probe bending, the analysis of experimental signals monitored during the backward and forward AFM tip-based machining and the inspection of the topography of produced grooves.

  19. Insight into mechanics of AFM tip-based nanomachining: bending of cantilevers and machined grooves.

    PubMed

    Al-Musawi, R S J; Brousseau, E B; Geng, Y; Borodich, F M

    2016-09-23

    Atomic force microscope (AFM) tip-based nanomachining is currently the object of intense research investigations. Values of the load applied to the tip at the free end of the AFM cantilever probe used for nanomachining are always large enough to induce plastic deformation on the specimen surface contrary to the small load values used for the conventional contact mode AFM imaging. This study describes an important phenomenon specific for AFM nanomachining in the forward direction: under certain processing conditions, the deformed shape of the cantilever probe may change from a convex to a concave orientation. The phenomenon can principally change the depth and width of grooves machined, e.g. the grooves machined on a single crystal copper specimen may increase by 50% on average following such a change in the deformed shape of the cantilever. It is argued that this phenomenon can take place even when the AFM-based tool is operated in the so-called force-controlled mode. The study involves the refined theoretical analysis of cantilever probe bending, the analysis of experimental signals monitored during the backward and forward AFM tip-based machining and the inspection of the topography of produced grooves. PMID:27532247

  20. Fabrication of carbon nanotube AFM probes using the Langmuir-Blodgett technique.

    PubMed

    Lee, Jae-Hyeok; Kang, Won-Seok; Choi, Bung-Sam; Choi, Sung-Wook; Kim, Jae-Ho

    2008-09-01

    Carbon nanotube (CNT)-tipped atomic force microscopy (AFM) probes have shown a significant potential for obtaining high-resolution imaging of nanostructure and biological materials. In this paper, we report a simple method to fabricate single-walled carbon nanotube (SWNT) nanoprobes for AFM using the Langmuir-Blodgett (LB) technique. Thiophenyl-modified SWNTs (SWNT-SHs) through amidation of SWNTs in chloroform allowed to be spread and form a stable Langmuir monolayer at the water/air interface. A simple two-step transfer process was used: (1) dipping conventional AFM probes into the Langmuir monolayer and (2) lifting the probes from the water surface. This results in the attachment of SWNTs onto the tips of AFM nanoprobes. We found that the SWNTs assembled on the nanoprobes were well-oriented and robust enough to maintain their shape and direction even after successive scans. AFM measurements of a nano-porous alumina substrate and deoxyribonucleic acid using SWNT-modified nanoprobes revealed that the curvature diameter of the nanoprobes was less than 3 nm and a fine resolution was obtained than that from conventional AFM probes. We also demonstrate that the LB method is a scalable process capable of simultaneously fabricating a large number of SWNT-modified nanoprobes.

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

    DOE PAGES

    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 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

  2. Targeting the extrinsic apoptotic pathway in cancer: lessons learned and future directions

    PubMed Central

    Ashkenazi, Avi

    2015-01-01

    Apoptosis is a metazoan process of controlled cell elimination that plays critical roles in embryonic development and adult tissue homeostasis. Apoptosis dysregulation contributes to several important diseases, including cancer. Two distinct yet interconnected signaling pathways control apoptosis by activating a core intracellular machinery of death proteases called caspases. The intrinsic apoptotic pathway engages caspases via members of the BCL-2 protein family and the mitochondria in reaction to severe cellular damage or stress. The extrinsic pathway activates caspases via cell-surface death receptors, which respond to cognate death ligands expressed on immune-effector cells. Tumor cells can acquire various apoptosis-evasion mechanisms; nevertheless, the transformed state of these cells makes them uniquely susceptible to apoptosis reactivation if resistance is circumvented. Molecular approaches to reengage the apoptotic pathways in cancer have been underway for over two decades. Gratifyingly, BCL-2 antagonists — which drive the intrinsic pathway — are beginning to bear clinical fruit. In contrast, clinical attempts to stimulate the extrinsic pathway with proapoptotic receptor agonists (PARAs) have been disappointing, despite compelling preclinical efficacy with this class of agents. Here, I discuss some of the possible reasons for this translational discrepancy and suggest strategies to overcome it with the next generation of PARAs. PMID:25642709

  3. AFM nanoindentations of diatom biosilica surfaces.

    PubMed

    Losic, Dusan; Short, Ken; Mitchell, James G; Lal, Ratnesh; Voelcker, Nicolas H

    2007-04-24

    Diatoms have intricately and uniquely nanopatterned silica exoskeletons (frustules) and are a common target of biomimetic investigations. A better understanding of the diatom frustule structure and function at the nanoscale could provide new insights for the biomimetic fabrication of nanostructured ceramic materials and lightweight, yet strong, scaffold architectures. Here, we have mapped the nanoscale mechanical properties of Coscinodiscus sp. diatoms using atomic force microscopy (AFM)-based nanoindentation. Mechanical properties were correlated with the frustule structures obtained from high-resolution AFM and scanning electron microscopy (SEM). Significant differences in the micromechanical properties for the different frustule layers were observed. A comparative study of other related inorganic material including porous silicon films and free-standing membranes as well as porous alumina was also undertaken.

  4. 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.

  5. 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. PMID:21965577

  6. 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.

  7. Formally direct pathways and low-temperature chain branching in hydrocarbon autoignition : the cyclohexyl + O2 reaction at high pressure.

    SciTech Connect

    Taatjes, Craig A.; Jusinski, Leonard E.; Zador, Judit; Fernandes, Ravi X.; Miller, James A.

    2008-10-01

    The OH concentration in the Cl-initiated oxidation of cyclohexane has been measured between 6.5-20.3 bar and in the 586-828 K temperature range by a pulsed-laser photolytic initiation--laser-induced fluorescence method. The experimental OH profiles are modeled by using a master-equation-based kinetic model as well as a comprehensive literature mechanism. Below 700 K OH formation takes place on two distinct time-scales, one on the order of microseconds and the other over milliseconds. Detailed modeling demonstrates that formally direct chemical activation pathways are responsible for the OH formation on short timescales. These results establish that formally direct pathways are surprisingly important even for relatively large molecules at the pressures of practical combustors. It is also shown that remaining discrepancies between model and experiment are attributable to low-temperature chain branching from the addition of the second oxygen to hydroperoxycyclohexyl radicals.

  8. Coding the direct/indirect pathways by D1 and D2 receptors is not valid for accumbens projections.

    PubMed

    Kupchik, Yonatan M; Brown, Robyn M; Heinsbroek, Jasper A; Lobo, Mary Kay; Schwartz, Danielle J; Kalivas, Peter W

    2015-09-01

    It is widely accepted that D1 dopamine receptor-expressing striatal neurons convey their information directly to the output nuclei of the basal ganglia, whereas D2-expressing neurons do so indirectly via pallidal neurons. Combining optogenetics and electrophysiology, we found that this architecture does not apply to mouse nucleus accumbens projections to the ventral pallidum. Thus, current thinking attributing D1 and D2 selectivity to accumbens projections akin to dorsal striatal pathways needs to be reconsidered.

  9. 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…

  10. 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

  11. 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.

  12. 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).

  13. 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

  14. TFPI inhibits lectin pathway of complement activation by direct interaction with MASP-2.

    PubMed

    Keizer, Mischa P; Pouw, Richard B; Kamp, Angela M; Patiwael, Sanne; Marsman, Gerben; Hart, Margreet H; Zeerleder, Sacha; Kuijpers, Taco W; Wouters, Diana

    2015-02-01

    The lectin pathway (LP) of complement has a protective function against invading pathogens. Recent studies have also shown that the LP plays an important role in ischemia/reperfusion (I/R)-injury. MBL-associated serine protease (MASP)-2 appears to be crucial in this process. The serpin C1-inhibitor is the major inhibitor of MASP-2. In addition, aprotinin, a Kunitz-type inhibitor, was shown to inhibit MASP-2 activity in vitro. In this study we investigated whether the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI) is also able to inhibit MASP-2. Ex vivo LP was induced and detected by C4-deposition on mannan-coated plates. The MASP-2 activity was measured in a fluid-phase chromogenic assay. rTFPI in the absence or presence of specific monoclonal antibodies was used to investigate which TFPI-domains contribute to MASP-2 inhibition. Here, we identify TFPI as a novel selective inhibitor of MASP-2, without affecting MASP-1 or the classical pathway proteases C1s and C1r. Kunitz-2 domain of TFPI is required for the inhibition of MASP-2. Considering the role of MASP-2 in complement-mediated I/R-injury, the inhibition of this protease by TFPI could be an interesting therapeutic approach to limit the tissue damage in conditions such as cerebral stroke, myocardial infarction or solid organ transplantation.

  15. Quantifying hepatic glycogen synthesis by direct and indirect pathways in rats under normal ad libitum feeding conditions.

    PubMed

    Soares, Ana F; Viega, Francisco J; Carvalho, Rui A; Jones, John G

    2009-01-01

    Hepatic glycogen synthesis from intact hexose (direct pathway) relative to that from gluconeogenic precursors (indirect pathway) was quantified in ad libitum-fed rats. Following (2)H(2)O administration and overnight feeding, the livers were removed and glycogen (2)H-enrichment was measured by (2)H NMR. Six controls and six rats rendered hyperglycemic by streptozotocin (STZ; fasting blood glucose = 385 +/- 31 mg/dl) were studied. The indirect pathway contribution, estimated as glycogen hydrogen 5 relative to hydrogen 2 enrichment, was 54% +/- 4% for control rats-similar to values from healthy, meal-fed humans. In STZ-treated rats, the indirect pathway contribution was significantly higher (68% +/- 4%, P < 0.05 vs. controls), similar to that of Type 1 diabetic (T1D) patients. In conclusion, sources of hepatic glycogen synthesis in rats during ad libitum nocturnal feeding were quantified by analysis of glycogen enrichment from (2)H(2)O. STZ caused alterations resembling the pathophysiology of hepatic glycogen synthesis in T1D patients.

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

    PubMed

    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

  17. 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.

  18. 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

  19. Common BRAF(V600E)-directed pathway mediates widespread epigenetic silencing in colorectal cancer and melanoma

    PubMed Central

    Fang, Minggang; Hutchinson, Lloyd; Deng, April

    2016-01-01

    During cancer development, it is well established that many genes, including tumor suppressor genes, are hypermethylated and transcriptionally repressed, a phenomenon referred to as epigenetic silencing. In general, the factors involved in, and the mechanistic basis of, epigenetic silencing during cancer development are not well understood. We have recently described an epigenetic silencing pathway, directed by the oncogenic B-Raf proto-oncogene (BRAF) variant BRAF(V600E), that mediates widespread epigenetic silencing in colorectal cancer (CRC). Notably, the BRAF(V600E) mutation is also present in 50–70% of melanomas. Here, we show that the same pathway we identified in CRC also directs epigenetic silencing of a similar set of genes in BRAF-positive melanoma. In both CRC and melanoma, BRAF(V600E) promotes epigenetic silencing through up-regulation of v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog G (MAFG), a transcriptional repressor with sequence-specific DNA-binding activity. The elevated concentration of MAFG drives DNA binding on the promoter. Promoter-bound MAFG recruits a set of corepressors that includes its heterodimeric partner BTB and CNC homology 1, basic leucine zipper transcription factor 1 (BACH1), the chromatin remodeling factor chromodomain helicase DNA-binding protein 8 (CHD8), and the DNA methyltransferase DNMT3B, resulting in hypermethylation and transcriptional silencing. Our results reveal a common BRAF(V600E)-directed transcriptional regulatory pathway that mediates epigenetic silencing in unrelated solid tumors and provide strong support for an instructive model of oncoprotein-directed epigenetic silencing. PMID:26787892

  20. Cells mechanics with AFM: problems and solutions

    NASA Astrophysics Data System (ADS)

    Sokolov, Igor; Dokukin, Maxim; Guz, Nataliia

    2012-02-01

    Atomic force microscopy (AFM) is used to study mechanics of cells. Cell is far from being a homogeneous medium. This creates a number of problems which will be discussed as well as the ways to solve them. We will focus on the following problems: 1. Cellular surface brush (microvilli, glycocalyx..) surrounds cells. A simple model to separate the brush and cell deformation will be overviewed. 2. Problem of nonlinearity of stress-strain relation. Although unsolved, this may indirectly tested: the rigidity modulus should be reasonably independent of penetration. We will show that it can be achieved when using relatively dull AFM probes (the radii of microns) only. 3. Heterogeneity of cell surface. Cells are not homogeneous over the surface. We will discuss the question how many points is enough to characterize the cell. We will exemplify the above with human cervical epithelial cells. We will demonstrate that without proper consideration of the above problems, the error in defining the modulus of rigidity can easily reach an order of magnitude.

  1. 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

  2. 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

  3. Structural insight into iodide uptake by AFm phases.

    PubMed

    Aimoz, Laure; Wieland, Erich; Taviot-Guého, Christine; Dähn, Rainer; Vespa, Marika; Churakov, Sergey V

    2012-04-01

    The ability of cement phases carrying positively charged surfaces to retard the mobility of (129)I, present as iodide (I(-)) in groundwater, was investigated in the context of safe disposal of radioactive waste. (125)I sorption experiments on ettringite, hydrotalcite, chloride-, carbonate- and sulfate-containing AFm phases indicated that calcium-monosulfate (AFm-SO(4)) is the only phase that takes up trace levels of iodide. The structures of AFm phases prepared by coprecipitating iodide with other anions were investigated in order to understand this preferential uptake mechanism. X-ray diffraction (XRD) investigations showed a segregation of monoiodide (AFm-I(2)) and Friedel's salt (AFm-Cl(2)) for I-Cl mixtures, whereas interstratifications of AFm-I(2) and hemicarboaluminate (AFm-OH-(CO(3))(0.5)) were observed for the I-CO(3) systems. In contrast, XRD measurements indicated the formation of a solid solution between AFm-I(2) and AFm-SO(4) for the I-SO(4) mixtures. Extended X-ray absorption fine structure spectroscopy showed a modification of the coordination environment of iodine in I-CO(3) and in I-SO(4) samples compared to pure AFm-I(2). This is assumed to be due to the introduction of stacking faults in I-CO(3) samples on one hand and due to the presence of sulfate and associated space-filling water molecules as close neighbors in I-SO(4) samples on the other hand. The formation of a solid solution between AFm-I(2) and AFm-SO(4), with a short-range mixing of iodide and sulfate, implies that AFm-SO(4) bears the potential to retard (129)I. PMID:22376086

  4. A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway.

    PubMed

    Lange, B M; Wildung, M R; McCaskill, D; Croteau, R

    1998-03-01

    Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms. However, based on in vivo feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors. Here we describe the cloning from peppermint (Mentha x piperita) and heterologous expression in Escherichia coli of 1-deoxy-D-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway. This synthase gene contains an ORF of 2,172 base pairs. When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa. The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants. PMID:9482845

  5. Immunomodulatory glycan LNFPIII alleviates hepatosteatosis and insulin resistance through direct and indirect control of metabolic pathways.

    PubMed

    Bhargava, Prerna; Li, Changlin; Stanya, Kristopher J; Jacobi, David; Dai, Lingling; Liu, Sihao; Gangl, Matthew R; Harn, Donald A; Lee, Chih-Hao

    2012-11-01

    Parasitic worms express host-like glycans to attenuate the immune response of human hosts. The therapeutic potential of this immunomodulatory mechanism in controlling the metabolic dysfunction that is associated with chronic inflammation remains unexplored. We demonstrate here that administration of lacto-N-fucopentaose III (LNFPIII), a Lewis(X)-containing immunomodulatory glycan found in human milk and on parasitic helminths, improves glucose tolerance and insulin sensitivity in diet-induced obese mice. This effect is mediated partly through increased interleukin-10 (Il-10) production by LNFPIII-activated macrophages and dendritic cells, which reduces white adipose tissue inflammation and sensitizes the insulin response of adipocytes. Concurrently, LNFPIII treatment upregulates nuclear receptor subfamily 1, group H, member 4 (Fxr-α, also known as Nr1h4) to suppress lipogenesis in the liver, conferring protection against hepatosteatosis. At the signaling level, the extracellular signal-regulated kinase (Erk)-activator protein 1 (Ap1) pathway seems to mediate the effects of LNFPIII on both inflammatory and metabolic pathways. Our results suggest that LNFPIII may provide new therapeutic approaches to treat metabolic diseases.

  6. 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.

  7. 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.

  8. 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. PMID:26745295

  9. 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).

  10. Direct evidences on bacterial growth pattern regulating pyrene degradation pathway and genotypic dioxygenase expression.

    PubMed

    Chen, Baowei; Huang, Jinyin; Yuan, Ke; Lin, Li; Wang, Xiaowei; Yang, Lihua; Luan, Tiangang

    2016-04-15

    Pyrene degradation by Mycobacterium sp. strain A1-PYR was investigated in the presence of nutrient broth, phenanthrene and fluoranthene, respectively. Fast bacterial growth in the nutrient broth considerably enhanced pyrene degradation rate, whereas degradation efficiency per cell was substantially decreased. The addition of nutrient broth could not alter the transcription levels of all dioxygenase genotypes. In the PAH-only substrates, bacterial growth completely relied on biological conversion of PAHs into the effective carbon sources, which led to a higher degradation efficiency of pyrene per cell than the case of nutrient broth. Significant correlations were only observed between nidA-related dioxygenase expression and pyrene degradation or bacterial growth. The highest pyrene degradation rate in the presence of phenanthrene was consistent with the highest transcription level of nidA and 4,5-pyrenediol as the sole initial metabolite. This study reveals that bacterial growth requirement can invigorate degradation of PAHs by regulating metabolic pathway and genotypic enzyme expression.

  11. [AFM fishing of proteins under impulse electric field].

    PubMed

    Ivanov, Yu D; Pleshakova, T O; Malsagova, K A; Kaysheva, A L; Kopylov, A T; Izotov, A A; Tatur, V Yu; Vesnin, S G; Ivanova, N D; Ziborov, V S; Archakov, A I

    2016-05-01

    A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell. PMID:27562998

  12. [AFM fishing of proteins under impulse electric field].

    PubMed

    Ivanov, Yu D; Pleshakova, T O; Malsagova, K A; Kaysheva, A L; Kopylov, A T; Izotov, A A; Tatur, V Yu; Vesnin, S G; Ivanova, N D; Ziborov, V S; Archakov, A I

    2016-05-01

    A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell.

  13. The Spn4 gene of Drosophila encodes a potent furin-directed secretory pathway serpin

    PubMed Central

    Richer, Martin J.; Keays, Clairessa A.; Waterhouse, Jennifer; Minhas, Jessey; Hashimoto, Carl; Jean, François

    2004-01-01

    Proprotein convertases (PCs) are an important class of host-cell serine endoproteases implicated in many physiological and pathological processes. Owing to their expanding roles in the proteolytic events required for generating infectious microbial pathogens and for tumor growth and invasiveness, there is increasing interest in identifying endogenous PC inhibitors. Here we report the identification of Spn4A, a previously uncharacterized secretory pathway serine protease inhibitor (serpin) from Drosophila melanogaster that contains a consensus furin cleavage site, -ArgP4-Arg-Lys-ArgP1↓-, in its reactive site loop (RSL). Our biochemical and kinetics analysis revealed that recombinant Spn4A inhibits human furin (Ki, 13 pM; kass, 3.2 × 107 M–1·s–1) and Drosophila PC2 (Ki, 3.5 nM; kass, 9.2 × 104 M–1·s–1) by a slow-binding mechanism characteristic of serpin molecules and forms a kinetically trapped SDS-stable complex with each enzyme. For both PCs, the stoichiometry of inhibition by Spn4A is nearly 1, which is characteristic of known physiological serpin–protease interactions. Mass analysis of furin–Spn4A reaction products identified the actual reactive site center of Spn4A to be -ArgP4-Arg-Lys-ArgP1↓-. Moreover, we demonstrate that Spn4A's highly effective PC inhibition properties are critically dependent on the unusual length of its RSL, which is composed of 18 aa instead of the typical 17-residue RSL found in most other inhibitory serpins. The identification of Spn4A, the most potent and effective natural serpin of PCs identified to date, suggests that Spn4A could be a prototype of endogenous serpins involved in the precise regulation of PC-dependent proteolytic cleavage events in the secretory pathway of eukaryotic cells. PMID:15247425

  14. Site-directed mutagenesis of aldehyde dehydrogenase-2 suggests three distinct pathways of nitroglycerin biotransformation.

    PubMed

    Wenzl, M Verena; Beretta, Matteo; Griesberger, Martina; Russwurm, Michael; Koesling, Doris; Schmidt, Kurt; Mayer, Bernd; Gorren, Antonius C F

    2011-08-01

    To elucidate the mechanism underlying reduction of nitroglycerin (GTN) to nitric oxide (NO) by mitochondrial aldehyde dehydrogenase (ALDH2), we generated mutants of the enzyme lacking the cysteines adjacent to reactive Cys302 (C301S and C303S), the glutamate that participates as a general base in aldehyde oxidation (E268Q) or combinations of these residues. The mutants were characterized regarding acetaldehyde dehydrogenation, GTN-triggered enzyme inactivation, GTN denitration, NO formation, and soluble guanylate cyclase activation. Lack of the cysteines did not affect dehydrogenase activity but impeded GTN denitration, aggravated GTN-induced enzyme inactivation, and increased NO formation. A triple mutant lacking the cysteines and Glu268 catalyzed sustained formation of superstoichiometric amounts of NO and exhibited slower rates of inactivation. These results suggest three alternative pathways for the reaction of ALDH2 with GTN, all involving formation of a thionitrate/sulfenyl nitrite intermediate at Cys302 as the initial step. In the first pathway, which predominates in the wild-type enzyme and reflects clearance-based GTN denitration, the thionitrate apparently reacts with one of the adjacent cysteine residues to yield nitrite and a protein disulfide. The predominant reaction catalyzed by the single and double cysteine mutants requires Glu268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu268 shifts the reaction toward formation of the free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Although the latter reaction accounts for less than 10% of total turnover of GTN metabolism catalyzed by wild-type ALDH2, it is most likely essential for vascular GTN bioactivation.

  15. Examining direct and indirect pathways to health behaviour: the influence of cognitive and affective probability beliefs.

    PubMed

    Janssen, Eva; van Osch, Liesbeth; de Vries, Hein; Lechner, Lilian

    2013-01-01

    This study aimed to extricate the influence of rational (e.g., 'I think …') and intuitive (e.g., 'I feel …') probability beliefs in the behavioural decision-making process regarding skin cancer prevention practices. Structural equation modelling was used in two longitudinal surveys (sun protection during winter sports [N = 491]; sun protection during summer [N = 277]) to examine direct and indirect behavioural effects of affective and cognitive likelihood (i.e. unmediated or mediated by intention), controlled for attitude, social influence and self-efficacy. Affective likelihood was directly related to sun protection in both studies, whereas no direct effects were found for cognitive likelihood. After accounting for past sun protective behaviour, affective likelihood was only directly related to sun protection in Study 1. No support was found for the indirect effects of affective and cognitive likelihood through intention. The findings underscore the importance of feelings of (cancer) risk in the decision-making process and should be acknowledged by health behaviour theories and risk communication practices. Suggestions for future research are discussed.

  16. 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

  17. 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).

  18. 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).

  19. Nuclear disconnection within the amygdala reveals a direct pathway to fear.

    PubMed

    Jimenez, Stephanie A; Maren, Stephen

    2009-12-01

    It is widely believed that a descending serial circuit consisting of neural projections from the basolateral complex (BLA) to the central nucleus (CEA) of the amygdala mediates fear expression. Here we directly test this hypothesis and show that disconnecting the BLA and CEA with asymmetric neurotoxic lesions after Pavlovian fear conditioning in rats completely abolishes the expression of conditional freezing. These results demonstrate that neural projections from the BLA to CEA are essential for the expression of learned fear responses. PMID:19933881

  20. Trypanosoma cruzi calreticulin inhibits the complement lectin pathway activation by direct interaction with L-Ficolin.

    PubMed

    Sosoniuk, Eduardo; Vallejos, Gerardo; Kenawy, Hany; Gaboriaud, Christine; Thielens, Nicole; Fujita, Teizo; Schwaeble, Wilhelm; Ferreira, Arturo; Valck, Carolina

    2014-07-01

    Trypanosoma cruzi, the agent of Chagas' disease, the sixth neglected tropical disease worldwide, infects 10-12 million people in Latin America. Differently from T. cruzi epimastigotes, trypomastigotes are complement-resistant and infective. CRPs, T-DAF, sialic acid and lipases explain at least part of this resistance. In vitro, T. cruzi calreticulin (TcCRT), a chaperone molecule that translocates from the ER to the parasite surface: (a) Inhibits the human classical complement activation, by interacting with C1, (b) As a consequence, an increase in infectivity is evident and, (c) It inhibits angiogenesis and tumor growth. We report here that TcCRT also binds to the L-Ficolin collagenous portion, thus inhibiting approximately between 35 and 64% of the human complement lectin pathway activation, initiated by L-Ficolin, a property not shared by H-Ficolin. While L-Ficolin binds to 60% of trypomastigotes and to 24% of epimastigotes, 50% of the former and 4% of the latter display TcCRT on their surfaces. Altogether, these data indicate that TcCRT is a parasite inhibitory receptor for Ficolins. The resulting evasive activities, together with the TcCRT capacity to inhibit C1, with a concomitant increase in infectivity, may represent T. cruzi strategies to inhibit important arms of the innate immune response.

  1. Targeted expression of μ-opioid receptors in a subset of striatal direct-pathway neurons restores opiate reward.

    PubMed

    Cui, Yijun; Ostlund, Sean B; James, Alex S; Park, Chang Sin; Ge, Weihong; Roberts, Kristofer W; Mittal, Nitish; Murphy, Niall P; Cepeda, Carlos; Kieffer, Brigitte L; Levine, Michael S; Jentsch, James David; Walwyn, Wendy M; Sun, Yi E; Evans, Christopher J; Maidment, Nigel T; Yang, X William

    2014-02-01

    μ-opioid receptors (MORs) are necessary for the analgesic and addictive effects of opioids such as morphine, but the MOR-expressing neuronal populations that mediate the distinct opiate effects remain elusive. Here we devised a new conditional bacterial artificial chromosome rescue strategy to show, in mice, that targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restores opiate reward and opiate-induced striatal dopamine release and partially restores motivation to self administer an opiate. However, these mice lack opiate analgesia or withdrawal. We used Cre-mediated deletion of the rescued MOR transgene to establish that expression of the MOR transgene in the striatum, rather than in extrastriatal sites, is needed for the restoration of opiate reward. Our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a new intersectional genetic approach to dissecting neurocircuit-specific gene function in vivo. PMID:24413699

  2. Targeted Expression of Mu-Opioid Receptors in a Subset of Striatal Direct-Pathway Neurons Restores Opiate Reward

    PubMed Central

    Cui, Yijun; Ostlund, Sean B.; James, Alex; Park, Chang Sin; Ge, Weihong; Roberts, Kristofer W.; Mittal, Nitish; Murphy, Niall P.; Cepeda, Carlos; Kieffer, Brigitte L.; Levine, Michael S.; Jentsch, J. David; Walwyn, Wendy M.; Sun, Yi E.; Evans, Christopher J.; Maidment, Nigel T.; Yang, X. William

    2014-01-01

    SUMMARY Mu-Opioid Receptors (MOR) are necessary for the analgesic and addictive effects of opioids such as morphine, but the MOR-expressing neuronal populations that mediate the distinct opiate effects remain elusive. Here we devised a novel conditional BAC rescue strategy to show that mice with targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restore opiate reward, opiate-induced striatal dopamine release, and partially restore motivation to self-administer opiates. However, they lack opiate analgesia or withdrawal. Importantly, we used Cre-mediated deletion of the rescued MOR transgene to establish that striatal, rather than a few extrastriatal sites of MOR transgene expression, is needed for the restoration of opiate reward. Together, our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a novel intersectional genetic approach to dissect neurocircuit-specific gene function in vivo. PMID:24413699

  3. Targeted expression of μ-opioid receptors in a subset of striatal direct-pathway neurons restores opiate reward.

    PubMed

    Cui, Yijun; Ostlund, Sean B; James, Alex S; Park, Chang Sin; Ge, Weihong; Roberts, Kristofer W; Mittal, Nitish; Murphy, Niall P; Cepeda, Carlos; Kieffer, Brigitte L; Levine, Michael S; Jentsch, James David; Walwyn, Wendy M; Sun, Yi E; Evans, Christopher J; Maidment, Nigel T; Yang, X William

    2014-02-01

    μ-opioid receptors (MORs) are necessary for the analgesic and addictive effects of opioids such as morphine, but the MOR-expressing neuronal populations that mediate the distinct opiate effects remain elusive. Here we devised a new conditional bacterial artificial chromosome rescue strategy to show, in mice, that targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restores opiate reward and opiate-induced striatal dopamine release and partially restores motivation to self administer an opiate. However, these mice lack opiate analgesia or withdrawal. We used Cre-mediated deletion of the rescued MOR transgene to establish that expression of the MOR transgene in the striatum, rather than in extrastriatal sites, is needed for the restoration of opiate reward. Our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a new intersectional genetic approach to dissecting neurocircuit-specific gene function in vivo.

  4. Mineral phosphate solubilization by rhizosphere bacteria and scope for manipulation of the direct oxidation pathway involving glucose dehydrogenase.

    PubMed

    Sashidhar, B; Podile, A R

    2010-07-01

    Microbial biodiversity in the soil plays a significant role in metabolism of complex molecules, helps in plant nutrition and offers countless new genes, biochemical pathways, antibiotics and other metabolites, useful molecules for agronomic productivity. Phosphorus being the second most important macro-nutrient required by the plants, next to nitrogen, its availability in soluble form in the soils is of great importance in agriculture. Microbes present in the soil employ different strategies to make use of unavailable forms of phosphate and in turn also help plants making phosphate available for plant use. Azotobacter, a free-living nitrogen fixer, is known to increase the fertility of the soil and in turn the productivity of different crops. The glucose dehydrogenase gene, the first enzyme in the direct oxidation pathway, contributes significantly to mineral phosphate solubilization ability in several Gram-negative bacteria. It is possible to enhance further the biofertilizer potential of plant growth-promoting rhizobacteria by introducing the genes involved mineral phosphate solubilization without affecting their ability to fix nitrogen or produce phytohormones for dual benefit to agricultural crops. Glucose dehydrogenases from Gram-negative bacteria can be engineered to improve their ability to use different substrates, function at higher temperatures and EDTA tolerance, etc., through site-directed mutagenesis.

  5. 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

  6. AFM studies of semicrystalline polymer/inorganic nanocomposites

    NASA Astrophysics Data System (ADS)

    Strawhecker, Kenneth E.

    2002-01-01

    vapor transmission properties superior to that of the neat polymer and its conventionally filled composites. For example, for a 5wt.% MMT exfoliated composite, the softening temperature increases by 25°C, the Young's modulus triples with a decrease of only 20% in toughness, whereas there is also a 60% reduction in the water permeability. Furthermore, due to the nanoscale dispersion of filler, the nanocomposites retain their optical clarity. Finally, to better connect changes in morphology with property enhancements, AFM methods were devised which allow direct imaging of morphology as well as local properties.

  7. 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

  8. Gene fusions for the directed modification of the carotenoid biosynthesis pathway in Mucor circinelloides.

    PubMed

    Iturriaga, Enrique A; Papp, Tamás; Alvarez, María Isabel; Eslava, Arturo P

    2012-01-01

    Several fungal species, particularly some included in the Mucorales, have been used to develop fermentation processes for the production of β-carotene. Oxygenated derivatives of β-carotene are more valuable products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use Mucor circinelloides as a model organism to develop strains able to produce new, more valuable, and with an increased content of carotenoids. In this chapter we describe part of our efforts to construct active gene fusions which could advance in the diversification of carotenoid production by this fungus. The main carotenoid accumulated by M. circinelloides is β-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. Two enzymatic activities are required for the production of astaxanthin from β-carotene: a hydroxylase and a ketolase. We used the ctrW gene of Paracoccus sp. N81106, encoding a bacterial β-carotene ketolase, to construct gene fusions with two fungal genes essential for the modification of the pathway in M. circinelloides. First we fused it to the carRP gene of M. circinelloides, which is responsible for the phytoene synthase and lycopene cyclase activities in this fungus. The expected activity of this fusion gene would be the accumulation by M. circinelloides of canthaxanthin and probably some astaxanthin. A second construction was the fusion of the crtW gene of Paracoccus sp. to the crtS gene of Xanthophyllomyces dendrorhous, responsible for the synthesis of astaxanthin from β-carotene in this fungus, but which was shown to have only hydroxylase activity in M. circinelloides. The expected result in M. circinelloides transformants was the accumulation of astaxanthin. Here we describe a detailed and empirically tested protocol for the construction of these gene fusions. PMID:22711120

  9. 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

    2016-09-01

    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. PMID:27532223

  10. TAK-063, a PDE10A Inhibitor with Balanced Activation of Direct and Indirect Pathways, Provides Potent Antipsychotic-Like Effects in Multiple Paradigms.

    PubMed

    Suzuki, Kazunori; Harada, Akina; Suzuki, Hirobumi; Miyamoto, Maki; Kimura, Haruhide

    2016-08-01

    Phosphodiesterase 10A (PDE10A) inhibitors are expected to be novel drugs for schizophrenia through activation of both direct and indirect pathway medium spiny neurons. However, excess activation of the direct pathway by a dopamine D1 receptor agonist SKF82958 canceled antipsychotic-like effects of a dopamine D2 receptor antagonist haloperidol in methamphetamine (METH)-induced hyperactivity in rats. Thus, balanced activation of these pathways may be critical for PDE10A inhibitors. Current antipsychotics and the novel PDE10A inhibitor TAK-063, but not the selective PDE10A inhibitor MP-10, produced dose-dependent antipsychotic-like effects in METH-induced hyperactivity and prepulse inhibition in rodents. TAK-063 and MP-10 activated the indirect pathway to a similar extent; however, MP-10 caused greater activation of the direct pathway than did TAK-063. Interestingly, the off-rate of TAK-063 from PDE10A in rat brain sections was faster than that of MP-10, and a slower off-rate PDE10A inhibitor with TAK-063-like chemical structure showed an MP-10-like pharmacological profile. In general, faster off-rate enzyme inhibitors are more sensitive than slower off-rate inhibitors to binding inhibition by enzyme substrates. As expected, TAK-063 was more sensitive than MP-10 to binding inhibition by cyclic nucleotides. Moreover, an immunohistochemistry study suggested that cyclic adenosine monophosphate levels in the direct pathway were higher than those in the indirect pathway. These data can explain why TAK-063 showed partial activation of the direct pathway compared with MP-10. The findings presented here suggest that TAK-063's antipsychotic-like efficacy may be attributable to its unique pharmacological properties, resulting in balanced activation of the direct and indirect striatal pathways. PMID:26849714

  11. Methoxychlor directly affects ovarian antral follicle growth and atresia through Bcl-2- and Bax-mediated pathways.

    PubMed

    Miller, Kimberly P; Gupta, Rupesh K; Greenfeld, Chuck R; Babus, Janice K; Flaws, Jodi A

    2005-11-01

    Methoxychlor (MXC) is an organochlorine pesticide and reproductive toxicant. While in vivo studies indicate that MXC exposure increases antral follicle atresia, in part by altering apoptotic regulators (Bcl-2 and Bax), they do not distinguish whether MXC does so via direct or indirect mechanisms. Therefore, we utilized an in vitro follicle culture system to test the hypothesis that MXC is directly toxic to antral follicles, and that overexpression of anti-apoptotic Bcl-2, or deletion of pro-apoptotic Bax, protects antral follicles from MXC-induced toxicity. Antral follicles were isolated from wild-type (WT), Bcl-2 overexpressing (Bcl-2 OE), or Bax deficient (BaxKO) mice, and exposed to dimethylsulfoxide (control) or MXC (1-100 microg/ml) for 96 h. Follicle diameters were measured every 24 h to assess growth. After 96 h, follicles were histologically evaluated for atresia or collected for quantitative PCR analysis of Bcl-2 and Bax mRNA levels. MXC (10-100 microg/ml) significantly inhibited antral follicle growth at 72 and 96 h, and increased atresia (100 microg/ml) compared to controls at 96 h. Furthermore, MXC increased Bax mRNA levels between 48-96 h and decreased Bcl-2 mRNA levels at 96 h. While MXC inhibited growth of WT antral follicles beginning at 72 h, it did not inhibit growth of Bcl-2 OE or BaxKO follicles until 96 h. MXC also increased atresia of small and large WT and BaxKO antral follicles over controls, but it did not increase atresia of large Bcl-2 OE antral follicles over controls. These data suggest that MXC directly inhibits follicle growth partly by Bcl-2 and Bax pathways, and increases atresia partly through Bcl-2 pathways.

  12. 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

  13. Longitudinal direct and indirect pathways linking older sibling competence to the development of younger sibling competence.

    PubMed

    Brody, Gene H; Kim, Sooyeon; Murry, Velma McBride; Brown, Anita C

    2003-05-01

    A 4-wave longitudinal model tested direct and indirect links between older sibling (OS; M = 11.7 years) and younger sibling (YS; M = 9.2 years) competence in 152 rural African American families. Data were collected at 1-year intervals. At each wave, different teachers assessed OS competence, YS competence, and YS self-regulation. Mothers reported their own psychological functioning; mothers and YSs reported parenting practices toward the YS. OS competence was stable across time and was linked with positive changes in mothers' psychological functioning from Wave 1 to Wave 2. Mothers' Wave 2 psychological functioning was associated with involved-supportive parenting of the YS at Wave 3. OS Wave 2 competence and Wave 3 parenting were indirectly linked with Wave 4 YS competence, through Wave 3 YS self-regulation. Structural equation modeling controlled for Wave 1 YS competence; thus, the model accounted for change in YS competence across 3 years.

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

    PubMed

    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.

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

    PubMed

    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. PMID:27083044

  16. Structural and Mechanical Mechanisms of Ocular Tissues Probed by AFM

    NASA Astrophysics Data System (ADS)

    Ziebarth, Noël M.; Rico, Felix; Moy, Vincent T.

    In recent years, the atomic force microscope (AFM) has become an important tool in ophthalmic research. It has gained popularity largely because AFM is not restricted by the diffraction limits of light microscopy and can be applied to resolve images with molecular resolution. AFM is a minimally invasive technique and can be used to visualize molecular structures under near-physiological conditions. In addition, the AFM can be employed as a force apparatus to characterize the viscoelastic properties of biomaterials on the micron level and at the level of individual proteins. In this article, we summarize recent AFM studies of ocular tissues, while highlighting the great potential of AFM technology in ophthalmic research. Previous research demonstrates the versatility of the AFM as high resolution imaging technique and as a sensitive force apparatus for probing the mechanical properties of ocular tissues. The structural and mechanical properties of ocular tissues are of major importance to the understanding of the optomechanical functions of the human eye. In addition, AFM has played an important role in the development and characterization of ocular biomaterials, such as contact lenses and intraocular lenses. Studying ocular tissues using Atomic Force Microscopy has enabled several advances in ophthalmic research.

  17. 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

  18. 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).

  19. 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).

  20. Arbuscular mycorrhizal colonization reduces arsenate uptake in barley via downregulation of transporters in the direct epidermal phosphate uptake pathway.

    PubMed

    Christophersen, H M; Smith, F A; Smith, S E

    2009-12-01

    *Here, we used barley (Hordeum vulgare) grown in normal and compartmented pots to investigate sensitivity to arsenic (As) in the absence of a positive growth response to arbuscular mycorrhizas (AM). *We tested the hypothesis that upon inoculation with AM fungi downregulation of HvPht1;1 and HvPht1;2 genes (encoding high-affinity inorganic orthophosphate (P(i))-uptake systems in a direct pathway via root epidermis and root hairs) and upregulation of the AM-induced HvPht1;8 (encoding the P(i)-uptake system responsible for transfer of P(i) from the symbiotic interface to cortical cells) play a role in decreased As uptake and hence reduced As sensitivity in AM plants. *Barley did not respond, or responded negatively to colonization by Glomus intraradices in terms of growth. In terms of specific phosphorus (P) uptake (P uptake per g of root) barley was nonresponsive. There was a significant interaction between As treatment and colonization, resulting in a lower As concentration and uptake in AM compared with nonmycorrhizal (NM) plants. *The decreased uptake of As and higher P : As molar ratios in the AM barley can be explained by the operation of the AM pathway as indicated by induction of HvPht1;8 and by down-regulation of HvPht1;1 and HvPht1;2.

  1. S(mu) mutation patterns suggest different progression pathways in follicular lymphoma: early direct or late from FL progenitor cells.

    PubMed

    Ruminy, Philippe; Jardin, Fabrice; Picquenot, Jean-Michel; Parmentier, Françoise; Contentin, Nathalie; Buchonnet, Gérard; Tison, Sandrine; Rainville, Vinciane; Tilly, Hervé; Bastard, Christian

    2008-09-01

    Follicular lymphoma (FL) is a B-cell malignancy characterized by the t(14;18) translocation. Although sensitive to treatment, the disease remains incurable and the reason why tumor cells invariably evade treatment, leading to clinical relapse, is still unknown. Here, we tracked the clonal history of tumor cells by studying mutations introduced by activation-induced cytidine deaminase on the switch mu region of the der(14)t(14;18) during the early phase of the class-switch recombination (CSR) process. We observed frequent intraclonal variations, suggesting that CSR often remains active after the acquisition of the fully transformed phenotype. However, mutations only rarely accumulated over time, but instead showed complex evolutionary scenarios and 2 different progression pathways. The first pathway was a direct and rapid evolution from the dominant clone. The second was indirect, arising from earlier subclones usually after years of remission. A better understanding of these mechanisms might influence the future choice of treatment strategies. PMID:18515657

  2. 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.

  3. Controlled AFM detachments and movement of nanoparticles: gold clusters on HOPG at different temperatures.

    PubMed

    Tripathi, Manoj; Paolicelli, Guido; D'Addato, Sergio; Valeri, Sergio

    2012-06-22

    The effect of temperature on the onset of movement of gold nanoclusters (diameter 27 nm) deposited on highly oriented pyrolytic graphite (HOPG) has been studied by atomic force microscopy (AFM) techniques. Using the AFM with amplitude modulation (tapping mode AFM) we have stimulated and controlled the movement of individual clusters. We show how, at room temperature, controlled detachments and smooth movements can be obtained for clusters having dimensions comparable to or smaller than the tip radius. Displacement is practically visible in real time and it can be started and stopped easily by adjusting only one parameter, the tip amplitude oscillation. Analysing the energy dissipation signal at the onset of nanocluster sliding we evaluated a detachment threshold energy as a function of temperature in the range 300-413 K. We also analysed single cluster thermal induced displacement and combining this delicate procedure with AFM forced movement behaviour we conclude that detachment threshold energy is directly related to the activation energy of nanocluster diffusion and it scales linearly with temperature as expected for a single-particle thermally activated process.

  4. Activation of direct and indirect pathways of glycogen synthesis by hepatic overexpression of protein targeting to glycogen

    PubMed Central

    O’Doherty, Robert M.; Jensen, Per B.; Anderson, Paul; Jones, John G.; Berman, Hal K.; Kearney, Denise; Newgard, Christopher B.

    2000-01-01

    Glycogen-targeting subunits of protein phosphatase-1, such as protein targeting to glycogen (PTG), direct the phosphatase to the glycogen particle, where it stimulates glycogenesis. We have investigated the metabolic impact of overexpressing PTG in liver of normal rats. After administration of PTG cDNA in a recombinant adenovirus, animals were fasted or allowed to continue feeding for 24 hours. Liver glycogen was nearly completely depleted in fasted control animals, whereas glycogen levels in fasted or fed PTG-overexpressing animals were 70% higher than in fed controls. Nevertheless, transgenic animals regulated plasma glucose, triglycerides, FFAs, ketones, and insulin normally in the fasted and fed states. Fasted PTG-overexpressing animals receiving an oral bolus of [U-13C]glucose exhibited a large increase in hepatic glycogen content and a 70% increase in incorporation of [13C]glucose into glycogen. However, incorporation of labeled glucose accounted for only a small portion of the glycogen synthesized in PTG-overexpressing animals, consistent with our earlier finding that PTG promotes glycogen synthesis from gluconeogenic precursors. We conclude that hepatic PTG overexpression activates both direct and indirect pathways of glycogen synthesis. Because of its ability to enhance glucose storage without affecting other metabolic indicators, the glycogen-targeting subunit may prove valuable in controlling blood glucose levels in diabetes. PMID:10683377

  5. SEM and AFM images of pyrite surfaces after bioleaching by the indigenous Thiobacillus thiooxidans.

    PubMed

    Liu, H-L; Chen, B-Y; Lan, Y-W; Cheng, Y-C

    2003-09-01

    The bioleaching mechanism of pyrite by the indigenous Thiobacillus thiooxidans was examined with the aid of scanning electron microscopy (SEM) and atomic force microscopy (AFM) images of the pyrite surface. The presence of pyrite eliminated the lag phase during growth of this microorganism. This was due to the stimulatory effect on cell growth of the slight amount of Cu2+ that had leached from the pyrite. Zn2+ was found to be much more readily solubilized than Cu2+. The efficiency of bioleaching was four times higher than that of chemical leaching. SEM images provided evidence of direct cell attachment onto the pyrite surface, thereby enhancing the bioleaching rate. Furthermore, extracellular polymeric substances (EPSs) were found on the pyrite surface after 4 days of oxidation. AFM images showed that the pyrite surface area positively correlated with the oxidation rate. A combination of direct and indirect mechanism is probably responsible for the oxidation of pyrite by T. thiooxidans.

  6. 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.

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

    SciTech Connect

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

    2013-02-05

    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 {sup 60}Co 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.

  8. Inhibition of PPAR alpha/RXR alpha-mediated direct hyperplasia pathways during griseofulvin-induced hepatocarcinogenesis.

    PubMed

    Nagao, Y; French, B A; Cai, Y; French, S W; Wan, Y J

    1998-05-01

    Chronic griseofulvin (GF) feeding induces preneoplastic foci followed by hepatocellular carcinoma in the mouse liver. Our previous study suggested that GF-induced hepatocellular proliferation had a different mechanism from that of peroxisome proliferator (PP)-induced direct hyperplasia. The GF-induced hepatocellular proliferation was mediated through activation of immediate early genes such as Fos, Jun, Myc, and NFKB. In contrast, PP-induced direct hyperplasia does not involve activation of any of these immediate early genes. It has been shown that nuclear hormone receptors including peroxisome proliferator activated receptors (PPARs) and retinoid x receptors (RXRs) play important roles in mediating the pleiotropic effects of PPs. To examine the possible roles of PPARs and RXRs during non-PP-induced hepatocellular proliferation and the interaction between PP and non-PP-induced proliferation, we have studied the expression of the PPAR and RXR genes in the GF model using northern blot hybridizations and gel retardation assays. The data showed that the expression of PPARalpha and RXRalpha genes was down-regulated in the livers containing preneoplastic nodules and in the liver tumors induced by GF. The mRNA down-regulation was accompanied by a decrease in the amount of nuclear protein-bound to peroxisome proliferator and retinoic acid responsive elements. Down-regulation was also associated with the suppressed expression of the PPARalpha/RXRalpha target genes (i.e., acyl-Co oxidase and cytochrome P450 4A1) and the catalase gene. The RXR-gamma gene was also down-regulated, but the RARalpha, beta, and gamma and PPARbeta and gamma genes were up-regulated. These results indicated that the hepatocarcinogenesis induced by GF is accompanied by suppression of the PPARalpha/RXRalpha-mediated direct hyperplasia pathway. The differential expression of these nuclear hormone receptors reveals a new aspect for understanding the individual roles and intercommunication of PPAR, RXR

  9. 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

  10. 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).

  11. 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).

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

    PubMed

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

    2013-11-21

    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. PMID:24056758

  13. Antibody directs properdin-dependent activation of the complement alternative pathway in a mouse model of abdominal aortic aneurysm.

    PubMed

    Zhou, Hui-Fang; Yan, Huimin; Stover, Cordula M; Fernandez, Tamara Montes; Rodriguez de Cordoba, Santiago; Song, Wen-Chao; Wu, Xiaobo; Thompson, Robert W; Schwaeble, Wilhelm J; Atkinson, John P; Hourcade, Dennis E; Pham, Christine T N

    2012-02-14

    Abdominal aortic aneurysm (AAA) is a complex inflammatory vascular disease. There are currently limited treatment options for AAA when surgery is inapplicable. Therefore, insights into molecular mechanisms underlying AAA pathogenesis may reveal therapeutic targets that could be manipulated pharmacologically or biologically to halt disease progression. Using an elastase-induced AAA mouse model, we previously established that the complement alternative pathway (AP) plays a critical role in the development of AAA. However, the mechanism by which complement AP is initiated remains undefined. The complement protein properdin, traditionally viewed as a positive regulator of the AP, may also initiate complement activation by binding directly to target surfaces. In this study, we sought to determine whether properdin serves as a focal point for the initiation of the AP complement activation in AAA. Using a properdin loss of function mutation in mice and a mutant form of the complement factor B protein that produces a stable, properdin-free AP C3 convertase, we show that properdin is required for the development of elastase-induced AAA in its primary role as a convertase stabilizer. Unexpectedly, we find that, in AAA, natural IgG antibodies direct AP-mediated complement activation. The absence of IgG abrogates C3 deposition in elastase-perfused aortic wall and protects animals from AAA development. We also determine that blockade of properdin activity prevents aneurysm formation. These results indicate that an innate immune response to self-antigens activates the complement system and initiates the inflammatory cascade in AAA. Moreover, the study suggests that properdin-targeting strategies may halt aneurysmal growth.

  14. 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

    2012-08-01

    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. PMID:22822216

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

    PubMed Central

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

    2012-01-01

    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. PMID:22822216

  16. Structure and Permeability of Ion-channels by Integrated AFM and Waveguide TIRF Microscopy

    PubMed Central

    Ramachandran, Srinivasan; Arce, Fernando Teran; Patel, Nirav R.; Quist, Arjan P.; Cohen, Daniel A.; Lal, Ratnesh

    2014-01-01

    Membrane ion channels regulate key cellular functions and their activity is dependent on their 3D structure. Atomic force microscopy (AFM) images 3D structure of membrane channels placed on a solid substrate. Solid substrate prevents molecular transport through ion channels thus hindering any direct structure-function relationship analysis. Here we designed a ~70 nm nanopore to suspend a membrane, allowing fluidic access to both sides. We used these nanopores with AFM and total internal reflection fluorescence microscopy (TIRFM) for high resolution imaging and molecular transport measurement. Significantly, membranes over the nanopore were stable for repeated AFM imaging. We studied structure-activity relationship of gap junction hemichannels reconstituted in lipid bilayers. Individual hemichannels in the membrane overlying the nanopore were resolved and transport of hemichannel-permeant LY dye was visualized when the hemichannel was opened by lowering calcium in the medium. This integrated technique will allow direct structure-permeability relationship of many ion channels and receptors. PMID:24651823

  17. 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.

  18. Crystallization of Probucol in Nanoparticles Revealed by AFM Analysis in Aqueous Solution.

    PubMed

    Egami, Kiichi; Higashi, Kenjirou; Yamamoto, Keiji; Moribe, Kunikazu

    2015-08-01

    The crystallization behavior of a pharmaceutical drug in nanoparticles was directly evaluated by atomic force microscopy (AFM) force curve measurements in aqueous solution. A ternary spray-dried sample (SPD) was prepared by spray drying the organic solvent containing probucol (PBC), hypromellose (HPMC), and sodium dodecyl sulfate (SDS). The amorphization of PBC in the ternary SPD was confirmed by powder X-ray diffraction (PXRD) and solid-state 13C NMR measurements. A nanosuspension containing quite small particles of 25 nm in size was successfully prepared immediately after dispersion of the ternary SPD into water. Furthermore, solution-state 1H NMR measurements revealed that a portion of HPMC coexisted with PBC as a mixed state in the freshly prepared nanosuspension particles. After storing the nanosuspension at 25 °C, a gradual increase in the size of the nanoparticles was observed, and the particle size changed to 93.9 nm after 7 days. AFM enabled the direct observation of the morphology and agglomeration behavior of the nanoparticles in water. Moreover, AFM force-distance curves were changed from (I) to (IV), depending on the storage period, as follows: (I) complete indentation within an applied force of 1 nN, (II) complete indentation with an applied force of 1-5 nN, (III) partial indentation with an applied force of 5 nN, and (IV) nearly no indentation with an applied force of 5 nN. This stiffness increase of the nanoparticles was attributed to gradual changes in the molecular state of PBC from the amorphous to the crystal state. Solid-state 13C NMR measurements of the freeze-dried samples demonstrated the presence of metastable PBC Form II crystals in the stored nanosuspension, strongly supporting the AFM results.

  19. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26443442

  1. 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.

  2. 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.

  3. 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.

  4. AFM investigation of Martian soil simulants on micromachined Si substrates.

    PubMed

    Vijendran, S; Sykulska, H; Pike, W T

    2007-09-01

    The micro and nanostructures of Martian soil simulants with particles in the micrometre-size range have been studied using a combination of optical and atomic force microscopy (AFM) in preparation for the 2007 NASA Phoenix Mars Lander mission. The operation of an atomic force microscope on samples of micrometre-sized soil particles is a poorly investigated area where the unwanted interaction between the scanning tip and loose particles results in poor image quality and tip contamination by the sample. In order to mitigate these effects, etched silicon substrates with a variety of features have been used to facilitate the sorting and gripping of particles. From these experiments, a number of patterns were identified that were particularly good at isolating and immobilizing particles for AFM imaging. This data was used to guide the design of micromachined substrates for the Phoenix AFM. Both individual particles as well as aggregates were successfully imaged, and information on sizes, shapes and surface morphologies were obtained. This study highlights both the strengths and weaknesses of AFM for the potential in situ investigation of Martian soil and dust. Also presented are more general findings of the limiting operational constraints that exist when attempting the AFM of high aspect ratio particles with current technology. The performance of the final designs of the substrates incorporated on Phoenix will be described in a later paper. PMID:17760618

  5. Extension of gravity center method for diameter calibration of polystyrene standard particles with a metrological AFM

    NASA Astrophysics Data System (ADS)

    Misumi, Ichiko; Takahata, Keiji; Sugawara, Kentaro; Gonda, Satoshi; Ehara, Kensei

    2012-06-01

    In the particle diameter calibration using metrological AFM, the distance between center points of neighboring two particles is referred to as "lateral diameter" when a single-layer close-packed structure of particles is successfully formed. The distance between an apex of a particle and a substrate is referred to as "vertical diameter." In the previous studies, lateral diameter was calculated by manually selecting and extracting a line profile from metrological AFM data and directly applying a method to calculate a pitch of one-dimensional grating. As the manual line profile extraction depends on who does it, however, there is a possibility that calculated lateral diameter is varied from person to person. We developed a technology to calibrate diameter of polystyrene latex (PSL) particles by using our metrological AFM. In this study, the gravity center method is extended to three dimensions to calculate position of and the center of gravity in each particle. Lateral diameter, which was defined as distance between gravity centers of neighboring two particles, is calibrated and uncertainty in the lateral diameter calibration is evaluated. Deformation of particles was also estimated by using Young's modulus of thin film PSL and bulk PSL.

  6. 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.

  7. Examination of dentin surface using AFM (our experience).

    PubMed

    Zapletalová, Zdenka; Kubínek, Roman; Vůjtek, Milan; Novotný, Radko

    2004-01-01

    Atomic force microscopy (AFM) as one the technique of Scanning Probe Microscopy is useful for imaging of surface structure. This method can yield three-dimensional high-resolution topographic images of sample surfaces by using a scanning technique for conductors and insulators on atomic scale. It is based upon mapping of atomic-forces on a surface of an investigated sample. The method is useful not only in physics and chemistry; it can be also applied in biological fields. Special construction of AFM scanner enables to follow biological samples in liquid environments. Artifacts caused by dehydration of samples are removed this way. Dentin of human teeth is a vital hydrated tissue. It is strongly sensitive to dehydration and drying that are commonly used in preparation of samples in examinations by Scanning Electron Microscopy (SEM). We describe our experience in examination of dentin surfaces of extracted human third molars using contact method of AFM under moist conditions.

  8. Optimization of phase contrast in bimodal amplitude modulation AFM.

    PubMed

    Damircheli, Mehrnoosh; Payam, Amir F; Garcia, Ricardo

    2015-01-01

    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.

  9. 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

  10. 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

  11. 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.

  12. The NTA-His6 bond is strong enough for AFM single-molecular recognition studies.

    PubMed

    Verbelen, Claire; Gruber, Hermann J; Dufrêne, Yves F

    2007-01-01

    There is a need in current atomic force microscopy (AFM) molecular recognition studies for generic methods for the stable, functional attachment of proteins on tips and solid supports. In the last few years, the site-directed nitrilotriacetic acid (NTA)-polyhistidine (Hisn) system has been increasingly used towards this goal. Yet, a crucial question in this context is whether the NTA-Hisn bond is sufficiently strong for ensuring stable protein immobilization during force spectroscopy measurements. Here, we measured the forces between AFM tips modified with NTA-terminated alkanethiols and solid supports functionalized with His6-Gly-Cys peptides in the presence of Ni2+. The force histogram obtained at a loading rate of 6600 pN s(-1) showed three maxima at rupture forces of 153 +/- 57 pN, 316 +/- 50 pN and 468 +/- 44 pN, that we attribute primarily to monovalent and multivalent interactions between a single His6 moiety and one, two and three NTA groups, respectively. The measured forces are well above the 50-100 pN unbinding forces typically observed by AFM for receptor-ligand pairs. The plot of adhesion force versus log (loading rate) revealed a linear regime, from which we deduced a kinetic off-rate constant of dissociation, k(off) approximately 0.07 s(-1). This value is in the range of that estimated for the multivalent interaction involving two NTA, using fluorescence measurements, and may account for an increased binding stability of the NTA-His6 bond. We conclude that the NTA-His6 system is a powerful, well-suited platform for the stable, oriented immobilization of proteins in AFM single-molecule studies. PMID:17712775

  13. CD-AFM reference metrology at NIST and SEMATECH

    NASA Astrophysics Data System (ADS)

    Dixson, Ronald; Fu, Joseph; Orji, Ndubuisi; Guthrie, William; Allen, Richard; Cresswell, Michael

    2005-05-01

    The National Institute of Standards and Technology (NIST) and SEMATECH have been working together to improve the traceability of critical dimension atomic force microscope (CD-AFM) dimensional metrology in semiconductor manufacturing. A major component of this collaboration has been the implementation of a Reference Measurement System (RMS) at SEMATECH using a current generation CD-AFM. An earlier tool, originally used at SEMATECH, has now been installed at NIST. Uncertainty budgets were developed for pitch, height, and CD measurements using both tools. At present, the standard uncertainties are approximately 0.2 % for pitch measurements and 0.4% for step height measurements. Prior to the current work, CD AFM linewidth measurements were limited to a standard uncertainty of about 5 nm. However, this limit can now be significantly reduced. This reduction results from the completion of the NIST/SEMATECH collaboration on the development of single crystal critical dimension reference materials (SCDDRM). A new generation of these reference materials was released to SEMATECH Member Companies during late 2004. The SEMATECH RMS was used to measure the linewidths of selected features on the distributed specimens. To reduce the uncertainty in tip width calibration, a separate transfer experiment was performed in which samples were measured by CD-AFM and then sent for high resolution transmission electron microscopy (HRTEM). In this manner, CD-AFM could be used to transfer the HRTEM width information to the distributed samples. Consequently, we are now able to reduce the limit on the standard uncertainty (k = 1) of CD-AFM width measurements to 1 nm.

  14. MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-β signalling pathway.

    PubMed

    Lynch, Jennifer; Fay, Joanna; Meehan, Maria; Bryan, Kenneth; Watters, Karen M; Murphy, Derek M; Stallings, Raymond L

    2012-05-01

    Transforming growth factor-β (TGF-β) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-β non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-β pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-β signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-β non-canonical signaling by miR-335, which in turn is downregulated by MYCN.

  15. AFM Investigation of mechanical properties of dentin

    SciTech Connect

    Cohen, Sidney R; Apter, Nathan; Jesse, Stephen; Kalinin, Sergei V; Barlam, David; Peretz, Adi Idit; Ziskind, Daniel; Wagner, H. Daniel

    2008-01-01

    Mechanical properties of peritubular dentin were investigated using scanning probe microscopy techniques, namely Nanoindentation and Band Excitation. Particular attention was directed to the possible existence of a gradient in these properties moving outward from the tubular lumen to the junction with the intertubular dentin. Finite element analysis showed that the influence of the boundaries is small relative to the effects observed. Thus, these results strongly support the concept of a lowering of modulus and hardness from the tubular exterior to its periphery, which appear to correlate with graded changes in the mineral content.

  16. Thermo-magnetic behaviour of AFM-MFM cantilevers

    NASA Astrophysics Data System (ADS)

    Kumar, M.; Arinero, R.; Bergez, W.; Tordjeman, Ph

    2015-08-01

    Atomic force microscopy (AFM) experiments were performed to study the behaviour of AFM cantilevers under an external magnetic field B and temperature field produced by a coil with an iron core. Four cantilever types were studied. Forces were measured for different B values and at various coil-to-cantilever separation distances. The results were analysed on the basis of a phenomenological model. This model contains the contribution of two terms, one monopole-monopole interaction at short distance, and one apparent paramagnetic interaction in \

  17. 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).

  18. 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.

  19. An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway.

    PubMed

    Robertson, Kevin A; Hsieh, Wei Yuan; Forster, Thorsten; Blanc, Mathieu; Lu, Hongjin; Crick, Peter J; Yutuc, Eylan; Watterson, Steven; Martin, Kimberly; Griffiths, Samantha J; Enright, Anton J; Yamamoto, Mami; Pradeepa, Madapura M; Lennox, Kimberly A; Behlke, Mark A; Talbot, Simon; Haas, Jürgen; Dölken, Lars; Griffiths, William J; Wang, Yuqin; Angulo, Ana; Ghazal, Peter

    2016-03-01

    In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway.

  20. An Interferon Regulated MicroRNA Provides Broad Cell-Intrinsic Antiviral Immunity through Multihit Host-Directed Targeting of the Sterol Pathway

    PubMed Central

    Robertson, Kevin A.; Hsieh, Wei Yuan; Forster, Thorsten; Blanc, Mathieu; Lu, Hongjin; Crick, Peter J.; Yutuc, Eylan; Watterson, Steven; Martin, Kimberly; Griffiths, Samantha J.; Enright, Anton J.; Yamamoto, Mami; Pradeepa, Madapura M.; Lennox, Kimberly A.; Behlke, Mark A.; Talbot, Simon; Haas, Jürgen; Dölken, Lars; Griffiths, William J.; Wang, Yuqin; Angulo, Ana; Ghazal, Peter

    2016-01-01

    In invertebrates, small interfering RNAs are at the vanguard of cell-autonomous antiviral immunity. In contrast, antiviral mechanisms initiated by interferon (IFN) signaling predominate in mammals. Whilst mammalian IFN-induced miRNA are known to inhibit specific viruses, it is not known whether host-directed microRNAs, downstream of IFN-signaling, have a role in mediating broad antiviral resistance. By performing an integrative, systematic, global analysis of RNA turnover utilizing 4-thiouridine labeling of newly transcribed RNA and pri/pre-miRNA in IFN-activated macrophages, we identify a new post-transcriptional viral defense mechanism mediated by miR-342-5p. On the basis of ChIP and site-directed promoter mutagenesis experiments, we find the synthesis of miR-342-5p is coupled to the antiviral IFN response via the IFN-induced transcription factor, IRF1. Strikingly, we find miR-342-5p targets mevalonate-sterol biosynthesis using a multihit mechanism suppressing the pathway at different functional levels: transcriptionally via SREBF2, post-transcriptionally via miR-33, and enzymatically via IDI1 and SC4MOL. Mass spectrometry-based lipidomics and enzymatic assays demonstrate the targeting mechanisms reduce intermediate sterol pathway metabolites and total cholesterol in macrophages. These results reveal a previously unrecognized mechanism by which IFN regulates the sterol pathway. The sterol pathway is known to be an integral part of the macrophage IFN antiviral response, and we show that miR-342-5p exerts broad antiviral effects against multiple, unrelated pathogenic viruses such Cytomegalovirus and Influenza A (H1N1). Metabolic rescue experiments confirm the specificity of these effects and demonstrate that unrelated viruses have differential mevalonate and sterol pathway requirements for their replication. This study, therefore, advances the general concept of broad antiviral defense through multihit targeting of a single host pathway. PMID:26938778

  1. Introduction to Atomic Force Microscopy (AFM) in Biology.

    PubMed

    Kreplak, Laurent

    2016-01-01

    The atomic force microscope (AFM) has the unique capability of imaging biological samples with molecular resolution in buffer solution over a wide range of time scales from milliseconds to hours. 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 nano-scale to the micro-scale. 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. © 2016 by John Wiley & Sons, Inc. PMID:27479503

  2. 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.

  3. Hydrodynamic effects in fast AFM single-molecule force measurements.

    PubMed

    Janovjak, Harald; Struckmeier, Jens; Müller, Daniel J

    2005-02-01

    Atomic force microscopy (AFM) allows the critical forces that unfold single proteins and rupture individual receptor-ligand bonds to be measured. To derive the shape of the energy landscape, the dynamic strength of the system is probed at different force loading rates. This is usually achieved by varying the pulling speed between a few nm/s and a few microm/s, although for a more complete investigation of the kinetic properties higher speeds are desirable. Above 10 microm/s, the hydrodynamic drag force acting on the AFM cantilever reaches the same order of magnitude as the molecular forces. This has limited the maximum pulling speed in AFM single-molecule force spectroscopy experiments. Here, we present an approach for considering these hydrodynamic effects, thereby allowing a correct evaluation of AFM force measurements recorded over an extended range of pulling speeds (and thus loading rates). To support and illustrate our theoretical considerations, we experimentally evaluated the mechanical unfolding of a multi-domain protein recorded at 30 microm/s pulling speed. PMID:15257425

  4. 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.

  5. 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

  6. 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.

  7. Novel Polymer Linkers for Single Molecule AFM Force Spectroscopy

    PubMed Central

    Tong, Zenghan; Mikheikin, Andrey; Krasnoslobodtsev, Alexey; Lv, Zhengjian; Lyubchenko, Yuri L.

    2013-01-01

    Flexible polymer linkers play an important role in various imaging and probing techniques that require surface immobilization, including atomic force microscopy (AFM). In AFM force spectroscopy, polymer linkers are necessary for the covalent attachment of molecules of interest to the AFM tip and the surface. The polymer linkers tether the molecules and provide their proper orientation in probing experiments. Additionally, the linkers separate specific interactions from nonspecific short-range adhesion and serve as a reference point for the quantitative analysis of single molecule probing events. In this report, we present our results on the synthesis and testing of a novel polymer linker and the identification of a number of potential applications for its use in AFM force spectroscopy experiments. The synthesis of the linker is based on the well-developed phosphoramidate (PA) chemistry that allows the routine synthesis of linkers with predetermined lengths and PA composition. These linkers are homogeneous in length and can be terminated with various functional groups. PA linkers with different functional groups were synthesized and tested in experimental systems utilizing different immobilization chemistries. We probed interactions between complementary DNA oligonucleotides; DNA and protein complexes formed by the site-specific binding protein SfiI; and interactions between amyloid peptide (Aβ42). The results of the AFM force spectroscopy experiments validated the feasibility of the proposed approach for the linker design and synthesis. Furthermore, the properties of the tether (length, functional groups) can be adjusted to meet the specific requirements for different force spectroscopy experiments and system characteristics, suggesting that it could be used for a large number of various applications. PMID:23624104

  8. 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.

  9. Oxide nanocrystal based nanocomposites for fabricating photoplastic AFM probes

    NASA Astrophysics Data System (ADS)

    Ingrosso, Chiara; Martin-Olmos, Cristina; Llobera, Andreu; Innocenti, Claudia; Sangregorio, Claudio; Striccoli, Marinella; Agostiano, Angela; Voigt, Anja; Gruetzner, Gabi; Brugger, Jürgen; Perez-Murano, Francesc; Curri, Maria Lucia

    2011-11-01

    We report on the synthesis, characterization and application of a novel nanocomposite made of a negative tone epoxy based photoresist modified with organic-capped Fe2O3 nanocrystals (NCs). The mechanical properties of the nanocomposite drastically improve upon incorporation of a suitable concentration of NCs in the polymer, without deteriorating its photolithography performance. High aspect ratio 3D microstructures made of the nanocomposite have been fabricated with a uniform surface morphology and with a resolution down to few micrometres. The embedded organic-capped Fe2O3 NCs drastically increase the stiffness and hardness of the epoxy based photoresist matrix, making the final material extremely interesting for manufacturing miniaturized polymer based mechanical devices and systems. In particular, the nanocomposite has been used as structural material for fabricating photoplastic Atomic Force Microscopy (AFM) probes with integrated tips showing outstanding mechanical response and high resolution imaging performance. The fabricated probes consist of straight cantilevers with low stress-gradient and high quality factors, incorporating sharp polymeric tips. They present considerably improved performance compared to pure epoxy based photoresist AFM probes, and to commercial silicon AFM probes.We report on the synthesis, characterization and application of a novel nanocomposite made of a negative tone epoxy based photoresist modified with organic-capped Fe2O3 nanocrystals (NCs). The mechanical properties of the nanocomposite drastically improve upon incorporation of a suitable concentration of NCs in the polymer, without deteriorating its photolithography performance. High aspect ratio 3D microstructures made of the nanocomposite have been fabricated with a uniform surface morphology and with a resolution down to few micrometres. The embedded organic-capped Fe2O3 NCs drastically increase the stiffness and hardness of the epoxy based photoresist matrix, making the

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

    PubMed

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

    2015-07-01

    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.

  11. 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

  12. 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.

  13. 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

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

    PubMed

    Mahon, Robert N; Hafner, Richard

    2015-10-15

    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.

  15. Starch Biosynthesis in Developing Wheat Grain : Evidence against the Direct Involvement of Triose Phosphates in the Metabolic Pathway.

    PubMed

    Keeling, P L; Wood, J R; Tyson, R H; Bridges, I G

    1988-06-01

    We have used (13)C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of (13)C between carbons atoms 1 and 6 of [1-(13)C] or [6-(13)C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the (13)C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the (13)C sugars were supplied in vivo to the intact plant. The (13)C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of (13)C in all glucosyl carbons was quantified by (13)C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background (13)C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate

  16. Starch Biosynthesis in Developing Wheat Grain : Evidence against the Direct Involvement of Triose Phosphates in the Metabolic Pathway.

    PubMed

    Keeling, P L; Wood, J R; Tyson, R H; Bridges, I G

    1988-06-01

    We have used (13)C-labeled sugars and nuclear magnetic resonance (NMR) spectrometry to study the metabolic pathway of starch biosynthesis in developing wheat grain (Triticum aestivum cv Mardler). Our aim was to examine the extent of redistribution of (13)C between carbons atoms 1 and 6 of [1-(13)C] or [6-(13)C]glucose (or fructose) incorporated into starch, and hence provide evidence for or against the involvement of triose phosphates in the metabolic pathway. Starch synthesis in the endosperm tissue was studied in two experimental systems. First, the (13)C sugars were supplied to isolated endosperm tissue incubated in vitro, and second the (13)C sugars were supplied in vivo to the intact plant. The (13)C starch produced by the endosperm tissue of the grain was isolated and enzymically degraded to glucose using amyloglucosidase, and the distribution of (13)C in all glucosyl carbons was quantified by (13)C-NMR spectrometry. In all of the experiments, irrespective of the incubation time or incubation conditions, there was a similar pattern of partial (between 15 and 20%) redistribution of label between carbons 1 and 6 of glucose recovered from starch. There was no detectable increase over background (13)C incidence in carbons 2 to 5. Within each experiment, the same pattern of partial redistribution of label was found in the glucosyl and fructosyl moieties of sucrose extracted from the tissue. Since it is unlikely that sucrose is present in the amyloplast, we suggest that the observed redistribution of label occurred in the cytosolic compartment of the endosperm cells and that both sucrose and starch are synthesized from a common pool of intermediates, such as hexose phosphate. We suggest that redistribution of label occurs via a cytosolic pathway cycle involving conversion of hexose phosphate to triose phosphate, interconversion of triose phosphate by triose phosphate isomerase, and resynthesis of hexose phosphate in the cytosol. A further round of triose phosphate

  17. Simultaneous AFM nano-patterning and imaging for photomask repair

    NASA Astrophysics Data System (ADS)

    Keyvani, Aliasghar; Tamer, Mehmet S.; van Es, Maarten H.; Sadeghian, Hamed

    2016-03-01

    In this paper we present a new AFM based nano-patterning technique that can be used for fast defect repairing of high resolution photomasks and possibly other high-speed nano-patterning applications. The proposed method works based on hammering the sample with tapping mode AFM followed by wet cleaning of the residuals. On the area where a specific pattern should be written, the tip-sample interaction force is tuned in a controlled manner by changing the excitation frequency of the cantilever without interrupting the imaging process. Using this method several patterns where transferred to different samples with imaging speed. While the pattern was transferred to the sample in each tracing scan line, the patterned sample was imaged in retracing scan line, thus the outcome was immediately visible during the experiment.

  18. 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).

  19. On CD-AFM bias related to probe bending

    NASA Astrophysics Data System (ADS)

    Ukraintsev, V. A.; Orji, N. G.; Vorburger, T. V.; Dixson, R. G.; Fu, J.; Silver, R. M.

    2012-03-01

    Critical Dimension AFM (CD-AFM) is a widely used reference metrology. To characterize modern semiconductor devices, very small and flexible probes, often 15 nm to 20 nm in diameter, are now frequently used. Several recent publications have reported on uncontrolled and significant probe-to-probe bias variation during linewidth and sidewall angle measurements [1,2]. Results obtained in this work suggest that probe bending can be on the order of several nanometers and thus potentially can explain much of the observed CD-AFM probe-to-probe bias variation. We have developed and experimentally tested one-dimensional (1D) and two-dimensional (2D) models to describe the bending of cylindrical probes. An earlier 1D bending model reported by Watanabe et al. [3] was refined. Contributions from several new phenomena were considered, including: probe misalignment, diameter variation near the carbon nanotube tip (CNT) apex, probe bending before snapping, distributed van der Waals-London force, etc. The methodology for extraction of the Hamaker probe-surface interaction energy from experimental probe bending data was developed. To overcome limitations of the 1D model, a new 2D distributed force (DF) model was developed. Comparison of the new model with the 1D single point force (SPF) model revealed about 27 % difference in probe bending bias between the two. A simple linear relation between biases predicted by the 1D SPF and 2D DF models was found. This finding simplifies use of the advanced 2D DF model of probe bending in various CD-AFM applications. New 2D and three-dimensional (3D) CDAFM data analysis software is needed to take full advantage of the new bias correction modeling capabilities.

  20. 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

  1. PKR is a novel functional direct player that coordinates skeletal muscle differentiation via p38MAPK/AKT pathways.

    PubMed

    Alisi, A; Spaziani, A; Anticoli, S; Ghidinelli, M; Balsano, C

    2008-03-01

    Myogenic differentiation is a highly orchestrated multistep process controlled by extracellular growth factors that modulate largely unknown signals into the cell affecting the muscle-transcription program. P38MAPK-dependent signalling, as well as PI3K/Akt pathway, has a key role in the control of muscle gene expression at different stages during the myogenic process. P38MAPK affects the activities of transcription factors, such as MyoD and myogenin, and contributes, together with PI3K/Akt pathway, to control the early and late steps of myogenic differentiation. The aim of our work was to better define the role of PKR, a dsRNA-activated protein kinase, as potential component in the differentiation program of C2C12 murine myogenic cells and to correlate its activity with p38MAPK and PI3K/Akt myogenic regulatory pathways. Here, we demonstrate that PKR is an essential component of the muscle development machinery and forms a functional complex with p38MAPK and/or Akt, contributing to muscle differentiation of committed myogenic cells in vitro. Inhibition of endogenous PKR activity by a specific (si)RNA and a PKR dominant-negative interferes with the myogenic program of C2C12 cells, causing a delay in activation of myogenic specific genes and inducing the formation of thinner myofibers. In addition, the construction of three PKR mutants allowed us to demonstrate that both N and C-terminal regions of PKR are critical for the interaction with p38MAPK and Akt. The novel discovered complex permits PKR to timely regulate the inhibition/activation of p38MAPK and Akt, controlling in this way the different steps characterizing skeletal muscle differentiation.

  2. Nanoscale fabrication of a peptide layer using an AFM probe

    NASA Astrophysics Data System (ADS)

    Nakamura, Chikashi; Miyamoto, Chie; Obataya, Ikuo; Nakamura, Noriyuki; Miyake, Jun

    2004-12-01

    Scanning probe microscopy has been applied in many studies to manipulate atoms or molecules. In particular, force spectroscopy using an atomic force microscope (AFM) is a powerful tool to elucidate intermolecular or intramolecular interactions and provide mechanical information. If enzymes could retain their activity when immobilized on probes, not only could enzyme-substrate interactions be investigated but also the probes could be used for precise biomolecular manipulation at the nano-scale. 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 fabricating activity of the protease immobilized on the AFM tip to peptides immobilized on a substrate, we designed and synthesized peptides that showed enzymatic action specific to the protease. When the protease digested the reporter peptide a quencher residue was released from the main flame of the peptide and resulted in fluorescence. In the designed 9 mer peptides, TAMRA functioned as a good quencher for FAM. After contact of the protease-immobilized tip to the reporter peptide layer, a fluorescent area was observed by microscopic imaging.

  3. In situ hydrodynamic lateral force calibration of AFM colloidal probes.

    PubMed

    Ryu, Sangjin; Franck, Christian

    2011-11-01

    Lateral force microscopy (LFM) is an application of atomic force microscopy (AFM) to sense lateral forces applied to the AFM probe tip. Recent advances in tissue engineering and functional biomaterials have shown a need for the surface characterization of their material and biochemical properties under the application of lateral forces. LFM equipped with colloidal probes of well-defined tip geometries has been a natural fit to address these needs but has remained limited to provide primarily qualitative results. For quantitative measurements, LFM requires the successful determination of the lateral force or torque conversion factor of the probe. Usually, force calibration results obtained in air are used for force measurements in liquids, but refractive index differences between air and liquids induce changes in the conversion factor. Furthermore, in the case of biochemically functionalized tips, damage can occur during calibration because tip-surface contact is inevitable in most calibration methods. Therefore, a nondestructive in situ lateral force calibration is desirable for LFM applications in liquids. Here we present an in situ hydrodynamic lateral force calibration method for AFM colloidal probes. In this method, the laterally scanned substrate surface generated a creeping Couette flow, which deformed the probe under torsion. The spherical geometry of the tip enabled the calculation of tip drag forces, and the lateral torque conversion factor was calibrated from the lateral voltage change and estimated torque. Comparisons with lateral force calibrations performed in air show that the hydrodynamic lateral force calibration method enables quantitative lateral force measurements in liquid using colloidal probes.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. Cell visco-elasticity measured with AFM and optical trapping at sub-micrometer deformations.

    PubMed

    Nawaz, Schanila; Sánchez, Paula; Bodensiek, Kai; Li, Sai; Simons, Mikael; Schaap, Iwan A T

    2012-01-01

    The measurement of the elastic properties of cells is widely used as an indicator for cellular changes during differentiation, upon drug treatment, or resulting from the interaction with the supporting matrix. Elasticity is routinely quantified by indenting the cell with a probe of an AFM while applying nano-Newton forces. Because the resulting deformations are in the micrometer range, the measurements will be affected by the finite thickness of the cell, viscous effects and even cell damage induced by the experiment itself. Here, we have analyzed the response of single 3T3 fibroblasts that were indented with a micrometer-sized bead attached to an AFM cantilever at forces from 30-600 pN, resulting in indentations ranging from 0.2 to 1.2 micrometer. To investigate the cellular response at lower forces up to 10 pN, we developed an optical trap to indent the cell in vertical direction, normal to the plane of the coverslip. Deformations of up to two hundred nanometers achieved at forces of up to 30 pN showed a reversible, thus truly elastic response that was independent on the rate of deformation. We found that at such small deformations, the elastic modulus of 100 Pa is largely determined by the presence of the actin cortex. At higher indentations, viscous effects led to an increase of the apparent elastic modulus. This viscous contribution that followed a weak power law, increased at larger cell indentations. Both AFM and optical trapping indentation experiments give consistent results for the cell elasticity. Optical trapping has the benefit of a lower force noise, which allows a more accurate determination of the absolute indentation. The combination of both techniques allows the investigation of single cells at small and large indentations and enables the separation of their viscous and elastic components. PMID:23028915

  9. 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.

  10. 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.

  11. The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis.

    PubMed

    Chen, Hongfeng; Fan, Meiyun; Pfeffer, Lawrence M; Laribee, R Nicholas

    2012-08-01

    Epigenetic changes in chromatin through histone post-translational modifications are essential for altering gene transcription in response to environmental cues. How histone modifications are regulated by environmental stimuli remains poorly understood yet this process is critical for delineating how epigenetic pathways are influenced by the cellular environment. We have used the target of rapamycin (TOR) pathway, which transmits environmental nutrient signals to control cell growth, as a model to delineate mechanisms underlying this phenomenon. A chemical genomics screen using the TOR inhibitor rapamycin against a histone H3/H4 mutant library identified histone H3 lysine 56 acetylation (H3K56ac) as a chromatin modification regulated by TOR signaling. We demonstrate this acetylation pathway functions in TOR-dependent cell growth in part by contributing directly to ribosomal RNA (rRNA) biogenesis. Specifically, H3K56ac creates a chromatin environment permissive to RNA polymerase I transcription and nascent rRNA processing by regulating binding of the high mobility group protein Hmo1 and the small ribosomal subunit (SSU) processome complex. Overall, these studies identify a novel chromatin regulatory role for TOR signaling and support a specific function for H3K56ac in ribosomal DNA (rDNA) gene transcription and nascent rRNA processing essential for cell growth.

  12. 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.

  13. 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.

  14. 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

  15. Dry powder inhaler: influence of humidity on topology and adhesion studied by AFM.

    PubMed

    Bérard, V; Lesniewska, E; Andrès, C; Pertuy, D; Laroche, C; Pourcelot, Y

    2002-01-31

    In the dry powder inhalers (DPIs), the adhesion results of the interactions between the active substance and the excipient. The carrier and the micronized drug particle morphologies are believed to affect the delivery of the drug. In this work, the couple studied was the lactose monohydrate and micronized zanamivir, used for the treatment of influenza. In a first approach, observations by scanning electron microscopy (SEM) have shown that the relative humidity (RH) greatly influenced the zanamivir amount fixed on the lactose monohydrate surface. This paper deals with the direct measurement in controlled atmosphere by atomic force microscopy (AFM) of the forces and the interaction ranges between a zanamivir probe and a lactose substrate. Selected zanamivir crystals were attached to the standard AFM probe. Different RH have been used in order to determine influent parameters permitting to identify the nature of adhesion forces between them. This study demonstrated that the increase of RH modified progressively the surface topology of the two components and increased the adhesion force.

  16. Fast image scanning method in liquid-AFM without image distortion.

    PubMed

    Choi, Inhee; Kim, Younghun; Kim, Jong Ho; Yang, Young In; Lee, Jeongjin; Lee, Suseung; Hong, Surin; Yi, Jongheop

    2008-11-01

    High speed imaging by atomic force microscopy (AFM) allows one to directly observe the dynamic behavior of a sample surface immersed in liquid media; thus, it has been considered to be an indispensable tool for nanobiotechnology and is used in many research fields, including molecular biology and surface science. For real-time observation of a certain behavior, the high speed imaging technique should be accompanied with a high resolution imaging technique to identify target materials. To improve the image quality at a high scanning rate, we developed a variable-controlled fast scanning method, which originated from the modified squeeze-drag superposition model in liquid media. A collection of non-distorted images was accomplished after proper modification of the operating conditions in a viscous fluid, via the simple handling of loading force and cantilever length. Consequently, a speeded-up AFM imaging process was achieved in the liquid environment at up to 200 µm s(-1), without attachment of additional devices. The reliability of the proposed method was verified by the characterization of a grating sample immersed in three types of liquid media. In addition, the results were visualized for elastic biomolecules submerged in a liquid with high kinematic viscosity. PMID:21832743

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

    SciTech Connect

    Seung-Ho-Baek

    2004-12-19

    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.

  18. Spontaneous aggregation of humic acid observed with AFM at different pH.

    PubMed

    Colombo, Claudio; Palumbo, Giuseppe; Angelico, Ruggero; Cho, Hyen Goo; Francioso, Ornella; Ertani, Andrea; Nardi, Serenella

    2015-11-01

    Atomic force microscopy in contact (AFM-C) mode was used to investigate the molecular dynamics of leonardite humic acid (HA) aggregate formed at different pH values. HA nanoparticles dispersed at pH values ranging from 2 to 12 were observed on a mica surface under dry conditions. The most clearly resolved and well-resulted AFM images of single particle were obtained at pH 5, where HA appeared as supramolecular particles with a conic shape and a hole in the centre. Those observations suggested that HA formed under these conditions exhibited a pseudo-amphiphilic nature, with secluded hydrophobic domains and polar subunits in direct contact with hydrophilic mica surface. Based on molecular simulation methods, a lignin-carbohydrate complex (LCC) model was proposed to explain the HA ring-like morphology. The LCC model optimized the parameters of β-O-4 linkages between 14 units of 1-4 phenyl propanoid, and resulted in an optimized structure comprising 45-50 linear helical molecules looped spirally around a central cavity. Those results added new insights on the adsorption mechanism of HA on polar surfaces as a function of pH, which was relevant from the point of view of natural aggregation in soil environment. PMID:26295541

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. A direct screen for c-di-GMP modulators reveals a Salmonella Typhimurium periplasmic ʟ-arginine-sensing pathway.

    PubMed

    Mills, Erez; Petersen, Erik; Kulasekara, Bridget R; Miller, Samuel I

    2015-06-01

    Cyclic-di-GMP (c-di-GMP) is a bacterial second messenger that transduces internal and external signals and regulates bacterial motility and biofilm formation. Some organisms encode more than 100 c-di-GMP-modulating enzymes, but only for a few has a signal been defined that modulates their activity. We developed and applied a high-throughput, real-time flow cytometry method that uses a fluorescence resonance energy transfer (FRET)-based biosensor of free c-di-GMP to screen for signals that modulate its concentration within Salmonella Typhimurium. We identified multiple compounds, including glucose, N-acetyl-d-glucosamine, salicylic acid, and ʟ-arginine, that modulated the FRET signal and therefore the free c-di-GMP concentration. By screening a library of mutants, we identified proteins required for the c-di-GMP response to each compound. Furthermore, low micromolar concentrations of ʟ-arginine induced a rapid translation-independent increase in c-di-GMP concentrations and c-di-GMP-dependent cellulose synthesis, responses that required the regulatory periplasmic domain of the diguanylate cyclase STM1987. ʟ-Arginine signaling also required the periplasmic putative ʟ-arginine-binding protein ArtI, implying that ʟ-arginine sensing occurred in the periplasm. Among the 20 commonly used amino acids, S. Typhimurium specifically responded to ʟ-arginine with an increase in c-di-GMP, suggesting that ʟ-arginine may serve as a signal during S. Typhimurium infection. Our results demonstrate that a second-messenger biosensor can be used to identify environmental signals and define pathways that alter microbial behavior.

  4. Performance improvement of a large range metrological AFM through parasitic interference feedback artifacts removing by using laser multimode modulation method

    NASA Astrophysics Data System (ADS)

    Li, Qi; Gao, Sitian; Li, Wei; Lu, Mingzhen; Shi, Yushu

    2013-05-01

    A large range multi-functional metrological atomic force microscope based on optical beam deflection method has been set up at NIM one year ago. Being designed intended to make a traceable measurement of standard samples, the machine uses three axes stacked piezoceramic actuators, each axis with a pair of push-pull piezo operated at opposite phases to make orthogonal scanning with maximized dimensional up to 50×50×2mm3. The stage displacement is measured by homodyne interferometer framework in x,y,z direction, from which beams are aligned to intersect at cantilever tip to avoid Abbe error, an eight times optical path multiplier interferometer mirror is researched to enhance fringe resolution. There is also a new compact AFM head integrated with LD, quadrant PD, cantilever, optical path and microscope, the head uses special track lens group to guarantee laser spot focused and static on the back of the cantilever, no matter whether or not the cantilever have lateral movements; similarly, reflect beam also focused and static in the center of quadrant detector through convergence lens group, assumed no cantilever bending on vertical direction. Attribute to above design, the AFM have a resolution up to 0.5nm. In the paper, further improvement is described to reduce the influence of parasitic interference caused by reflection from sample surface using laser multimode modulation, the results shows metrological AFM have a better performance in measuring step, lateral pitch, line width, nanoroughness and other nanoscale structures.

  5. AFM and electroanalytical studies of synthetic oligonucleotide hybridization.

    PubMed

    Chiorcea Paquim, A-M; Diculescu, V C; Oretskaya, T S; Oliveira Brett, A M

    2004-11-15

    The first and most important step in the development and manufacture of a sensitive DNA-biosensor for hybridization detection is the immobilization procedure of the nucleic acid probe on the transducer surface, maintaining its mobility and conformational flexibility. MAC Mode AFM images were used to demonstrate that oligonucleotide (ODN) molecules adsorb spontaneously at the electrode surface. After adsorption, the ODN layers were formed by molecules with restricted mobility, as well as by superposed molecules, which can lead to reduced hybridization efficiency. The images also showed the existence of pores in the adsorbed ODN film that revealed large parts of the electrode surface, and enabled non-specific adsorption of other ODNs on the uncovered areas. Electrostatic immobilization onto a clean glassy carbon electrode surface was followed by hybridization with complementary sequences and by control experiments with non-complementary sequences, studied using differential pulse voltammetry. The data obtained showed that non-specific adsorption strongly influenced the results, which depended on the sequence of the ODNs. In order to reduce the contribution of non-specific adsorbed ODNs during hybridization experiments, the carbon electrode surface was modified. After modification, the AFM images showed an electrode completely covered by the ODN probe film, which prevented the undesirable binding of target ODN molecules to the electrode surface. The changes of interfacial capacitance that took place after hybridization or control experiments showed the formation of a mixed multilayer that strongly depended on the local environment of the immobilized ODN.

  6. 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.

  7. 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.

  8. 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.

  9. Viscoelasticity of gelatin surfaces probed by AFM noise analysis.

    PubMed

    Benmouna, Farida; Johannsmann, Diethelm

    2004-01-01

    The viscoelastic properties of surfaces of swollen gelatin were investigated by analyzing the Brownian motion of an atomic force microscopy (AFM) cantilever in contact with the gel surface. A micron-sized glass sphere attached to the AFM cantilever is used as the dynamic probe. When the sphere approaches the gelatin surface, there is a static repulsive force without a jump into contact. The cantilever's Brownian movement is monitored in parallel, providing access to the dynamic sphere-surface interaction as quantified by the dynamic spring constant, kappa, and the drag coefficient, xi. Gelatin is used as a model substance for a variety of other soft surfaces, where the stiffness of the gel can be varied via the solvent quality, the bloom number, and the pH. The modulus derived from the static force-distance curve is in the kPa range, consistent with the literature. However, the dynamic spring constant as derived from the Brownian motion is much larger than the static differential spring constant dF/dz. On retraction, one observes a rather strong adhesion hysteresis. The strength of the bridge (as given by the dynamic spring constant and the drag coefficient) is very small. PMID:15745019

  10. Analysis of AFM cantilever dynamics close to sample surface

    NASA Astrophysics Data System (ADS)

    Habibnejad Korayem, A.; Habibnejad Korayem, Moharam; Ghaderi, Reza

    2013-07-01

    For imaging and manipulation of biological specimens application of atomic force microscopy (AFM) in liquid is necessary. In this paper, tapping-mode AFM cantilever dynamics in liquid close to sample surface is modeled and simulated by well defining the contact forces. The effect of cantilever tilting angle has been accounted carefully. Contact forces have some differences in liquid in comparison to air or vacuum in magnitude or formulation. Hydrodynamic forces are also applied on the cantilever due to the motion in liquid. A continuous beam model is used with its first mode and forward-time simulation method for simulation of its hybrid dynamics and the frequency response and amplitude versus separation diagrams are extracted. The simulation results show a good agreement with experimental results. The resonance frequency in liquid is so small in comparison to air due to additional mass and also additional damping due to the viscosity of the liquid around. The results show that the effect of separation on free vibration amplitude is great. Its effect on resonance frequency is considerable too.

  11. 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.

  12. 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.

  13. Corticotropin-releasing hormone directly stimulates cortisol and the cortisol biosynthetic pathway in human fetal adrenal cells.

    PubMed

    Sirianni, Rosa; Rehman, Khurram S; Carr, Bruce R; Parker, C Richard; Rainey, William E

    2005-01-01

    Near term the human fetal adrenals (HFAs) initiate production of cortisol, which promotes organ maturation and acts to increase placental CRH biosynthesis. The objective of the present study was to determine whether CRH directly stimulates both cortisol production and expression of the steroidogenic enzymes in HFA-definitive zone cells. CRH stimulated the production of cortisol in a time- and dose-dependent manner, with an effective concentration of as low as 0.01 nm. In real-time RT-PCR experiments, CRH treatment increased the mRNA levels of steroidogenic acute regulatory protein and each of the enzymes needed to produce cortisol. CRH induced 3beta-hydroxysteroid dehydrogenase type II (HSD3B2) by 34-fold, 21-hydroxylase (CYP21) by 55-fold, and 11beta-hydroxylase by 41-fold. Induction of steroidogenic acute regulatory protein, cholesterol side chain cleavage (CYP11A), and 17alpha-hydroxylase (CYP17) mRNA by CRH was 6-, 4-, and 6-fold, respectively. We also demonstrated that submaximal concentrations of CRH (30 pm) and ACTH (30 pm) that are seen in fetal circulation were additive on cortisol biosynthesis and 3beta-hydroxysteroid dehydrogenase type II mRNA induction. We suggest that CRH may play an important role in the late gestational rise in cortisol secretion from the HFAs, which may serve to augment placental CRH production and therefore participate in the endocrine cascade that is involved in fetal organ maturation and potentially in the timing of human parturition.

  14. 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. PMID:24086378

  15. Extracellular hydrogen peroxide, produced through a respiratory burst oxidase/superoxide dismutase pathway, directs ingrowth wall formation in epidermal transfer cells of Vicia faba cotyledons.

    PubMed

    Xia, Xue; Zhang, Hui-Ming; Andriunas, Felicity A; Offler, Christina E; Patrick, John W

    2012-09-01

    The intricate, and often polarized, ingrowth walls of transfer cells (TCs) amplify their plasma membrane surface areas to confer a transport function of supporting high rates of nutrient exchange across apo-/symplasmic interfaces. The TC ingrowth wall comprises a uniform wall layer on which wall ingrowths are deposited. Signals and signal cascades inducing trans-differentiation events leading to formation of TC ingrowth walls are poorly understood. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, when placed into culture, their adaxial epidermal cells rapidly (h) and synchronously form polarized ingrowth walls accessible for experimental observations. Using this model, we recently reported findings consistent with extracellular hydrogen peroxide, produced through a respiratory burst oxidase homolog/superoxide dismutase pathway, initiating cell wall biosynthetic activity and providing directional information guiding deposition of the polarized uniform wall. Our conclusions rested on observations derived from pharmacological manipulations of hydrogen peroxide production and correlative gene expression data sets. A series of additional studies were undertaken, the results of which verify that extracellular hydrogen peroxide contributes to regulating ingrowth wall formation and is generated by a respiratory burst oxidase homolog/superoxide dismutase pathway.

  16. The Wnt and Delta-Notch signalling pathways interact to direct pair-rule gene expression via caudal during segment addition in the spider Parasteatoda tepidariorum.

    PubMed

    Schönauer, Anna; Paese, Christian L B; Hilbrant, Maarten; Leite, Daniel J; Schwager, Evelyn E; Feitosa, Natália Martins; Eibner, Cornelius; Damen, Wim G M; McGregor, Alistair P

    2016-07-01

    In short-germ arthropods, posterior segments are added sequentially from a segment addition zone (SAZ) during embryogenesis. Studies in spiders such as Parasteatoda tepidariorum have provided insights into the gene regulatory network (GRN) underlying segment addition, and revealed that Wnt8 is required for dynamic Delta (Dl) expression associated with the formation of new segments. However, it remains unclear how these pathways interact during SAZ formation and segment addition. Here, we show that Delta-Notch signalling is required for Wnt8 expression in posterior SAZ cells, but represses the expression of this Wnt gene in anterior SAZ cells. We also found that these two signalling pathways are required for the expression of the spider orthologues of even-skipped (eve) and runt-1 (run-1), at least in part via caudal (cad). Moreover, it appears that dynamic expression of eve in this spider does not require a feedback loop with run-1, as is found in the pair-rule circuit of the beetle Tribolium Taken together, our results suggest that the development of posterior segments in Parasteatoda is directed by dynamic interactions between Wnt8 and Delta-Notch signalling that are read out by cad, which is necessary but probably not sufficient to regulate the expression of eve and run-1 Our study therefore provides new insights towards better understanding the evolution and developmental regulation of segmentation in other arthropods, including insects. PMID:27287802

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

    PubMed

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

    2016-05-01

    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

  18. 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.

  19. HEAT-INDUCED TAS1 TARGET1 Mediates Thermotolerance via HEAT STRESS TRANSCRIPTION FACTOR A1a–Directed Pathways in Arabidopsis[C][W

    PubMed Central

    Li, Shuxia; Liu, Jinxin; Liu, Zhongyuan; Li, Xiaorong; Wu, Feijie; He, Yuke

    2014-01-01

    Many heat stress transcription factors (Hsfs) and heat shock proteins (Hsps) have been identified to play important roles in the heat tolerance of plants. However, many of the key factors mediating the heat response pathways remain unknown. Here, we report that two genes, which are targets of TAS1 (trans-acting siRNA precursor 1)–derived small interfering RNAs that we named HEAT-INDUCED TAS1 TARGET1 (HTT1) and HTT2, are involved in thermotolerance. Microarray analysis revealed that the HTT1 and HTT2 genes were highly upregulated in Arabidopsis thaliana seedlings in response to heat shock. Overexpression of TAS1a, whose trans-acting small interfering RNAs target the HTT genes, elevated accumulation of TAS1-siRNAs and reduced expression levels of the HTT genes, causing weaker thermotolerance. By contrast, overexpression of HTT1 and HTT2 upregulated several Hsf genes, leading to stronger thermotolerance. In heat-tolerant plants overexpressing HsfA1a, the HTT genes were upregulated, especially at high temperatures. Meanwhile, HsfA1a directly activated HTT1 and HTT2 through binding to their promoters. HTT1 interacted with the heat shock proteins Hsp70-14 and Hsp40 and NUCLEAR FACTOR Y, SUBUNIT C2. Taken together, these results suggest that HTT1 mediates thermotolerance pathways because it is targeted by TAS1a, mainly activated by HsfA1a, and acts as cofactor of Hsp70-14 complexes. PMID:24728648

  20. The Wnt and Delta-Notch signalling pathways interact to direct pair-rule gene expression via caudal during segment addition in the spider Parasteatoda tepidariorum.

    PubMed

    Schönauer, Anna; Paese, Christian L B; Hilbrant, Maarten; Leite, Daniel J; Schwager, Evelyn E; Feitosa, Natália Martins; Eibner, Cornelius; Damen, Wim G M; McGregor, Alistair P

    2016-07-01

    In short-germ arthropods, posterior segments are added sequentially from a segment addition zone (SAZ) during embryogenesis. Studies in spiders such as Parasteatoda tepidariorum have provided insights into the gene regulatory network (GRN) underlying segment addition, and revealed that Wnt8 is required for dynamic Delta (Dl) expression associated with the formation of new segments. However, it remains unclear how these pathways interact during SAZ formation and segment addition. Here, we show that Delta-Notch signalling is required for Wnt8 expression in posterior SAZ cells, but represses the expression of this Wnt gene in anterior SAZ cells. We also found that these two signalling pathways are required for the expression of the spider orthologues of even-skipped (eve) and runt-1 (run-1), at least in part via caudal (cad). Moreover, it appears that dynamic expression of eve in this spider does not require a feedback loop with run-1, as is found in the pair-rule circuit of the beetle Tribolium Taken together, our results suggest that the development of posterior segments in Parasteatoda is directed by dynamic interactions between Wnt8 and Delta-Notch signalling that are read out by cad, which is necessary but probably not sufficient to regulate the expression of eve and run-1 Our study therefore provides new insights towards better understanding the evolution and developmental regulation of segmentation in other arthropods, including insects.

  1. Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG.

    PubMed

    Seemann, Myriam; Tse Sum Bui, Bernadette; Wolff, Murielle; Miginiac-Maslow, Myroslawa; Rohmer, Michel

    2006-03-01

    In the methylerythritol phosphate pathway for isoprenoid biosynthesis, the GcpE/IspG enzyme catalyzes the conversion of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate. This reaction requires a double one-electron transfer involving a [4Fe-4S] cluster. A thylakoid preparation from spinach chloroplasts was capable in the presence of light to act as sole electron donor for the plant GcpE Arabidopsis thaliana in the absence of any pyridine nucleotide. This is in sharp contrast with the bacterial Escherichia coli GcpE, which requires flavodoxin/flavodoxin reductase and NADPH as reducing system and represents the first proof that the electron flow from photosynthesis can directly act in phototrophic organisms as reducer in the 2-C-methyl-d-erythritol 4-phosphate pathway, most probably via ferredoxin, in the absence of any reducing cofactor. In the dark, the plant GcpE catalysis requires in addition of ferredoxin NADP(+)/ferredoxin oxido-reductase and NADPH as electron shuttle.

  2. 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

  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. Acute Cardiac Rejection Requires Directly Cytotoxic CD4 T cells: A Parallel Pathway between Fas and Perforin1

    PubMed Central

    Grazia, Todd J.; Plenter, Robert J.; Weber, Sarah M.; Lepper, Helen M.; Victorino, Francisco; Zamora, Martin R.; Pietra, Biagio A.; Gill, Ronald G.

    2009-01-01

    Background CD4 T cells can suffice as effector cells to mediate primary acute cardiac allograft rejection. While CD4 T cells can readily kill appropriate target cells in vitro, the corresponding role of such cytolytic activity for mediating allograft rejection in vivo is unknown. Therefore, we determined whether the cytolytic effector molecules perforin and/or FasL (CD95L) were necessary for CD4 T cell-mediated rejection in vivo. Methods Wild type C3H(H-2k) or Fas (CD95)-deficient C3Hlpr (H-2k) hearts were transplanted into immune-deficient C57B6rag−/− (H-2b) mice. Recipients then were reconstituted with naïve purified CD4 T cells from either wild-type, perforin (pfp)-deficient, or FasL (gld)-deficient T cell donors. Results In vitro, alloreactive CD4 T cells were competent to lyse donor MHC class II+ target cells, largely by a Fas-dependent mechanism. In vivo, the individual disruption of either donor Fas expression (lpr) or CD4 T cell-derived perforin had no signifcant impact on acute rejection. However, FasL-deficient (gld) CD4 T cells demonstrated delayed allograft rejection. Importantly, the simultaneous removal of both donor Fas expression and CD4 T cell perforin completely abrograted acute rejection, despite the persistence of CD4 T cells within the graft. Conclusions Results demonstrate that the direct rejection of cardiac allografts by CD4 effector T cells requires the alternative contribution of graft Fas expression and T cell perforin expression. To our knowledge, this is the first demonstration that cytolytic activity by CD4 T cells can play an obligate role for primary acute allograft rejection in vivo. PMID:20061916

  5. 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. PMID

  6. 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.

  7. 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

    2016-08-01

    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. PMID:27403761

  8. AFM CHARACTERIZATION OF RAMAN LASER INDUCED DAMAGE ON CDZNTECRYSTAL SURFACES

    SciTech Connect

    Teague, L.; Duff, M.

    2008-10-07

    High quality CdZnTe (or CZT) crystals have the potential for use in room temperature gamma-ray and X-ray spectrometers. Over the last decade, the methods for growing high quality CZT have improved the quality of the produced crystals however there are material features that can influence the performance of these materials as radiation detectors. The presence of structural heterogeneities within the crystals, such as twinning, pipes, grain boundaries (polycrystallinity), and secondary phases (SPs) can have an impact on the detector performance. There is considerable need for reliable and reproducible characterization methods for the measurement of crystal quality. With improvements in material characterization and synthesis, these crystals may become suitable for widespread use in gamma radiation detection. Characterization techniques currently utilized to test for quality and/or to predict performance of the crystal as a gamma-ray detector include infrared (IR) transmission imaging, synchrotron X-ray topography, photoluminescence spectroscopy, transmission electron microscopy (TEM), atomic force microscopy (AFM) and Raman spectroscopy. In some cases, damage caused by characterization methods can have deleterious effects on the crystal performance. The availability of non-destructive analysis techniques is essential to validate a crystal's quality and its ability to be used for either qualitative or quantitative gamma-ray or X-ray detection. The work presented herein discusses the damage that occurs during characterization of the CZT surface by a laser during Raman spectroscopy, even at minimal laser powers. Previous Raman studies have shown that the localized annealing from tightly focused, low powered lasers results in areas of higher Te concentration on the CZT surface. This type of laser damage on the surface resulted in decreased detector performance which was most likely due to increased leakage current caused by areas of higher Te concentration. In this study

  9. Intrinsically high-Q dynamic AFM imaging in liquid with a significantly extended needle tip

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

    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 operational frequency of over 200 kHz was demonstrated. The method has the potential to be extended to acquire viscoelastic material properties and provide truly gentle imaging of soft biological samples in physiological environments.

  10. Effect of tip mass on frequency response and sensitivity of AFM cantilever in liquid.

    PubMed

    Farokh Payam, Amir; Fathipour, Morteza

    2015-03-01

    The effect of tip mass on the frequency response and sensitivity of atomic force microscope (AFM) cantilever in the liquid environment is investigated. For this purpose, using Euler-Bernoulli beam theory and considering tip mass and hydrodynamic functions in a liquid environment, an expression for the resonance frequencies of AFM cantilever in liquid is derived. Then, based on this expression, the effect of the surface contact stiffness on the flexural mode of a rectangular AFM cantilever in fluid is investigated and compared with the case where the AFM cantilever operates in the air. The results show that in contrast with an air environment, the tip mass has no significant impact on the resonance frequency and sensitivity of the AFM cantilever in the liquid. Hence, analysis of AFM behaviour in liquid environment by neglecting the tip mass is logical. PMID:25562584

  11. Application of the Discrete Wavelet Transform to SEM and AFM Micrographs for Quantitative Analysis of Complex Surfaces.

    PubMed

    Workman, Michael J; Serov, Alexey; Halevi, Barr; Atanassov, Plamen; Artyushkova, Kateryna

    2015-05-01

    The discrete wavelet transform (DWT) has found significant utility in process monitoring, filtering, and feature isolation of SEM, AFM, and optical images. Current use of the DWT for surface analysis assumes initial knowledge of the sizes of the features of interest in order to effectively isolate and analyze surface components. Current methods do not adequately address complex, heterogeneous surfaces in which features across multiple size ranges are of interest. Further, in situations where structure-to-property relationships are desired, the identification of features relevant for the function of the material is necessary. In this work, the DWT is examined as a tool for quantitative, length-scale specific surface metrology without prior knowledge of relevant features or length-scales. A new method is explored for determination of the best wavelet basis to minimize variation in roughness and skewness measurements with respect to change in position and orientation of surface features. It is observed that the size of the wavelet does not directly correlate with the size of features on the surface, and a method to measure the true length-scale specific roughness of the surface is presented. This method is applied to SEM and AFM images of non-precious metal catalysts, yielding new length-scale specific structure-to-property relationships for chemical speciation and fuel cell performance. The relationship between SEM and AFM length-scale specific roughness is also explored. Evidence is presented that roughness distributions of SEM images, as measured by the DWT, is representative of the true surface roughness distribution obtained from AFM.

  12. Mechanical properties study of SW480 cells based on AFM.

    PubMed

    Liu, Xiaogang; Song, Zhengxun; Qu, Yingmin; Wang, Guoliang; Wang, Zuobin

    2015-08-01

    Since the invention of the atomic force microscope (AFM), it has been widely applied in biomedicine. One of the most important applications is used as an indenter tool to do the indentation experiment in order to get the mechanical properties of cells. In this paper, SW480 cells were used as the test subjects. Through the analysis of the contact and indentation, Young's modulus (E), which is an important parameter of cancer cells, has been estimated. Experimental results show that different mechanical models should be chosen to calculate the E in different indentation depths. Here, the E of SW480 cells was (2.5 ± 0.8) KPa at the indentation depth of 99 nm.

  13. 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

    2015-09-01

    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. PMID:26005137

  14. Mechanical Characterization of Photo-crosslinkable Hydrogels with AFM

    NASA Astrophysics Data System (ADS)

    McKenna, Alyssa; Byun, Myunghwan; Hayward, Ryan; Aidala, Katherine

    2012-02-01

    Stimuli-responsive hydrogel films formed from photo-crosslinkable polymers are versatile materials for controlled drug delivery devices, three-dimensional micro-assemblies, and components in microfluidic systems. For such applications, it is important to understand both the mechanical properties and the dynamics responses of these materials. We describe the use of atomic force microscope (AFM) based indentation experiments to characterize the properties of poly(N-isopropylacrylamide) copolymer films, crosslinked by activation of pendent benzophenone units using ultraviolet light. In particular, we study how the elastic modulus of the material, determined using the Johnson, Kendall, and Roberts model, depends on UV dose, and simultaneously investigate stress relaxation in these materials in the context of viscoelastic and poroelastic relaxation models.

  15. AFM of self-assembled lambda DNA-histone networks.

    PubMed

    Liu, YuYing; Guthold, Martin; Snyder, Matthew J; Lu, HongFeng

    2015-10-01

    Atomic force microscopy (AFM) was used to investigate the self-assembly behavior of λ-DNA and histones at varying histone:DNA ratios. Without histones and at the lowest histone:DNA ratio (less than one histone per 1000 base pairs of DNA), the DNA appeared as individual (uncomplexed), double-stranded DNA molecules. At increasing histone concentrations (one histone per 500, 250 and 167 base pairs of DNA), the DNA molecules started to form extensive polygonal networks of mostly pentagons and hexagons. The observed networks might be one of the naturally occurring, stable DNA-histone structures. The condensing effects of the divalent cations Mg(2+) and Ca(2+) on the DNA-histone complexes were also investigated. The networks persisted at high Mg(2+) concentration (20mM) and the highest histone concentration. At high Ca(2+) concentration and the highest histone concentration, the polygonal network disappeared and, instead, individual, tightly condensed aggregates were formed. PMID:26141439

  16. Modeling AFM Induced Mechanical Deformation of Living Cells

    SciTech Connect

    Rudd, R E; McElfresh, M; Balhorn, R; Allen, M J; Belak, J

    2002-11-15

    Finite element modeling has been applied to study deformation of living cells in Atomic Force Microscopy (AFM) and particularly Recognition Force Microscopy (RFM). The abstract mechanical problem of interest is the response to RFM point loads of an incompressible medium enclosed in a fluid membrane. Cells are soft systems, susceptible to large deformations in the course of an RFM measurement. Often the local properties such as receptor anchoring forces, the reason for the measurement, are obscured by the response of the cell as a whole. Modeling can deconvolute these effects. This facilitates experimental efforts to have reproducible measurements of mechanical and chemical properties at specific kinds of receptor sites on the membrane of a living cell. In this article we briefly review the RFM technique for cells and the problems it poses, and then report on recent progress in modeling the deformation of cells by a point load.

  17. 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. PMID:23721486

  18. In situ AFM crystal growth and dissolution study of calcite in the presence of aqueous fluoride

    NASA Astrophysics Data System (ADS)

    Vavouraki, A.; Putnis, C. V.; Putnis, A.; Koutsoukos, P. G.

    2009-04-01

    Fluoride is naturally abundant, encountered in rocks, soil and fresh and ocean water. Calcite crystals, during crystal growth may incorporate fluoride ions into their lattice (Okumura et al., 1983). In situ atomic force microscopy (AFM) has been used to study the growth and dissolution of calcite {104} surfaces in aqueous solutions in the presence of fluoride, using a fluid cell in which the supersaturated and the understaturated solutions respectively, flow over a freshly cleaved calcite crystal. For growth experiments, supersaturation index (S.I.) with respect to calcite was equal to 0.89 and the initial solution pH 10.2. The crystal growth rates were measured from the closure of the rhombohedral etch pits along the [010] direction induced by an initial dissolution step using pure water. The spreading rate of 2-dimensional nuclei was also measured along the same direction. In the presence of low fluoride concentrations (≤0.33 mM), the crystal growth rate of calcite was unaffected. At higher concentrations (up to 5 mM) growth rate decreased substantially to 50% of the rate in the absence of fluoride. Potential fluoride sorption over the calcite surface may ascribe the decrease of growth rates. Dissolution experiments were conducted at pH= 7.2 and dissolution rates of calcite were measured from the spreading of rhombohedral etch pits along both [010] and [42] directions. The presence of low concentrations of fluoride (≤1.1 mM) in the undersaturated solutions enhanced the dissolution rate along the [42] direction by 50% in comparison with pure water. The morphology of rhombohedral etch pits changed to hexagonal in the presence of fluoride in the undersaturated solutions. The AFM dissolution experiments suggested that the fluoride ions adsorbed onto the calcite surface. Further increase of fluoride concentrations (up to 1.6 mM) resulted in the decrease of the calcite dissolution rate by 60% in both [010] and [42] directions. Reference: Okumura, M, Kitano, Y

  19. Shewanella oneidensis MR-1 Uses Overlapping Pathways for Iron Reduction at a Distance and by Direct Contact under Conditions Relevant for Biofilms

    PubMed Central

    Lies, Douglas P.; Hernandez, Maria E.; Kappler, Andreas; Mielke, Randall E.; Gralnick, Jeffrey A.; Newman, Dianne K.

    2005-01-01

    We developed a new method to measure iron reduction at a distance based on depositing Fe(III) (hydr)oxide within nanoporous glass beads. In this “Fe-bead” system, Shewanella oneidensis reduces at least 86.5% of the iron in the absence of direct contact. Biofilm formation accompanies Fe-bead reduction and is observable both macro- and microscopically. Fe-bead reduction is catalyzed by live cells adapted to anaerobic conditions, and maximal reduction rates require sustained protein synthesis. The amount of reactive ferric iron in the Fe-bead system is available in excess such that the rate of Fe-bead reduction is directly proportional to cell density; i.e., it is diffusion limited. Addition of either lysates prepared from anaerobic cells or exogenous electron shuttles stimulates Fe-bead reduction by S. oneidensis, but iron chelators or additional Fe(II) do not. Neither dissolved Fe(III) nor electron shuttling activity was detected in culture supernatants, implying that the mediator is retained within the biofilm matrix. Strains with mutations in omcB or mtrB show about 50% of the wild-type levels of reduction, while a cymA mutant shows less than 20% of the wild-type levels of reduction and a menF mutant shows insignificant reduction. The Fe-bead reduction defect of the menF mutant can be restored by addition of menaquinone, but menaquinone itself cannot stimulate Fe-bead reduction. Because the menF gene encodes the first committed step of menaquinone biosynthesis, no intermediates of the menaquinone biosynthetic pathway are used as diffusible mediators by this organism to promote iron reduction at a distance. CymA and menaquinone are required for both direct and indirect mineral reduction, whereas MtrB and OmcB contribute to but are not absolutely required for iron reduction at a distance. PMID:16085832

  20. Mechanism for the Direct Synthesis of H2O2 on Pd Clusters: Heterolytic Reaction Pathways at the Liquid-Solid Interface.

    PubMed

    Wilson, Neil M; Flaherty, David W

    2016-01-20

    Direct synthesis (H2 + O2 → H2O2) is a promising reaction for producing H2O2, which can replace chlorinated oxidants in industrial processes. The mechanism of this reaction and the reasons for the importance of seemingly unrelated factors (e.g., Pd cluster size and solvent pH) remain unclear despite significant research. We propose a mechanism for H2O2 formation on Pd clusters consistent with steady-state H2O2 and H2O formation rates measured as functions of reactant pressures and temperature and the interpretations of proton concentration effects. H2O2 forms by sequential proton-electron transfer to O2 and OOH surface intermediates, whereas H2O forms by O-O bond rupture within OOH surface species. Direct synthesis, therefore, does not proceed by the Langmuir-Hinshelwood mechanism often invoked. Rather, H2O2 forms by heterolytic reaction pathways resembling the two-electron oxygen reduction reaction (ORR); however, the chemical potential of H2 replaces an external electrical potential as the thermodynamic driving force. Activation enthalpies (ΔH(⧧)) for H2O formation increase by 14 kJ mol(-1) when Pd cluster diameters increase from 0.7 to 7 nm because changes in the electronic structure of Pd surface atoms decrease their propensity to cleave O-O bonds. ΔH(⧧) values for H2O2 remain nearly constant because barriers for proton-electron transfer depend weakly on the coordinative saturation of Pd surface atoms. Collectively, these results provide a self-consistent mechanism, which clarifies many studies in which H2O2 rates and selectivities were shown to depend on the concentration of acid/halide additives and Pd cluster size. These findings will guide the rational design of selective catalysts for direct synthesis.

  1. Mechanism for the Direct Synthesis of H2O2 on Pd Clusters: Heterolytic Reaction Pathways at the Liquid-Solid Interface.

    PubMed

    Wilson, Neil M; Flaherty, David W

    2016-01-20

    Direct synthesis (H2 + O2 → H2O2) is a promising reaction for producing H2O2, which can replace chlorinated oxidants in industrial processes. The mechanism of this reaction and the reasons for the importance of seemingly unrelated factors (e.g., Pd cluster size and solvent pH) remain unclear despite significant research. We propose a mechanism for H2O2 formation on Pd clusters consistent with steady-state H2O2 and H2O formation rates measured as functions of reactant pressures and temperature and the interpretations of proton concentration effects. H2O2 forms by sequential proton-electron transfer to O2 and OOH surface intermediates, whereas H2O forms by O-O bond rupture within OOH surface species. Direct synthesis, therefore, does not proceed by the Langmuir-Hinshelwood mechanism often invoked. Rather, H2O2 forms by heterolytic reaction pathways resembling the two-electron oxygen reduction reaction (ORR); however, the chemical potential of H2 replaces an external electrical potential as the thermodynamic driving force. Activation enthalpies (ΔH(⧧)) for H2O formation increase by 14 kJ mol(-1) when Pd cluster diameters increase from 0.7 to 7 nm because changes in the electronic structure of Pd surface atoms decrease their propensity to cleave O-O bonds. ΔH(⧧) values for H2O2 remain nearly constant because barriers for proton-electron transfer depend weakly on the coordinative saturation of Pd surface atoms. Collectively, these results provide a self-consistent mechanism, which clarifies many studies in which H2O2 rates and selectivities were shown to depend on the concentration of acid/halide additives and Pd cluster size. These findings will guide the rational design of selective catalysts for direct synthesis. PMID:26597848

  2. 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

    2010-04-01

    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.

  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

    2010-04-01

    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. PMID:20106967

  4. 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.

  5. 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

  6. Beyond topography - enhanced imaging of cometary dust with the MIDAS AFM

    NASA Astrophysics Data System (ADS)

    Bentley, M. S.; Torkar, K.; Jeszenszky, H.; Romstedt, J.

    2013-09-01

    The MIDAS atomic force microscope (AFM) onboard the Rosetta spacecraft is primarily designed to return the 3D shape and structure of cometary dust particles collected at comet 67P/Churyumov-Gerasimenko [1]. Commercial AFMs have, however, been further developed to measure many other sample properties. The possibilities to make such measurements with MIDAS are explored here.

  7. A new nano-accuracy AFM system for minimizing Abbe errors and the evaluation of its measuring uncertainty.

    PubMed

    Kim, Dongmin; Lee, Dong Yeon; Gweon, Dae Gab

    2007-01-01

    A new AFM system was designed for the establishment of a standard technique of nano-length measurement in a 2D plane. In a long range (about several tens of micrometers), measurement uncertainty is dominantly affected by the Abbe error of the XY scanning stage. No linear stage is perfectly straight; in other words, every scanning stage is subject to tilting, pitch and yaw motions. In this paper, an AFM system with minimum offsets of XY sensing is designed. Moreover, the XY scanning stage is designed to minimize the rotation angle, as Abbe errors occur through multiple combination of the offset and the rotation angle. To minimize the rotation angle, an optimal design is performed by maximizing the ratio of the stiffness of the parasitic direction to the motion direction of each stage. This paper describes a design scheme of a full AFM system, in particular, the XY scanner. The full range of a fabricated XY scanner is 100 microm x 100 microm. The tilting, pitch and yaw motions are measured by an autocollimator to evaluate the performance of the XY stage. The results show that the XY scanner have a 0.75 arcsec parasitic rotation about the maximum range, thus the uncertainty in terms of the Abbe errors are very small relative to other standard equipment. Using this AFM system, a 3mum pitch specimen was measured. The measurement uncertainty of the total system was evaluated especially about pitch length. For a 1D evaluation, Abbe errors are the most dominant factor, and the expanded combined uncertainty (k = 2) of system was square root (4.13)(2)+(5.07 x 10(-5)xp)(2)(nm). For a 2D evaluation, mirror non-orthogonality and Abbe errors are dominant factors, and expanded combined uncertainty (k = 2) of the system was square root (4.13)(2)+(1.228 x 10(-4)xp)(2) in the X direction, and square root (6.28)(2)+(1.266 x 10(-4)xp)(2) in the Y direction (the unit is nanometers), where p is the measured length in nm.

  8. 3D force and displacement sensor for SFA and AFM measurements.

    PubMed

    Kristiansen, Kai; McGuiggan, Patricia; Carver, Greg; Meinhart, Carl; Israelachvili, Jacob

    2008-02-19

    A new device has been designed, and a prototype built and tested, that can simultaneously measure the displacements and/or the components of a force in three orthogonal directions. The "3D sensor" consists of four or eight strain gauges attached to the four arms of a single cross-shaped force-measuring cantilever spring. Finite element modeling (FEM) was performed to optimize the design configuration to give desired sensitivity of force, displacement, stiffness, and resonant frequency in each direction (x, y, and z) which were tested on a "mesoscale" device and found to agree with the predicted values to within 4-10%. The device can be fitted into a surface forces apparatus (SFA), and a future smaller "microscale" microfabricated version can be fitted into an atomic force microscope (AFM) for simultaneous measurements of the normal and lateral (friction) forces between a tip (or colloidal bead probe) and a surface, and the topography of the surface. Results of the FEM analysis are presented, and approximate equations derived using linear elasticity theory are given for the sensitivity in each direction. Initial calibrations and measurements of thin film rheology (lubrication forces) using the "mesoscale" prototype show the device to function as expected.

  9. 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.

  10. AFM imaging of functionalized double-walled carbon nanotubes.

    PubMed

    Lamprecht, C; Danzberger, J; Lukanov, P; Tîlmaciu, C-M; Galibert, A-M; Soula, B; Flahaut, E; Gruber, H J; Hinterdorfer, P; Ebner, A; Kienberger, F

    2009-07-01

    We present a comparative study of several non-covalent approaches to disperse, debundle and non-covalently functionalize double-walled carbon nanotubes (DWNTs). We investigated the ability of bovine serum albumin (BSA), phospholipids grafted onto amine-terminated polyethylene glycol (PL-PEG(2000)-NH(2)), as well as a combination thereof, to coat purified DWNTs. Topographical imaging with the atomic force microscope (AFM) was used to assess the coating of individual DWNTs and the degree of debundling and dispersion. Topographical images showed that functionalized DWNTs are better separated and less aggregated than pristine DWNTs and that the different coating methods differ in their abilities to successfully debundle and disperse DWNTs. Height profiles indicated an increase in the diameter of DWNTs depending on the functionalization method and revealed adsorption of single molecules onto the nanotubes. Biofunctionalization of the DWNT surface was achieved by coating DWNTs with biotinylated BSA, providing for biospecific binding of streptavidin in a simple incubation step. Finally, biotin-BSA-functionalized DWNTs were immobilized on an avidin layer via the specific avidin-biotin interaction. PMID:19375857

  11. Conductive probe AFM measurements of conjugated molecular wires.

    PubMed

    Ishida, Takao; Mizutani, Wataru; Liang, Tien-Tzu; Azehara, Hiroaki; Miyake, Koji; Sasaki, Shinya; Tokumoto, Hiroshi

    2003-12-01

    The electrical conduction of self-assembled monolayers (SAMs) made from conjugated molecules was measured using conductive probe atomic force microscopy (CP-AFM), with a focus on the molecular structural effect on conduction. First, the electrical conduction of SAMs made from phenylene oligomer SAMs was measured. The resistances through the monolayers increased exponentially with an increase in molecular length and the decay constants of transconductance beta were about 0.45 to 0.61 A(-1) measured at lower bias region. We further investigated the influence of applied load on the resistances. The resistances through terphenyl SAMs increased with an increase in the applied load up to 14 nN. Second, using an insertion technique into insulating alkanethiol SAMs, the electrical conduction of single conjugated terphenyl methanethiol and oligo(para-phenylenevinylene) (OPV) molecules embedded into insulating alkanethiol SAMs were measured. Electrical currents through these single molecules of OPVs were estimated to be larger than those through single terphenyl molecules, suggesting that the OPV structure can increase the electrical conduction of single molecules. Third, apparent negative differential resistance (NDR) was observed at higher bias measurements of SAMs. The appearance of NDR might be related to roughness of SAM surface, because apparent NDR was often observed on rough surfaces. In any case, the tip-molecule contact condition strongly affected carrier transport through metal tip/SAM/metal junction.

  12. AFM Investigation of Liquid-Filled Polymer Microcapsules Elasticity.

    PubMed

    Sarrazin, Baptiste; Tsapis, Nicolas; Mousnier, Ludivine; Taulier, Nicolas; Urbach, Wladimir; Guenoun, Patrick

    2016-05-10

    Elasticity of polymer microcapsules (MCs) filled with a liquid fluorinated core is studied by atomic force microscopy (AFM). Accurately characterized spherical tips are employed to obtain the Young's moduli of MCs having four different shell thicknesses. We show that those moduli are effective ones because the samples are composites. The strong decrease of the effective MC elasticity (from 3.0 to 0.1 GPa) as the shell thickness decreases (from 200 to 10 nm) is analyzed using a novel numerical approach. This model describes the evolution of the elasticity of a coated half-space according to the contact radius, the thickness of the film, and the elastic moduli of bulk materials. This numerical model is consistent with the experimental data and allows simulating the elastic behavior of MCs at high frequencies (5 MHz). While the quasi-static elasticity of the MCs is found to be very dependent on the shell thickness, the high frequency (5 MHz) elastic behavior of the core leads to a stable behavior of the MCs (from 2.5 to 3 GPa according to the shell thickness). Finally, the effect of thermal annealing on the MCs elasticity is investigated. The Young's modulus is found to decrease because of the reduction of the shell thickness due to the loss of the polymer. PMID:27058449

  13. AFM-based force microsensor for a microrobot

    NASA Astrophysics Data System (ADS)

    Fatikow, Sergej; Fahlbusch, Stephan

    2001-10-01

    Microrobots are the result of increasing research activities at the border between microsystem technology and robotics. Today already, robots with dimensions of a few cubic- centimeters can be developed. Like conventional robots, microrobots represent a complex system that usually contains several different types of actuators and sensors. The measurement of gripping forces is the most important sensor application in micromanipulation besides visual servoing to protect the parts from too high surface pressures and thereby damage during the assembly process. Very small forces in the range of 200 (mu) N down to 0.1 (mu) N or even less have to be sensed. Thus, the aim of our current research activities is the development of a high-resolution integrated force microsensor for measuring gripping forces in a microhandling robot. On the one hand, the sensor should be a device for teleoperated manipulation tasks in a flexible microhandling station. On the other hand, typical microhandling operations should to a large extend be automated with the aid of computer-based signal processing of sensor information. The user should be provided with an interface for teleoperated manipulation and an interface for partially automated manipulation of microobjects. In this paper, a concept for the measurement of gripping forces in microrobotics using piezoresistive AFM (atomic force microscope) cantilevers is introduced. Further on, the concept of a microrobot-based SEM station and its applications are presented.

  14. 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.

  15. Comparison of the somatosensory evoked potential and the direct cortical response following severe incomplete global ischemia: selective vulnerability of the white matter conduction pathways.

    PubMed

    Lesnick, J E; Coyer, P E; Michele, J J; Welsh, F A; Simeone, F A

    1986-01-01

    Eight cats were subjected to graded hemorrhagic hypotension following bilateral carotid ligation to produce incomplete global cerebral ischemia. Three additional cats served as controls. The somatosensory evoked potential (SEP) and direct cortical response (DCR) were monitored in all animals and in each case, the cortical component of the SEP was abolished during progressive ischemia while the morphology of the DCR was well-preserved but with reduced amplitude. Determinations of adenosine triphosphate (ATP), phosphocreatine (PCr), and lactate levels in cerebral cortex and white matter were made in five experimental cats and the three controls. At the time of failure of the cortical SEP, PCr was dramatically reduced and lactate moderately elevated in the white matter while ATP remained unchanged. Cortical lactate was only mildly elevated and PCr and ATP were unchanged accounting for preservation of the DCR. In this model of global ischemia, abolition of the cortical SEP is due to a block of stimulus conduction in white matter projection pathways. A hypothesis to explain the observed metabolic changes is presented and correlation is made to clinical situations. PMID:3810728

  16. The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins

    NASA Astrophysics Data System (ADS)

    Beedle, Amy E. M.; Lezamiz, Ainhoa; Stirnemann, Guillaume; Garcia-Manyes, Sergi

    2015-08-01

    Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.

  17. The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins

    PubMed Central

    Beedle, Amy E. M.; Lezamiz, Ainhoa; Stirnemann, Guillaume; Garcia-Manyes, Sergi

    2015-01-01

    Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs. PMID:26235284

  18. 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

  19. 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

    2014-10-01

    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.

  20. AFM and uni-axial testing of pericardium exposed to radiotherapy doses

    NASA Astrophysics Data System (ADS)

    Daar, Eman; Kaabar, W.; Lei, C.; Keddie, J. L.; Nisbet, A.; Bradley, D. A.

    2011-10-01

    The pericardium, a double-layered sac that encloses the heart, is made up of collagen and elastin fibres embedded in an amorphous matrix (forming the extracellular matrix). Collagen fibres are aligned in multidirectional orientation layers. This free arrangement of fibres gives the pericardium its viscoelastic properties and the ability to deform in all directions. This is an important mechanical property for the heart to perform its physiological functions, acknowledging the fact that the heart is attached to different ligaments and muscles in all directions. The present study aims to investigate the effect of penetrating photon ionising radiation on bovine pericardium tissue. This links to an interest in seeking to understand possible mechanisms underlying cardiac complications following treatment of the left breast in radiotherapy regimes. Pericardium samples were subjected to doses in the range 0-80 Gy. Atomic force microscopy (AFM) has been applied in characterising changes in the infrastructural and mechanical properties of the tissues. Preliminary data for doses of 80 Gy shows there was no significant change in the D-spacing period of the banded structure collagen type I but a significant increase is observed in the FWHM of the fibril widths (by between 25% and 27%) over that of unirradiated pericardium tissue.

  1. Dissolution of uranophane: An AFM, XPS, SEM and ICP study

    NASA Astrophysics Data System (ADS)

    Schindler, Michael; Freund, Michael; Hawthorne, Frank C.; Burns, Peter C.; Maurice, Patricia A.

    2009-05-01

    Dissolution experiments on single crystals of uranophane and uranophane-β, Ca(H 2O) 5[(UO 2)(SiO 3(OH)] 2, from the Shinkolobwe mine of the Democratic Republic of Congo, were done in an aqueous HCl solution of pH 3.5 for 3 h, in HCl solutions of pH 2 for 5, 10 and 30 min, and in Pb 2+-, Ba-, Sr-, Ca- and Mg-HCl solutions of pH 2 for 30 min. The basal surfaces of the treated uranophane crystals were examined using atomic-force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Solutions after dissolution experiments on single crystals and synthetic powders were analysed with inductively coupled plasma-optical emission spectroscopy (ICP-OES) and mass spectroscopy (ICP-MS). The morphology of the observed etch pits (measured by AFM) were compared to the morphology, predicted on the basis of the bond-valence deficiency of polyhedron chains along the edges of the basal surface. Etch pits form in HCl solutions of pH 2. Their decrease in depth with the duration of the dissolution experiment is explained with the stepwave dissolution model, which describes the lowering of the surrounding area of an etch pit with continuous waves of steps emanated from the etch pit into the rest of the crystal surface. Hillocks form in an HCl solution of pH 3.5, and the chemical composition of the surface (as indicated by XPS) shows that these hillocks are the result of the precipitation of a uranyl-hydroxy-hydrate phase. Well-orientated hillocks form on the surface of uranophane in a SrCl 2-HCl solution of pH 2. They are part of an aged silica coating of composition Si 2O 2(OH) 4(H 2O) n. An amorphous layer forms on the surface of uranophane in a MgCl 2-HCl solution of pH 2, which has a composition and structure similar to silicic acid. Small crystallites of uranyl-hydroxy-hydrate phases form on the surface of uranophane after treatment in Pb(NO 3) 2-HCl and BaCl 2-HCl solutions of pH 2. Dissolution experiments on synthetic uranophane powders

  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. 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.

  4. 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

  5. Direct AFM force measurements between air bubbles in aqueous monodisperse sodium poly(styrene sulfonate) solutions.

    PubMed

    Browne, Christine; Tabor, Rico F; Grieser, Franz; Dagastine, Raymond R

    2015-08-01

    Structural forces play an important role in the rheology, processing and stability of colloidal systems and complex fluids, with polyelectrolytes representing a key class of structuring colloids. Here, we explore the interactions between soft colloids, in the form of air bubbles, in solutions of monodisperse sodium poly(styrene sulfonate) as a model polyelectrolyte. It is found that by self-consistently modelling the force oscillations due to structuring of the polymer chains along with deformation of the bubbles, it is possible to precisely predict the interaction potential between approaching bubbles. In line with polyelectrolyte scaling theory, two distinct regimes of behaviour are seen, corresponding to dilute and semi-dilute polymer solutions. It is also seen that by blending monodisperse systems to give a bidisperse sample, the interaction forces between soft colloids can be controlled with a high degree of precision. At increasing bubble collision velocity, it is revealed that hydrodynamic flow overwhelms oscillatory structural interactions, showing the important disparity between equilibrium behaviour and dynamic interactions.

  6. 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

  7. Tumor Suppressor Density-enhanced Phosphatase-1 (DEP-1) Inhibits the RAS Pathway by Direct Dephosphorylation of ERK1/2 Kinases*

    PubMed Central

    Sacco, Francesca; Tinti, Michele; Palma, Anita; Ferrari, Emanuela; Nardozza, Aurelio P.; van Huijsduijnen, Rob Hooft; Takahashi, Takamune; Castagnoli, Luisa; Cesareni, Gianni

    2009-01-01

    Density-enhanced phosphatase-1 (DEP-1) is a trans-membrane receptor protein-tyrosine phosphatase that plays a recognized prominent role as a tumor suppressor. However, the mechanistic details underlying its function are poorly understood because its primary physiological substrate(s) have not been firmly established. To shed light on the mechanisms underlying the anti-proliferative role of this phosphatase, we set out to identify new DEP-1 substrates by a novel approach based on screening of high density peptide arrays. The results of the array experiment were combined with a bioinformatics filter to identify eight potential DEP-1 targets among the proteins annotated in the MAPK pathway. In this study we show that one of these potential targets, the ERK1/2, is indeed a direct DEP-1 substrate in vivo. Pulldown and in vitro dephosphorylation assays confirmed our prediction and demonstrated an overall specificity of DEP-1 in targeting the phosphorylated tyrosine 204 of ERK1/2. After epidermal growth factor stimulation, the phosphorylation of the activation loop of ERK1/2 can be modulated by changing the concentration of DEP-1, without affecting the activity of the upstream kinase MEK. In addition, we show that DEP-1 contains a KIM-like motif to recruit ERK1/2 proteins by a docking mechanism mediated by the common docking domain in ERK1/2. ERK proteins that are mutated in the conserved docking domain become insensitive to DEP-1 de-phosphorylation. Overall this study provides novel insights into the anti-proliferative role of this phosphatase and proposes a new mechanism that may also be relevant for the regulation of density-dependent growth inhibition. PMID:19494114

  8. 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.

  9. The direct pathway from the brainstem reticular formation to the cerebral cortex in the ascending reticular activating system: A diffusion tensor imaging study.

    PubMed

    Jang, Sung Ho; Kwon, Hyeok Gyu

    2015-10-01

    Precise evaluation of the ascending reticular activating system (ARAS) is important for diagnosis, prediction of prognosis, and management of patients with disorders of impaired consciousness. In the current study, we attempted to reconstruct the direct neural pathway between the brainstem reticular formation (RF) and the cerebral cortex in normal subjects, using diffusion tensor imaging (DTI). Forty-one healthy subjects were recruited for this study. DTIs were performed using a sensitivity-encoding head coil at 1.5Tesla with FMRIB Software Library. For connectivity of the brainstem RF, we used two regions of interest (ROIs) for the brainstem RF (seed ROI) and the thalamus and hypothalamus (exclusion ROI). Connectivity was defined as the incidence of connection between the brainstem RF and target brain regions at the threshold of 5 and 50 streamlines. Regarding the thresholds of 5 and 50, the brainstem RF showed high connectivity to the lateral prefrontal cortex (lPFC, 67.1% and 20.7%) and ventromedial prefrontal cortex (vmPFC, 50.0% and 18.3%), respectively. In contrast, the brainstem RF showed low connectivity to the primary motor cortex (31.7% and 3.7%), premotor cortex (24.4% and 3.7%), primary somatosensory cortex (23.2% and 2.4%), orbitofrontal cortex (17.1% and 7.3%), and posterior parietal cortex (12.2% and 0%), respectively. The brainstem RF was mainly connected to the prefrontal cortex, particularly lPFC and vmPFC. We believe that the methodology and results of this study would be useful to clinicians involved in the care of patients with impaired consciousness and researchers in studies of the ARAS. PMID:26363340

  10. Exenatide exerts direct protective effects on endothelial cells through the AMPK/Akt/eNOS pathway in a GLP-1 receptor-dependent manner.

    PubMed

    Wei, Rui; Ma, Shifeng; Wang, Chen; Ke, Jing; Yang, Jin; Li, Weihong; Liu, Ye; Hou, Wenfang; Feng, Xinheng; Wang, Guang; Hong, Tianpei

    2016-06-01

    Glucagon-like peptide-1 (GLP-1) may have direct favorable effects on cardiovascular system. The aim of this study was to investigate the effects of the GLP-1 analog exenatide on improving coronary endothelial function in patients with type 2 diabetes and to investigate the underlying mechanisms. The newly diagnosed type 2 diabetic subjects were enrolled and given either lifestyle intervention or lifestyle intervention plus exenatide treatment. After 12-wk treatment, coronary flow velocity reserve (CFVR), an important indicator of coronary endothelial function, was improved significantly, and serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were remarkably decreased in the exenatide treatment group compared with the baseline and the control group. Notably, CFVR was correlated inversely with hemoglobin A1c (Hb A1c) and positively with high-density lipoprotein cholesterol (HDL-C). In human umbilical vein endothelial cells, exendin-4 (a form of exenatide) significantly increased NO production, endothelial NO synthase (eNOS) phosphorylation, and GTP cyclohydrolase 1 (GTPCH1) level in a dose-dependent manner. The GLP-1 receptor (GLP-1R) antagonist exendin (9-39) or GLP-1R siRNA, adenylyl cyclase inhibitor SQ-22536, AMPK inhibitor compound C, and PI3K inhibitor LY-294002 abolished the effects of exendin-4. Furthermore, exendin-4 reversed homocysteine-induced endothelial dysfunction by decreasing sICAM-1 and reactive oxygen species (ROS) levels and upregulating NO production and eNOS phosphorylation. Likewise, exendin (9-39) diminished the protective effects of exendin-4 on the homocysteine-induced endothelial dysfunction. In conclusion, exenatide significantly improves coronary endothelial function in patients with newly diagnosed type 2 diabetes. The effect may be mediated through activation of AMPK/PI3K-Akt/eNOS pathway via a GLP-1R/cAMP-dependent mechanism. PMID:27072494

  11. 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.

  12. MetaRep, an extended CMAS 3D program to visualize mafic (CMAS, ACF-S, ACF-N) and pelitic (AFM-K, AFM-S, AKF-S) projections

    NASA Astrophysics Data System (ADS)

    France, Lydéric; Nicollet, Christian

    2010-06-01

    MetaRep is a program based on our earlier program CMAS 3D. It is developed in MATLAB ® script. MetaRep objectives are to visualize and project major element compositions of mafic and pelitic rocks and their minerals in the pseudo-quaternary projections of the ACF-S, ACF-N, CMAS, AFM-K, AFM-S and AKF-S systems. These six systems are commonly used to describe metamorphic mineral assemblages and magmatic evolutions. Each system, made of four apices, can be represented in a tetrahedron that can be visualized in three dimensions with MetaRep; the four tetrahedron apices represent oxides or combination of oxides that define the composition of the projected rock or mineral. The three-dimensional representation allows one to obtain a better understanding of the topology of the relationships between the rocks and minerals and relations. From these systems, MetaRep can also project data in ternary plots (for example, the ACF, AFM and AKF ternary projections can be generated). A functional interface makes it easy to use and does not require any knowledge of MATLAB ® programming. To facilitate the use, MetaRep loads, from the main interface, data compiled in a Microsoft Excel ™ spreadsheet. Although useful for scientific research, the program is also a powerful tool for teaching. We propose an application example that, by using two combined systems (ACF-S and ACF-N), provides strong confirmation in the petrological interpretation.

  13. 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

  14. Fabrication of cone-shaped boron doped diamond and gold nanoelectrodes for AFM-SECM

    NASA Astrophysics Data System (ADS)

    Avdic, A.; Lugstein, A.; Wu, M.; Gollas, B.; Pobelov, I.; Wandlowski, T.; Leonhardt, K.; Denuault, G.; Bertagnolli, E.

    2011-04-01

    We demonstrate a reliable microfabrication process for a combined atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) measurement tool. Integrated cone-shaped sensors with boron doped diamond (BDD) or gold (Au) electrodes were fabricated from commercially available AFM probes. The sensor formation process is based on mature semiconductor processing techniques, including focused ion beam (FIB) machining, and highly selective reactive ion etching (RIE). The fabrication approach preserves the geometry of the original AFM tips resulting in well reproducible nanoscaled sensors. The feasibility and functionality of the fully featured tips are demonstrated by cyclic voltammetry, showing good agreement between the measured and calculated currents of the cone-shaped AFM-SECM electrodes.

  15. Fabrication of cone-shaped boron doped diamond and gold nanoelectrodes for AFM-SECM.

    PubMed

    Avdic, A; Lugstein, A; Wu, M; Gollas, B; Pobelov, I; Wandlowski, T; Leonhardt, K; Denuault, G; Bertagnolli, E

    2011-04-01

    We demonstrate a reliable microfabrication process for a combined atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) measurement tool. Integrated cone-shaped sensors with boron doped diamond (BDD) or gold (Au) electrodes were fabricated from commercially available AFM probes. The sensor formation process is based on mature semiconductor processing techniques, including focused ion beam (FIB) machining, and highly selective reactive ion etching (RIE). The fabrication approach preserves the geometry of the original AFM tips resulting in well reproducible nanoscaled sensors. The feasibility and functionality of the fully featured tips are demonstrated by cyclic voltammetry, showing good agreement between the measured and calculated currents of the cone-shaped AFM-SECM electrodes.

  16. Fabrication of cone-shaped boron doped diamond and gold nanoelectrodes for AFM-SECM.

    PubMed

    Avdic, A; Lugstein, A; Wu, M; Gollas, B; Pobelov, I; Wandlowski, T; Leonhardt, K; Denuault, G; Bertagnolli, E

    2011-04-01

    We demonstrate a reliable microfabrication process for a combined atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) measurement tool. Integrated cone-shaped sensors with boron doped diamond (BDD) or gold (Au) electrodes were fabricated from commercially available AFM probes. The sensor formation process is based on mature semiconductor processing techniques, including focused ion beam (FIB) machining, and highly selective reactive ion etching (RIE). The fabrication approach preserves the geometry of the original AFM tips resulting in well reproducible nanoscaled sensors. The feasibility and functionality of the fully featured tips are demonstrated by cyclic voltammetry, showing good agreement between the measured and calculated currents of the cone-shaped AFM-SECM electrodes. PMID:21368355

  17. Advances in CO2 cryogenic aerosol technology for photomask post AFM repair

    NASA Astrophysics Data System (ADS)

    Bowers, Charles; Varghese, Ivin; Balooch, Mehdi; Rodriguez, Jaime

    2009-10-01

    As the mask technology moves towards production of 36 nm and 22 nm DRAM half pitch nodes, printing features and sub-resolution assist features (SRAF) shrink below 80 nm. These narrow features become more fragile and place new demands on cleaning processes for a physically non damaging solution. These challenges include compatibility with new materials, oxidation, chemical contamination sensitivity, proportionally decreasing printable defect size, and a requirement for a damage-free clean. CO2 cryogenic aerosol cleaning has, for many years, shown potential to offer a wide process window for meeting some of these new challenges. CO2 cryogenic aerosol cleaning for post AFM repair debris cleaning has been used for many years on masks greater than 90 nm DRAM half pitch nodes. Until recently, CO2 purity and delivery hardware issues resulted in foreign material adder (FMACO2) contamination and SRAF damage below 150 nm critical feature size. Some key desirable properties of CO2 cryogenic aerosol cleaning are the non-oxidizing and non-etching properties when compared to current chemical wet clean processes. In this paper, recent advancements of CO2 cryogenic aerosol cleaning technology are presented, highlighting improvements in the areas of FMACO2 reduction, lowering the critical feature size without damage, and electrostatic discharge (ESD) mitigation. Key aspects of successful CO2 cryogenic aerosol cleaning include the spray nozzle design, CO2 liquid purity, and integrated system design. The design of the nozzle directly controls the size, flux, and velocity of the CO2 snow particles. Methodology and measurements of the solid CO2 particle size and velocity distributions will be presented, and their responses to various control parameters will be discussed. FMACO2 mitigation can be achieved only through use of highly purified CO2 and careful materials selection of the delivery hardware. Recent advances in CO2 purity will be discussed and data shown. The mask cleaning

  18. Characterization of local elastic modulus in confined polymer films via AFM indentation.

    PubMed

    Cheng, Xu; Putz, Karl W; Wood, Charles D; Brinson, L Catherine

    2015-02-01

    The properties of polymers near an interface are altered relative to their bulk value due both to chemical interaction and geometric confinement effects. For the past two decades, the dynamics of polymers in confined geometries (thin polymer film or nanocomposites with high-surface area particles) has been studied in detail, allowing progress to be made toward understanding the origin of the dynamic effects near interfaces. Observations of mechanical property enhancements in polymer nanocomposites have been attributed to similar origins. However, the existing measurement methods of these local mechanical properties have resulted in a variety of conflicting results on the change of mechanical properties of confined polymers. Here, an atomic force microscopy (AFM)-based method is demonstrated that directly measures the mechanical properties of polymers adjacent to a substrate with nanometer resolution. This method allows us to consistently observe the gradient in mechanical properties away from a substrate in various materials systems, and paves the way for a unified understanding of thermodynamic and mechanical response of polymers. This gradient is both longer (up to 170 nm) and of higher magnitude (50% increase) than expected from prior results. Through the use of this technique, we will be better able to understand how to design polymer nanocomposites and polymeric structures at the smallest length scale, which affects the fields of structures, electronics, and healthcare.

  19. Characterization of local elastic modulus in confined polymer films via AFM indentation.

    PubMed

    Cheng, Xu; Putz, Karl W; Wood, Charles D; Brinson, L Catherine

    2015-02-01

    The properties of polymers near an interface are altered relative to their bulk value due both to chemical interaction and geometric confinement effects. For the past two decades, the dynamics of polymers in confined geometries (thin polymer film or nanocomposites with high-surface area particles) has been studied in detail, allowing progress to be made toward understanding the origin of the dynamic effects near interfaces. Observations of mechanical property enhancements in polymer nanocomposites have been attributed to similar origins. However, the existing measurement methods of these local mechanical properties have resulted in a variety of conflicting results on the change of mechanical properties of confined polymers. Here, an atomic force microscopy (AFM)-based method is demonstrated that directly measures the mechanical properties of polymers adjacent to a substrate with nanometer resolution. This method allows us to consistently observe the gradient in mechanical properties away from a substrate in various materials systems, and paves the way for a unified understanding of thermodynamic and mechanical response of polymers. This gradient is both longer (up to 170 nm) and of higher magnitude (50% increase) than expected from prior results. Through the use of this technique, we will be better able to understand how to design polymer nanocomposites and polymeric structures at the smallest length scale, which affects the fields of structures, electronics, and healthcare. PMID:25537230

  20. 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.

  1. Colloid-probe AFM studies of the interaction forces of proteins adsorbed on colloidal crystals.

    PubMed

    Singh, Gurvinder; Bremmell, Kristen E; Griesser, Hans J; Kingshott, Peter

    2015-04-28

    In recent years, colloid-probe AFM has been used to measure the direct interaction forces between colloidal particles of different size or surface functionality in aqueous media, as one can study different forces in symmerical systems (i.e., sphere-sphere geometry). The present study investigates the interaction between protein coatings on colloid probes and hydrophilic surfaces decorated with hexagonally close packed single particle layers that are either uncoated or coated with proteins. Controlled solvent evaporation from aqueous suspensions of colloidal particles (coated with or without lysozyme and albumin) produces single layers of close-packed colloidal crystals over large areas on a solid support. The measurements have been carried out in an aqueous medium at different salt concentrations and pH values. The results show changes in the interaction forces as the surface charge of the unmodified or modified particles, and ionic strength or pH of the solution is altered. At high ionic strength or pH, electrostatic interactions are screened, and a strong repulsive force at short separation below 5 nm dominates, suggesting structural changes in the absorbed protein layer on the particles. We also study the force of adhesion, which decreases with an increment in the salt concentration, and the interaction between two different proteins indicating a repulsive interaction on approach and adhesion on retraction. PMID:25758979

  2. 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).

  3. 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.

  4. 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. PMID:23465925

  5. A Multifunctional Frontloading Approach for Repeated Recycling of a Pressure-Controlled AFM Micropipette

    PubMed Central

    Roder, Phillip; Hille, Carsten

    2015-01-01

    Fluid force microscopy combines the positional accuracy and force sensitivity of an atomic force microscope (AFM) with nanofluidics via a microchanneled cantilever. However, adequate loading and cleaning procedures for such AFM micropipettes are required for various application situations. Here, a new frontloading procedure is described for an AFM micropipette functioning as a force- and pressure-controlled microscale liquid dispenser. This frontloading procedure seems especially attractive when using target substances featuring high costs or low available amounts. Here, the AFM micropipette could be filled from the tip side with liquid from a previously applied droplet with a volume of only a few μL using a short low-pressure pulse. The liquid-loaded AFM micropipettes could be then applied for experiments in air or liquid environments. AFM micropipette frontloading was evaluated with the well-known organic fluorescent dye rhodamine 6G and the AlexaFluor647-labeled antibody goat anti-rat IgG as an example of a larger biological compound. After micropipette usage, specific cleaning procedures were tested. Furthermore, a storage method is described, at which the AFM micropipettes could be stored for a few hours up to several days without drying out or clogging of the microchannel. In summary, the rapid, versatile and cost-efficient frontloading and cleaning procedure for the repeated usage of a single AFM micropipette is beneficial for various application situations from specific surface modifications through to local manipulation of living cells, and provides a simplified and faster handling for already known experiments with fluid force microscopy. PMID:26636981

  6. 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.

  7. A Multifunctional Frontloading Approach for Repeated Recycling of a Pressure-Controlled AFM Micropipette.

    PubMed

    Roder, Phillip; Hille, Carsten

    2015-01-01

    Fluid force microscopy combines the positional accuracy and force sensitivity of an atomic force microscope (AFM) with nanofluidics via a microchanneled cantilever. However, adequate loading and cleaning procedures for such AFM micropipettes are required for various application situations. Here, a new frontloading procedure is described for an AFM micropipette functioning as a force- and pressure-controlled microscale liquid dispenser. This frontloading procedure seems especially attractive when using target substances featuring high costs or low available amounts. Here, the AFM micropipette could be filled from the tip side with liquid from a previously applied droplet with a volume of only a few μL using a short low-pressure pulse. The liquid-loaded AFM micropipettes could be then applied for experiments in air or liquid environments. AFM micropipette frontloading was evaluated with the well-known organic fluorescent dye rhodamine 6G and the AlexaFluor647-labeled antibody goat anti-rat IgG as an example of a larger biological compound. After micropipette usage, specific cleaning procedures were tested. Furthermore, a storage method is described, at which the AFM micropipettes could be stored for a few hours up to several days without drying out or clogging of the microchannel. In summary, the rapid, versatile and cost-efficient frontloading and cleaning procedure for the repeated usage of a single AFM micropipette is beneficial for various application situations from specific surface modifications through to local manipulation of living cells, and provides a simplified and faster handling for already known experiments with fluid force microscopy. PMID:26636981

  8. 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.

  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. 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. PMID:26618709

  11. 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

  12. Physical properties of poly(lactic-co-glycolic) and poly(ethylene glycol) nanoparticles for drug delivery using atomic force microscopy (AFM) and electrostatic nanolithography

    NASA Astrophysics Data System (ADS)

    Lyuksyutov, Sergei; Fedin, Igor; Nedashkivska, Victoria; Lyuksyutova, Caterina; Geldenhuys, Werner; Sutariya, Vijay

    2010-03-01

    Nanoparticles (NP) of biodegradable polymers poly(lactic-co-glycolic)(PLGA) and poly(ethylene glycol) (PEG) are potential drug delivery components for biomedical applications. The NP based on PLGA or PEG can be directed to accumulate in cancer tumor cells with the use of anti-bodies which are conjugated to the NP. The NP's size distribution is the critical property for biochemical affinity and therefore delivery to the specific target organs. We used an atomic force microscopy (AFM) to characterize the NP size and AFM electrostatic nanolithography (AFMEN) to study the behavior of PEG-PLGA NP under the extreme electric fields exceeding 10^9 V m-1. AFMEN allows the displacement of molecules along the lines of the electric field due to electrostatic polarization. This study has an important practical application for the optimum design of NP with the correct characteristics for drug delivery.

  13. A rapid and automated relocation method of an AFM probe for high-resolution imaging

    NASA Astrophysics Data System (ADS)

    Zhou, Peilin; Yu, Haibo; Shi, Jialin; Jiao, Niandong; Wang, Zhidong; Wang, Yuechao; Liu, Lianqing

    2016-09-01

    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.

  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. PMID:27559679

  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. Strength by atomic force microscopy (AFM): Molecular dynamics of water layer squeezing on magnesium oxide

    NASA Astrophysics Data System (ADS)

    Kendall, K.; Dhir, Aman; Yong, Chin W.

    2010-11-01

    Localised strength testing of materials is often carried out in an atomic force microscope (AFM), as foreseen by Kelly in his book Strong Solids (Clarendon Press, Oxford, 1966). During AFM indentation experiments, contamination can strongly influence the observed strength and theoretical interpretation of the results is a major problem. Here, we use molecular dynamics computer modelling to describe the contact of NaCl and MgO crystal probes onto surfaces, comparable to an AFM experiment. Clean NaCl gave elastic, brittle behaviour in contact simulations at 300 K, whereas MgO was more plastic, leading to increased toughness. This paper also considers the strength of an oxide substrate contaminated by water molecules and tested by indentation with a pyramidal probe of oxide crystal. Recent theory on the effect of liquid contaminant layers on surface strength has been mainly focussed on Lennard Jones (LJ) molecules with some studies on alcohols and water, described by molecular dynamics, which allows the molecules to be squeezed out as the crystal lattice is deformed. In this work, we have focused on water by studying the forces between a magnesium oxide (MgO) atomic force microscope (AFM) probe and an MgO slab. Force versus separation has been plotted as the AFM probe was moved towards and away from the substrate. Simulation results showed that the water layers could be removed in steps, giving up to four force peaks. The last monolayer of water could not be squeezed out, even at pressures where MgO deformed plastically. Interestingly, with water present, strength was reduced, but more in tensile than compressive measurements. In conclusion, water contaminating the oxide surface in AFM strength testing is structured. Water layer squeezing removal can be predicted by molecular modelling, which may be verified by AFM experiments to show that water can influence the strength of perfect crystals at the nanometre scale.

  17. A software tool for STED-AFM correlative super-resolution microscopy

    NASA Astrophysics Data System (ADS)

    Koho, Sami; Deguchi, Takahiro; Löhmus, Madis; Näreoja, Tuomas; Hänninen, Pekka E.

    2015-03-01

    Multi-modal correlative microscopy allows combining the strengths of several imaging techniques to provide unique contrast. However it is not always straightforward to setup instruments for such customized experiments, as most microscope manufacturers use their own proprietary software, with limited or no capability to interface with other instruments - this makes correlation of the multi-modal data extremely challenging. We introduce a new software tool for simultaneous use of a STimulated Emission Depletion (STED) microscope with an Atomic Force Microscope (AFM). In our experiments, a Leica TCS STED commercial super-resolution microscope, together with an Agilent 5500ilm AFM microscope was used. With our software, it is possible to synchronize the data acquisition between the STED and AFM instruments, as well as to perform automatic registration of the AFM images with the super-resolution STED images. The software was realized in LabVIEW; the registration part was also implemented as an ImageJ script. The synchronization was realized by controlling simple trigger signals, also available in the commercial STED microscope, with a low-cost National Instruments USB-6501 digital I/O card. The registration was based on detecting the positions of the AFM tip inside the STED fieldof-view, which were then used as registration landmarks. The registration should work on any STED and tip-scanning AFM microscope combination, at nanometer-scale precision. Our STED-AFM correlation method has been tested with a variety of nanoparticle and fixed cell samples. The software will be released under BSD open-source license.

  18. AFM probes fabricated with masked maskless combined anisotropic etching and p+ surface doping

    NASA Astrophysics Data System (ADS)

    Han, Jianqiang; Li, Xinxin; Bao, Haifei; Zuo, Guomin; Wang, Yuelin; Feng, Fei; Yu, Zhenyin; Ge, Xiaohong

    2006-02-01

    The paper presents a newly developed high-yield micro-fabrication technology for single-crystalline silicon atomic force microscope (AFM) probes. Both the tips and the cantilevers are simultaneously formed by a masked-maskless combined anisotropic etching process. Compared to a conventional tip-to-cantilever sequential fabrication scheme, this tip-and-cantilever simultaneous formation can effectively increase fabrication yield by avoiding the tips damaged during the following processed photolithographic steps for defining the cantilevers. By heavy boron doping at the surface, the conductive AFM probe provides an electrical path to the electric ground of the AFM that helps to eliminate the electrostatic accumulation of charges and, therefore, eliminate undesirable electrostatic forces between the probes and the samples. A fabrication yield as high as 90% has been obtained for the AFM probes for 4 inch wafers. The tips after oxidation-sharpening treatment generally have a radius of 10-30 nm. The cantilever spring constant can be well controlled in the range of 0.025-40 N m-1. High-quality sample scanning results with the formed AFM probes are obtained with a slightly better resolution than that from commercial probes without surface conductive treatment.

  19. AFM Bio-Mechanical Investigation of the Taxol Treatment of Breast Cancer Cells

    NASA Astrophysics Data System (ADS)

    Smith, Dylan; Patel, Dipika; Monjaraz, Fernando; Park, Soyeun

    2009-10-01

    Cancerous cells are known to be softer and easier to deform than normal cells. Changes in mechanical properties originate from the alteration of the actin cytoskeleton. The mechanism of cancer treatment using Taxol is related to the stabilization of microtubules. It has been shown that Taxol binds to polymerized tublin, stabilizes it against disassembly, and consequently inhibits cell division. An accurate quantitative study still lacks to relate the microtubule stabilizing effect with the cellular mechanical properties. We utilized our AFM to study changes in elastic properties of treated breast cancer cells. The AFM has several advantages for precise force measurements on a localized region with nanometer lateral dimension. In previous AFM studies, measurable contributions from the underlying hard substrate have been an obstacle to accurately determine the properties on thin samples. We modified our AFM tip to obtain the exact deformation profile as well as reducing the high stresses produced. We have probed depth profiles of mechanical properties of the taxol-treated and untreated cells by varying the indentation depth of the AFM-nanoindenting experiments.

  20. 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.

  1. 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.

  2. 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.

  3. AFM study of the plastic deformation of polysynthetically-twinned (PST) TiAl crystals in soft orientation.

    PubMed

    Chen, Yali; Pope, David P

    2006-05-01

    PST TiAl samples with a nominal composition of Ti52Al48 were deformed at room temperature with compression axis inclined to the lamellar interfaces by 45 degrees and one of the side surface normal directions set to be (112). The deformation structures on the free surfaces of the deformed samples were investigated using Atomic Force Microscope (AFM). It was found that in-plane shear (shear in planes parallel to lamellar interfaces) is the dominant deformation mode in all gamma domains and most of the deformation traces on the free surfaces are parallel to lamellar interfaces. Out-of-plane shear (shear in planes inclined to lamellar interfaces) also occurs but contributes much less to the macroscopic strain. This selective activation of deformation modes leads to a highly anisotropic deformation behavior in PST crystals with this orientation. PMID:16646014

  4. The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding.

    PubMed

    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.

  5. 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.

  6. GGAP2/PIKE-A directly activates both the Akt and NF-κB pathways and promotes prostate cancer progression

    PubMed Central

    Cai, Yi; Wang, Jianghua; Li, Rile; Ayala, Gustavo; Ittmann, Michael; Liu, Mingyao

    2009-01-01

    GGAP2/PIKE-A is a GTP-binding protein which can enhance Akt activity. Increased activation of the AKT and NF-κB pathways have been identified as critical steps in cancer initiation and progression in a variety of human cancers. We have found significantly increased expression GGAP2 in the majority of human prostate cancers and GGAP2 expression increases Akt activation in prostate cancer cells. Thus increased GGAP2 expression is a common mechanism for enhancing the activity of the Akt pathway in prostate cancers. In addition, we have found that activated Akt can bind and phosphorylate GGAP2 at serine 629, which enhances GTP binding by GGAP2. Phosphorylated GGAP2 can bind the p50 subunit of NF-κB and enhances NF-κB transcriptional activity. When expressed in prostate cancer cells, GGAP2 enhances proliferation, foci formation and tumor progression in vivo. Thus increased GGAP2 expression, which is present in three quarters of human prostate cancers, can activate two critical pathways that have been linked to prostate cancer initiation and progression. PMID:19176382

  7. WT1 is involved in the Akt-JNK pathway dependent autophagy through directly regulating Gas1 expression in human osteosarcoma cells.

    PubMed

    Mo, Hao; He, Juliang; Yuan, Zhenchao; Mo, Ligen; Wu, Zhenjie; Lin, Xiang; Liu, Bin; Guan, Jian

    2016-09-01

    Macroautophagy (herein termed autophagy) works as a protective mechanism in tumorigenesis and development under metabolic stress condition. Multitudes of genes have been found involved in this process during past decades. In the present study, we report that Wilm's tumor suppressor1 (WT1) is involved in autophagy in osteosarcoma (OS) cells. WT1, a transcription factor with multitude of target genes, expresses in a majority of cancer types. Though wide-ranging effect of WT1 is now well documented, the function of WT1 in tumors remains poorly defined. In this chapter, it is found that high expression of WT1 positively correlates with active autophagy in human osteosarcoma cells. And further study on cell signaling pathway illustrates that Akt/JNK pathway acts as a positive regulator of autophagy induced by WT1. Here, we present evidence that WT1 modulates Akt/JNK signaling pathway mediated autophagy by controlling the expression of growth arrest-specific 1 (Gas1). We show that WT1 is required for Gas1 transcription in osteosarcoma cells. And Gas1 is upregulated followed WT1 overexpression in a time-dependent manner. Loss of Gas1 results in a reduction of WT1-induced autophagy. PMID:27453337

  8. Advances in post AFM repair cleaning of photomask with CO2 cryogenic aerosol technology

    NASA Astrophysics Data System (ADS)

    Bowers, Charles; Varghese, Ivin; Balooch, Mehdi; Brandt, Werner

    2009-04-01

    As the mask technology matures, critical printing features and sub-resolution assist features (SRAF) shrink below 100 nm, forcing critical cleaning processes to face significant challenges. These challenges include use of new materials, oxidation, chemical contamination sensitivity, proportionally decreasing printable defect size, and a requirement for a damage-free clean. CO2 cryogenic aerosol cleaning has the potential to offer a wide process window for meeting these new challenges, if residue adder issues and damage can be eliminated. Some key differentiations of CO2 cryogenic aerosol cleaning are the non-oxidizing and non-etching properties compared to conventional chemical wet clean processes with or without megasonics. In prior work, the feasibility of CO2 cryogenic aerosol in post AFM repair photomask cleaning was demonstrated. In this paper, recent advancements of CO2 cryogenic aerosol cleaning technology are presented, focusing on the traditional problem areas of particle adders, electrostatic discharge (ESD), and mask damage mitigation. Key aspects of successful CO2 cryogenic aerosol cleaning include the spray nozzle design, CO2 liquid purity, and system design. The design of the nozzle directly controls the size, density, and velocity of the CO2 snow particles. Methodology and measurements of the solid CO2 particle size and velocity distributions will be presented, and their responses to various control parameters will be discussed. Adder control can be achieved only through use of highly purified CO2 and careful materials selection. Recent advances in CO2 purity will be discussed and data shown. The mask cleaning efficiency by CO2 cryogenic aerosol and damage control is essentially an optimization of the momentum of the solid CO2 particles and elimination of adders. The previous damage threshold of 150 nm SRAF structures has been reduced to 70nm and data will be shown indicating 60 nm is possible in the near future. Data on CO2 tribocharge mitigation

  9. Tendon glycosaminoglycan proteoglycan sidechains promote collagen fibril sliding-AFM observations at the nanoscale.

    PubMed

    Rigozzi, S; Müller, R; Stemmer, A; Snedeker, J G

    2013-02-22

    The extracellular matrix of tendon is mainly composed of discontinuous Type-I collagen fibrils and small leucine rich proteoglycans (PG). Macroscopic tendon behaviors like stiffness and strength are determined by the ultrastructural arrangement of these components. When a tendon is submitted to load, the collagen fibrils both elongate and slide relative to their neighboring fibrils. The role of PG glycosaminoglycan (GAG) sidechains in mediating inter-fibril load sharing remains controversial, with competing structure-function theories suggesting that PGs may mechanically couple neighboring collagen fibrils (cross-linking them to facilitate fibril stretch) or alternatively isolating them (promoting fibril gliding). In this study, we sought to clarify the functional role of GAGs in tensile tendon mechanics by directly investigating the mechanical response of individual collagen fibrils within their collagen network in both native and GAG depleted tendons. A control group of Achilles tendons from adult mice was compared with tendons in which GAGs were enzymatically depleted using chondroitinase ABC. Tendons were loaded to specific target strains, chemically fixed under constant load, and later sectioned for morphological analysis by an atomic force microscope (AFM). Increases in periodic banding of the collagen fibrils (D-period) or decreases in fibril diameter was considered to be representative of collagen fibril elongation and the mechanical contribution of GAGs at the ultrascale was quantified on this basis. At high levels of applied tendon strain (10%), GAG depleted tendons showed increased collagen stretch (less fibril sliding). We conclude that the hydrophilic GAGs seem thus not to act as mechanical crosslinks but rather act to promote collagen fibril sliding under tension.

  10. Interaction Mechanism of Oil-in-Water Emulsions with Asphaltenes Determined Using Droplet Probe AFM.

    PubMed

    Shi, Chen; Zhang, Ling; Xie, Lei; Lu, Xi; Liu, Qingxia; Mantilla, Cesar A; van den Berg, Frans G A; Zeng, Hongbo

    2016-03-15

    Emulsions with interface-active components at the oil/water interface have long been of fundamental and practical interest in many fields. In this work, the interaction forces between two oil droplets in water in the absence/presence of asphaltenes were directly measured using droplet probe atomic force microscopy (AFM) and analyzed using a theoretical model based on Reynolds lubrication theory and the augmented Young-Laplace equation by including the effects of disjoining pressure. It was revealed that the interaction forces measured between two pristine oil droplets (i.e., toluene) could be well described by the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, while an additional steric interaction should be included in the presence of asphaltenes in the oil. The surface interaction and the stability of oil droplets in aqueous solution were demonstrated to be significantly influenced by the asphaltenes concentration in oil, salt concentration, pH, and presence of divalent ions (Ca(2+)) in water. Adsorbed asphaltenes at the oil/water interface led to more negative surface potential of the oil/water interface and also induced steric repulsion between oil droplets, inhibiting the drop coalescence and stabilizing the oil-in-water emulsion. Lower pH of aqueous solution could lead to less negative surface potential and weaken the repulsion between oil droplets. Addition of divalent ions (Ca(2+)) was found to disrupt the protecting effects of adsorbed asphaltenes at oil/water interface and induce coalescence of oil droplets. Our results provide a useful methodology for quantifying the interaction forces and investigating the properties of asphaltenes at the oil/water interfaces and provide insights into the stabilization mechanism of oil-in-water emulsions due to asphaltenes in oil production and water treatment.

  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. 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.

  13. 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.

  14. Mechanism of immonoglobulin G adsorption on mica-AFM and electrokinetic studies.

    PubMed

    Dąbkowska, Maria; Adamczyk, Zbigniew

    2014-06-01

    Adsorption of immunoglobulin G (IgG) from aqueous NaCl solutions of the concentration 10(-3)-0.15M on mica was studied. Initially, the kinetics was evaluated at pH 3.5 by direct AFM imaging. A monotonic increase in the maximum coverage of IgG with NaCl concentration was observed. These results were interpreted in terms of the theoretical model postulating an irreversible adsorption of the protein governed by the random sequential adsorption (RSA) model. Additionally, IgG adsorption and desorption was studied under in situ conditions, with streaming potential measurements. These measurements revealed that the maximum coverage of irreversibly adsorbed IgG varies from 0.37mgm(-2) for 10(-3)M, NaCl to 1.2mgm(-2) for 0.15M, NaCl. The significant role of ionic strength was attributed to the lateral electrostatic repulsion among adsorbed IgG molecules, positively charged at this pH value. These experimental results confirmed that monolayers of irreversibly bound IgG can be produced by adjusting ionic strength of the protein solution. In further experiments the stability and acid base properties of such monolayers were studied using the streaming potential method. It revealed that the monolayers were stable against pH cycling for the range from 3.5 to 9.5. The isoelectric point of mica supported IgG monolayers was 5.9, similar to derived from the micro-electrophoretic measurements in the bulk (5.8). Beside significance for basic sciences, the results indicate that thorough characteristics of IgG can be acquired via streaming potential measurements using microgram quantities of the protein.

  15. 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. PMID:25679491

  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. Quantitative atomic resolution force imaging on epitaxial graphene with reactive and nonreactive AFM probes.

    PubMed

    Boneschanscher, Mark P; van der Lit, Joost; Sun, Zhixiang; Swart, Ingmar; Liljeroth, Peter; Vanmaekelbergh, Daniël

    2012-11-27

    Atomic force microscopy (AFM) images of graphene and graphite show contrast with atomic periodicity. However, the contrast patterns vary depending on the atomic termination of the AFM tip apex and the tip-sample distance, hampering the identification of the atomic positions. Here, we report quantitative AFM imaging of epitaxial graphene using inert (carbon-monoxide-terminated) and reactive (iridium-terminated) tips. The atomic image contrast is markedly different with these tip terminations. With a reactive tip, we observe an inversion from attractive to repulsive atomic contrast with decreasing tip-sample distance, while a nonreactive tip only yields repulsive atomic contrast. We are able to identify the atoms with both tips at any tip-sample distance. This is a prerequisite for future structural and chemical analysis of adatoms, defects, and the edges of graphene nanostructures, crucial for understanding nanoscale graphene devices.

  18. Custom AFM for X-ray beamlines: in situ biological investigations under physiological conditions.

    PubMed

    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

    2015-11-01

    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

  19. 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

  20. 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

  1. Review and perspectives of AFM application on the study of deformable drop/bubble interactions.

    PubMed

    Wang, Wei; Li, Kai; Ma, Mengyu; Jin, Hang; Angeli, Panagiota; Gong, Jing

    2015-11-01

    The applications of Atomic Force Microscopy (AFM) on the study of dynamic interactions and film drainage between deformable bodies dispersed in aqueous solutions are reviewed in this article. Novel experimental designs and recent advances in experimental methodologies are presented, which show the advantage of using AFM as a tool for probing colloidal interactions. The effects of both DLVO and non-DLVO forces on the colloid stabilization mechanism are discussed. Good agreement is found between the force - drop/bubble deformation behaviour revealed by AFM measurements and the theoretical modeling of film drainage process, giving a convincing explanation of the occurrence of certain phenomenon. However, the behaviour and shape of deformable drops as they approach or retract is still not well resolved. In addition, when surfactants are present further research is needed on the absorption of surfactant molecules into the interfaces, their mobility and the effects on interfacial film properties.

  2. AFM-assisted fabrication of thiol SAM pattern with alternating quantified surface potential

    PubMed Central

    2011-01-01

    Thiol self-assembled monolayers (SAMs) are widely used in many nano- and bio-technology applications. We report a new approach to create and characterize a thiol SAMs micropattern with alternating charges on a flat gold-coated substrate using atomic force microscopy (AFM) and Kelvin probe force microscopy (KPFM). We produced SAMs-patterns made of alternating positively charged, negatively charged, and hydrophobic-terminated thiols by an automated AFM-assisted manipulation, or nanografting. We show that these thiol patterns possess only small topographical differences as revealed by AFM, and distinguished differences in surface potential (20-50 mV), revealed by KPFM. The pattern can be helpful in the development of biosensor technologies, specifically for selective binding of biomolecules based on charge and hydrophobicity, and serve as a model for creating surfaces with quantified alternating surface potential distribution. PMID:21711703

  3. Direct improvement of quality of life using a tailored quality of life diagnosis and therapy pathway: randomised trial in 200 women with breast cancer

    PubMed Central

    Klinkhammer-Schalke, M; Koller, M; Steinger, B; Ehret, C; Ernst, B; Wyatt, J C; Hofstädter, F; Lorenz, W

    2012-01-01

    Background: Despite thousands of papers, the value of quality of life (QoL) in curing disease remains uncertain. Until now, we lacked tools for the diagnosis and specific treatment of diseased QoL. We approached this problem stepwise by theory building, modelling, an exploratory trial and now a definitive randomised controlled trial (RCT) in breast cancer, whose results we report here. Methods: In all, 200 representative Bavarian primary breast cancer patients were recruited by five hospitals and treated by 146 care professionals. Patients were randomised to either (1) a novel care pathway including diagnosis of ‘diseased' QoL (any QoL measure below 50 points) using a QoL profile and expert report sent to the patient's coordinating practitioner, who arranged QoL therapy consisting of up to five standardised treatments for specific QoL defects or (2) standard postoperative care adhering to the German national guideline for breast cancer. The primary end point was the proportion of patients in each group with diseased QoL 6 months after surgery. Patients were blinded to their allocated group. Results: At 0 and 3 months after surgery, diseased QoL was diagnosed in 70% of patients. The QoL pathway reduced rates of diseased QoL to 56% at 6 months, especially in emotion and coping, compared with 71% in controls (P=0.048). Relative risk reduction was 21% (95% confidence interval (CI): 0–37), absolute risk reduction 15% (95% CI: 0.3–29), number needed to treat (NNT)=7 (95% CI: 3–37). When QoL therapy finished after successful treatment, diseased QoL often returned again, indicating good responsiveness of the QoL pathway. Conclusion: A three-component outcome system including clinician-derived objective, patient-reported subjective end points and qualitative analysis of clinical relevance was developed in the last 10 years for cancer as a complex intervention. A separate QoL pathway was implemented for the diagnosis and treatment of diseased QoL and its

  4. FRAME (Force Review Automation Environment): MATLAB-based AFM data processor.

    PubMed

    Partola, Kostyantyn R; Lykotrafitis, George

    2016-05-01

    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.

  5. FRAME (Force Review Automation Environment): MATLAB-based AFM data processor.

    PubMed

    Partola, Kostyantyn R; Lykotrafitis, George

    2016-05-01

    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. PMID:26972765

  6. Coexistence of orbital and CE-AFM orders in colossal magnetoresistance manganites: A symmetry perspective

    NASA Astrophysics Data System (ADS)

    Ribeiro, J. L.

    2016-07-01

    The complex interplay between order parameters of different nature that dominates the physics of colossal magnetoresistance manganites is analysed from a symmetry based perspective. Phenomenological energies are given for the different competing phases. It is shown that the general trends observed in different systems, such as the mutual exclusion of orbital order and A-AFM order and the related stabilization of the CE-AFM order, stem to large extend from the symmetry of the parameters involved. The possible stabilization of complex phases where charge and orbital order coexist with magnetic and ferroelectric states is also anticipated.

  7. Probing the PEDOT:PSS/cell interface with conductive colloidal probe AFM-SECM

    NASA Astrophysics Data System (ADS)

    Knittel, P.; Zhang, H.; Kranz, C.; Wallace, G. G.; Higgins, M. J.

    2016-02-01

    Conductive colloidal probe Atomic Force-Scanning Electrochemical Microscopy (AFM-SECM) is a new approach, which employs electrically insulated AFM probes except for a gold-coated colloid located at the end of the cantilever. Hence, force measurements can be performed while biasing the conductive colloid under physiological conditions. Moreover, such colloids can be modified by electrochemical polymerization resulting, e.g. in conductive polymer-coated spheres, which in addition may be loaded with specific dopants. In contrast to other AFM-based single cell force spectroscopy measurements, these probes allow adhesion measurements at the cell-biomaterial interface on multiple cells in a rapid manner while the properties of the polymer can be changed by applying a bias. In addition, spatially resolved electrochemical information e.g., oxygen reduction can be obtained simultaneously. Conductive colloid AFM-SECM probes modified with poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) are used for single cell force measurements in mouse fibroblasts and single cell interactions are investigated as a function of the applied potential.Conductive colloidal probe Atomic Force-Scanning Electrochemical Microscopy (AFM-SECM) is a new approach, which employs electrically insulated AFM probes except for a gold-coated colloid located at the end of the cantilever. Hence, force measurements can be performed while biasing the conductive colloid under physiological conditions. Moreover, such colloids can be modified by electrochemical polymerization resulting, e.g. in conductive polymer-coated spheres, which in addition may be loaded with specific dopants. In contrast to other AFM-based single cell force spectroscopy measurements, these probes allow adhesion measurements at the cell-biomaterial interface on multiple cells in a rapid manner while the properties of the polymer can be changed by applying a bias. In addition, spatially resolved electrochemical

  8. Resonance Frequency Analysis for Surface-Coupled AFM Cantilever in Liquids

    SciTech Connect

    Mirman, B; Kalinin, Sergei V

    2008-01-01

    Shifts in the resonance frequencies of surface-coupled atomic force microscope (AFM) probes are used as the basis for the detection mechanisms in a number of scanning probe microscopy techniques including atomic force acoustic microscopy (AFAM), force modulation microscopy, and resonance enhanced piezoresponse force microscopy (PFM). Here, we analyze resonance characteristics for AFM cantilever coupled to surface in liquid environment, and derive approximate expressions for resonant frequencies as a function of vertical and lateral spring constant of the tip-surface junction. This analysis provides a simplified framework for the interpretation of AFAM and PFM data in ambient, liquid, and vacuum environments.

  9. 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

  10. AFM and XPA data on structural features and properties of films and powders based on naphthalocyanines

    NASA Astrophysics Data System (ADS)

    Ramonova, A. G.; Nakusov, A. T.; Sozanov, V. G.; Bliev, A. P.; Magkoev, T. T.

    2015-06-01

    The template synthesis is used to produce powders and films based on naphthalocyanines and the corresponding metal complexes (Pc, CuPc, and NiPc). The atomic-force microscopy (AFM) and X-ray phase analysis (XPA) are employed in the study of structure and phase of fine powders and nanostructured films. The AFM data are used to determine the orientation and density of primary particles packed in the film. The XPA method is used to study the chemical composition and crystal structure of the synthesized samples. The regularities related to the structural features that affect the electrophysical properties of the films under study are revealed.

  11. Study on the AFM Force Spectroscopy method for elastic modulus measurement of living cells

    NASA Astrophysics Data System (ADS)

    Demichelis, A.; Pavarelli, S.; Mortati, L.; Sassi, G.; Sassi, M.

    2013-09-01

    The cell elasticity gives information about its pathological state and metastatic potential. The aim of this paper is to study the AFM Force Spectroscopy technique with the future goal of realizing a reference method for accurate elastic modulus measurement in the elasticity range of living cells. This biological range has not been yet explored with a metrological approach. Practical hints are given for the realization of a Sylgard elasticity scale. Systematic effects given by the sample curing thickness and nanoindenter geometry have been found with regards of the measured elastic modulus. AFM measurement reproducibility better than 20% is obtained in the entire investigated elastic modulus scale of 101 - 104 kPa.

  12. 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.

  13. 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.

  14. 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

  15. The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling

    PubMed Central

    Cruse, Glenn; Beaven, Michael A.; Music, Stephen C.; Bradding, Peter; Gilfillan, Alasdair M.; Metcalfe, Dean D.

    2015-01-01

    MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1–enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases. PMID:25717186

  16. Diamond-modified AFM probes: from diamond nanowires to atomic force microscopy-integrated boron-doped diamond electrodes.

    PubMed

    Smirnov, Waldemar; Kriele, Armin; Hoffmann, René; Sillero, Eugenio; Hees, Jakob; Williams, Oliver A; Yang, Nianjun; Kranz, Christine; Nebel, Christoph E

    2011-06-15

    In atomic force microscopy (AFM), sharp and wear-resistant tips are a critical issue. Regarding scanning electrochemical microscopy (SECM), electrodes are required to be mechanically and chemically stable. Diamond is the perfect candidate for both AFM probes as well as for electrode materials if doped, due to diamond's unrivaled mechanical, chemical, and electrochemical properties. In this study, standard AFM tips were overgrown with typically 300 nm thick nanocrystalline diamond (NCD) layers and modified to obtain ultra sharp diamond nanowire-based AFM probes and probes that were used for combined AFM-SECM measurements based on integrated boron-doped conductive diamond electrodes. Analysis of the resonance properties of the diamond overgrown AFM cantilevers showed increasing resonance frequencies with increasing diamond coating thicknesses (i.e., from 160 to 260 kHz). The measured data were compared to performed simulations and show excellent correlation. A strong enhancement of the quality factor upon overgrowth was also observed (120 to 710). AFM tips with integrated diamond nanowires are shown to have apex radii as small as 5 nm and where fabricated by selectively etching diamond in a plasma etching process using self-organized metal nanomasks. These scanning tips showed superior imaging performance as compared to standard Si-tips or commercially available diamond-coated tips. The high imaging resolution and low tip wear are demonstrated using tapping and contact mode AFM measurements by imaging ultra hard substrates and DNA. Furthermore, AFM probes were coated with conductive boron-doped and insulating diamond layers to achieve bifunctional AFM-SECM probes. For this, focused ion beam (FIB) technology was used to expose the boron-doped diamond as a recessed electrode near the apex of the scanning tip. Such a modified probe was used to perform proof-of-concept AFM-SECM measurements. The results show that high-quality diamond probes can be fabricated, which are

  17. Toward an uncertainty budget for measuring nanoparticles by AFM

    NASA Astrophysics Data System (ADS)

    Delvallée, A.; Feltin, N.; Ducourtieux, S.; Trabelsi, M.; Hochepied, J. F.

    2016-02-01

    This article reports on the evaluation of an uncertainty budget associated with the measurement of the mean diameter of a nanoparticle (NP) population by Atomic Force Microscopy. The measurement principle consists in measuring the height of a spherical-like NP population to determine the mean diameter and the size distribution. This method assumes that the NPs are well-dispersed on the substrate and isolated enough to avoid measurement errors due to agglomeration phenomenon. Since the measurement is directly impacted by the substrate roughness, the NPs have been deposited on a mica sheet presenting a very low roughness. A complete metrological characterization of the instrument has been carried out and the main error sources have been evaluated. The measuring method has been tested on a population of SiO2 NPs. Homemade software has been used to build the height distribution histogram taking into account only isolated NP. Finally, the uncertainty budget including main components has been established for the mean diameter measurement of this NP population. The most important components of this uncertainty budget are the calibration process along Z-axis, the scanning speed influence and then the vertical noise level.

  18. 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. PMID:26517548

  19. 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.

  20. Tapping and contact mode imaging of native chromosomes and extraction of genomic DNA using AFM tips

    NASA Astrophysics Data System (ADS)

    Sun, Yingchun; Arakawa, Hideo; Osada, Toshiya; Ikai, Atsushi

    2002-03-01

    It is very important both in medicine and biology to clarify the chromosomal structure to understand its functions. In a standard cytogenetic procedure, chromosomes are often fixed in a mixture of acetic acid and methanol. This process most likely changes the mechanical property of chromosomes. We adopted a method to prepare native and unfixed chromosomes from mouse 3T3 cells and used tapping and contact mode atomic force microscopy (AFM) to image and manipulate them. Modified AFM tips were used to image chromosomes in contact mode in air, and then the chromosome samples were immobilized on a substrate and placed in a buffer solution to pull out DNA-histone complexes from them after they were optimally treated with trypsin. From the AFM images, we could see several bands and granular structures on chromosomes. We obtained force curves indicating long fiber extensions from native chromosomes both with low (in high concentration of NaCl) and high forces (physiological conditions). The result suggested that the degree of chromosome condensation decreased in high concentration of salt. It agrees with the known fact of histone H1 dissociation in a high concentration of salt. We intend to pull out DNA-histone complexes from chromosomes for later molecular operations on them using an AFM.

  1. Nanomechanical probing of soft matter through hydrophobic AFM tips fabricated by two-photon polymerization

    NASA Astrophysics Data System (ADS)

    Suriano, Raffaella; Zandrini, Tommaso; De Marco, Carmela; Osellame, Roberto; Turri, Stefano; Bragheri, Francesca

    2016-04-01

    Atomic force microscopy (AFM) nanoindentation of soft materials is a powerful tool for probing mechanical properties of biomaterials. Though many results have been reported in this field over the last decade, adhesion forces between the tip and the sample hinder the elastic modulus measurement when hydrophilic soft samples are investigated. Here, two-photon polymerization (2PP) technology was used to fabricate hydrophobic perfluoropolyether-based AFM tips. The hydrophobic 2PP tips allowed us to overcome the limitations of commercial and functionalized tips as well as to successfully measure the elastic modulus of medically relevant soft materials in air. Our results obtained in the characterization of poly(dimethyl siloxane) and polyethylene glycol hydrogels showed lower adhesion forces over a larger measurement range when compared to measurements performed with commercial tips. The elastic moduli measured by means of hydrophobic 2PP AFM tips were also found to be comparable to those obtained using conventional techniques for macroscopic samples. We successfully showed that the hydrophobic AFM tips developed by this highly versatile technology enable the study of mechanical properties of soft matter, benefiting from reduced sample-tip interactions, and a custom-made shape and dimension of the tips.

  2. The structure of high-methoxyl sugar acid gels of citrus pectin as determined by AFM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Images of native high methoxyl sugar acid gels (HMSAG) were obtained by atomic force microscopy (AFM) in the Tapping ModeTM. Electronic thinning of the pectin strands to one pixel wide allowed the pectin network to be viewed in the absence of variable strand widths related to preferentially solvate...

  3. Difference in cellular mechanics of cancer and normal cervical cells as seen with the AFM

    NASA Astrophysics Data System (ADS)

    Sokolov, Igor; Iyer, Swaminathan; Subba-Rao, Venkatesh; Woodworth, Craig

    2006-03-01

    Oncogenically transformed cells differ from their normal counter parts in many aspects, including organization and the amount of cytoskeleton. Consequently it is natural to expect to see the difference in cellular mechanics. Here we will present the study of such differences by using atomic force microscopy (AFM) in-vitro. So far the present research is the first study of mechanics of cervical cells, and the third comparative study of differences between mechanics of cancer and normal cells down with the help of AFM. Using a micron size silica ball as the AFM probe, we presumably do not overstress the cell surface as it can be in the case of the sharp AFM tip, and consequently, we may use the classical Hertz model. In contrast to the reported previously studies (bladder and fibroblast cells), we found that oncogenically transformed cervical cells are more rigid than the normal cells. The reason for such difference will be discussed. To demonstrate the complexity of the problem, we study cell mechanics in detail. The Young's modulus of rigidity clearly shows two separate regions of rigidity depending on the depth of the probe penetration. There may be two alternative explanations of the difference in rigidity of this top layer: it is either the cell membrane layer or detected long-range (presumably steric) forces due to the molecular ``brush'' of glycocalyx molecules. Experiments and modal calculations will be presented to choose between these two possibilities.

  4. Accurate calibration and uncertainty estimation of the normal spring constant of various AFM cantilevers.

    PubMed

    Song, Yunpeng; Wu, Sen; Xu, Linyan; Fu, Xing

    2015-03-10

    Measurement of force on a micro- or nano-Newton scale is important when exploring the mechanical properties of materials in the biophysics and nanomechanical fields. The atomic force microscope (AFM) is widely used in microforce measurement. The cantilever probe works as an AFM force sensor, and the spring constant of the cantilever is of great significance to the accuracy of the measurement results. This paper presents a normal spring constant calibration method with the combined use of an electromagnetic balance and a homemade AFM head. When the cantilever presses the balance, its deflection is detected through an optical lever integrated in the AFM head. Meanwhile, the corresponding bending force is recorded by the balance. Then the spring constant can be simply calculated using Hooke's law. During the calibration, a feedback loop is applied to control the deflection of the cantilever. Errors that may affect the stability of the cantilever could be compensated rapidly. Five types of commercial cantilevers with different shapes, stiffness, and operating modes were chosen to evaluate the performance of our system. Based on the uncertainty analysis, the expanded relative standard uncertainties of the normal spring constant of most measured cantilevers are believed to be better than 2%.

  5. High spatial resolution surface imaging and analysis of fungal cells using SEM and AFM.

    PubMed

    Kaminskyj, Susan G W; Dahms, Tanya E S

    2008-06-01

    We review the use of scanning electron microscopy (SEM), atomic force microscopy (AFM) and force spectroscopy (FS) for probing the ultrastructure, chemistry, physical characteristics and motion of fungal cells. When first developed, SEM was used to image fixed/dehydrated/gold coated specimens, but here we describe more recent SEM developments as they apply to fungal cells. CryoSEM offers high resolution for frozen fungal samples, whereas environmental SEM allows the analysis of robust samples (e.g. spores) under ambient conditions. Dual beam SEM, the most recently developed, adds manipulation capabilities along with element detection. AFM has similar lateral and better depth resolution compared to SEM, and can image live cells including growing fungal hyphae. FS can analyze cell wall chemistry, elasticity and dynamic cell characteristics. The integration of AFM with optical microscopy will allow examination of individual molecules or cellular structures in the context of fungal cell architecture. SEM and AFM are complementary techniques that are clarifying our understanding of fungal biology. PMID:18068995

  6. Study of Thin Oxide Films with NC-AFM: Atomically Resolved Imaging and Beyond

    NASA Astrophysics Data System (ADS)

    Heyde, M.; Simon, G. H.; König, T.

    Results presented in the following show structural analysis of metaloxide surfaces and the extraction of physical quantities from the force field above such a surface by noncontact atomic force microscopy (NC-AFM). The measurements have been performed with our dual mode NC-AFM/STM in ultrahigh vacuum at 5 K. The introduction will be followed by a description of the experimental setup, including the ultrahigh vacuum cryogenic environment and our tuning fork tunneling current and force sensor. The sensor parameters affecting the measurements are given together with an amplitude characterization method. In the next section, a structure determination of ultrathin Alumina/NiAl(110) is shown. Atomic resolution could be achieved throughout both reflection domain unit cells. NC-AFM reveals details of morphological features, interconnections to substrate-film interactions, and comparability to theory also with respect to topographic height. In the last section, we present measurements beyond imaging, namely spectroscopy data taken on thin MgO films grown on Ag(001). Force-distance measurements based on atomically resolved NC-AFM images of these films have been taken. Inequivalent sites could be resolved and their effect on nucleation and adsorption processes is debated. Furthermore, work function shift measurements on different MgO film thicknesses grown on Ag(001) are studied and the impact of this shift on the catalytic properties of adsorbed metal species is discussed.

  7. Afm Measrurements of Martian Soil Particles Using Mems Technology - Results from the PHOENIX Mission

    NASA Astrophysics Data System (ADS)

    Gautsch, S.; Parrat, D.; de Rooij, N. F.; Staufer, U.; Morookian, J. M.; Hecht, M. H.; Vijendran, S.; Sykulska, H.; Pike, W. T.

    2011-12-01

    Light scattering experiments conducted on Mars indicated that soil particles have dimensions around 1 μm. Particles in that range play an important role in the gas exchange between sub-surface water ice and the atmosphere. Their shape can help tracing the geological history and may indicate past presence of liquid water. NASA's Phoenix mission therefore decided to analyze soil and dust particles in the sub-micrometer to a few micrometer range using an atomic force microscope (AFM) for the first time on another planet. The co-axially mounted AFM was capable of resolving particles with 10nm lateral resolution. A MEMS approach combined with mechatronic concepts for the scanner was selected for implementing the AFM. For redundancy, the sensor chip featured eight silicon cantilevers each with a 7 to 8 μm high tip. The cantilevers could be cleaved off if contaminated. During NASA's Phoenix Mission, which operated on the red planet from May to October 2008, we could demonstrate successful AFM operations. The instrument has executed 85 experiments of which 26 were needed for calibration. Of the remaining experiments about half (28) returned images where signatures of particles could be discerned.

  8. 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.

  9. Implications of the contact radius to line step (CRLS) ratio in AFM for nanotribology measurements.

    PubMed

    Helt, James M; Batteas, James D

    2006-07-01

    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

  10. Implications of the contact radius to line step (CRLS) ratio in AFM for nanotribology measurements.

    PubMed

    Helt, James M; Batteas, James D

    2006-07-01

    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

  11. AFM Studies of Lunar Soils and Application to the Mars 2001 Mission

    NASA Technical Reports Server (NTRS)

    Weitz, C. M.; Anderson, M. S.; Marshall, J.

    1999-01-01

    The upcoming Mars 01 mission will carry an Atomic Force Microscope (AFM) as part of the Mars Environmental Compatibility Assessment (MECA) instrument. By operating in a tapping mode, the AFM is capable of sub-nanometer resolution in three dimensions and can distinguish between substances of different compositions by employing phase contrast imaging. To prepare for the Mars 01 mission, we are testing the AFM on a lunar soil to determine its ability to define particle shapes and sizes and grain-surface textures. The test materials are from the Apollo 17 soil 79221, which is a mixture of agglutinates, impact and volcanic beads, and mare and highland rock and mineral fragments. The majority of the lunar soil particles are less than 100 microns in size, comparable to the sizes estimated for martian dust. We have used the AFM to examine several different soil particles at various resolutions. The instrument has demonstrated the ability to identify parallel ridges characteristic of twinning on a 150 micron plagioclase feldspar particle. Extremely small (10-100 nanometer) adhering particles are visible on the surface of the feldspar grain, and they appear elongate with smooth surfaces. Phase contrast imaging of the nanometer particles shows several compositions to be present. When the AFM was applied to a 100 micron glass spherule, it was possible to define an extremely smooth surface; this is in clear contrast to results from a basalt fragment which exhibited a rough surface texture. Also visible on the surface of the glass spherule were chains of 100 nanometer and smaller impact melt droplets. For the '01 Mars mission, the AFM is intended to define the size and shape distributions of soil particles, in combination with the NMCA optical microscope system and images from the Robot Arm Camera (RAC). These three data sets will provide a means of assessing potentially hazardous soil and dust properties. The study that we have conducted on the lunar soils now suggests that the

  12. Rapid actions of plasma membrane estrogen receptors regulate motility of mouse embryonic stem cells through a profilin-1/cofilin-1-directed kinase signaling pathway.

    PubMed

    Yun, Seung Pil; Ryu, Jung Min; Kim, Mi Ok; Park, Jae Hong; Han, Ho Jae

    2012-08-01

    Long-term estrogen actions are vital for driving cell growth, but more recent evidence suggests that estrogen mediates more rapid cellular effects. However, the function of estradiol-17β (E(2))-BSA in mouse embryonic stem cells has not been reported. Therefore, we examined the role of E(2)-BSA in mouse embryonic stem cell motility and its related signal pathways. E(2)-BSA (10(-8) m) significantly increased motility after 24 h incubation and increased filamentous (F)-actin expression; these effects were inhibited by the estrogen receptor antagonist ICI 182,780, indicating that E(2)-BSA bound membrane estrogen receptors and initiated a signal. E(2)-BSA increased c-Src and focal adhesion kinase (FAK) phosphorylation, which was attenuated by ICI 182,780. The E(2)-BSA-induced increase in epidermal growth factor receptor (EGFR) phosphorylation was inhibited by Src inhibitor PP2. As a downstream signal molecule, E(2)-BSA activated cdc42 and increased formation of a complex with the neural Wiskott-Aldrich syndrome protein (N-WASP)/cdc42/transducer of cdc42-dependent actin assembly-1 (TOCA-1), which was inhibited by FAK small interfering RNA (siRNA) and EGFR inhibitor AG 1478. In addition, E(2)-BSA increased profilin-1 expression and cofilin-1 phosphorylation, which was blocked by cdc42 siRNA. Subsequently, E(2)-BSA induced an increase in F-actin expression, and cell motility was inhibited by each signal pathway-related siRNA molecule or inhibitors but not by cofilin-1 siRNA. A combined treatment of cofilin-1 siRNA and E(2)-BSA increased F-actin expression and cell motility more than that of E(2)-BSA alone. These data demonstrate that E(2)-BSA stimulated motility by interacting with profilin-1/cofilin-1 and F-actin through FAK- and c-Src/EGFR transactivation-dependent N-WASP/cdc42/TOCA-1 complex.

  13. TLR9 activation induces normal neutrophil responses in a child with IRAK-4 deficiency: involvement of the direct PI3K pathway.

    PubMed

    Hoarau, Cyrille; Gérard, Bénédicte; Lescanne, Emmanuel; Henry, Dominique; François, Stéphanie; Lacapère, Jean-Jacques; El Benna, Jamel; Dang, Pham My-Chan; Grandchamp, Bernard; Lebranchu, Yvon; Gougerot-Pocidalo, Marie-Anne; Elbim, Carole

    2007-10-01

    Polymorphonuclear neutrophils (PMN) play a key role in innate immunity. Their activation and survival are tightly regulated by microbial products via pattern recognition receptors such as TLRs, which mediate recruitment of the IL-1R-associated kinase (IRAK) complex. We describe a new inherited IRAK-4 deficiency in a child with recurrent pyogenic bacterial infections. Analysis of the IRAK4 gene showed compound heterozygosity with two mutations: a missense mutation in the death domain of the protein (pArg12Cys) associated in cis-with a predicted benign variant (pArg391His); and a splice site mutation in intron 7 that led to the skipping of exon 7. A nontruncated IRAK-4 protein was detected by Western blotting. The patient's functional deficiency of IRAK-4 protein was confirmed by the absence of IRAK-1 phosphorylation after stimulation with all TLR agonists tested. The patient's PMNs showed strongly impaired responses (L-selectin and CD11b expression, oxidative burst, cytokine production, cell survival) to TLR agonists which engage TLR1/2, TLR2/6, TLR4, and TLR7/8; in contrast, the patient's PMN responses to CpG-DNA (TLR9) were normal, except for cytokine production. The surprisingly normal effect of CpG-DNA on PMN functions and apoptosis disappeared after pretreatment with PI3K inhibitors. Together, these results suggest the existence of an IRAK-4-independent TLR9-induced transduction pathway leading to PI3K activation. This alternative pathway may play a key role in PMN control of infections by microorganisms other than pyogenic bacteria in inherited IRAK-4 deficiency.

  14. Targeting the CD80/CD86 costimulatory pathway with CTLA4-Ig directs microglia toward a repair phenotype and promotes axonal outgrowth.

    PubMed

    Louveau, Antoine; Nerrière-Daguin, Véronique; Vanhove, Bernard; Naveilhan, Philippe; Neunlist, Michel; Nicot, Arnaud; Boudin, Hélène

    2015-12-01

    Among the costimulatory factors widely studied in the immune system is the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA4)-CD80/CD86 pathway, which critically controls the nature and duration of the T-cell response. In the brain, up-regulated expression of CD80/CD86 during inflammation has consistently been reported in microglia. However, the role of CD80/CD86 molecules has mainly been studied in a context of microglia-T cell interactions in pathological conditions, while the function of CD80/CD86 in the regulation of intrinsic brain cells remains largely unknown. In this study, we used a transgenic pig line in which neurons express releasable CTLA4-Ig, a synthetic molecule mimicking CTLA4 and binding to CD80/CD86. The effects of CTLA4-Ig on brain cells were analyzed after intracerebral transplantation of CTLA4-Ig-expressing neurons or wild-type neurons as control. This model provided in vivo evidence that CTLA4-Ig stimulated axonal outgrowth, in correlation with a shift of the nearby microglia from a compact to a ramified morphology. In a culture system, we found that the CTLA4-Ig-induced morphological change of microglia was mediated through CD86, but not CD80. This was accompanied by microglial up-regulated expression of the anti-inflammatory molecule Arginase 1 and the neurotrophic factor BDNF, in an astrocyte-dependent manner through the purinergic P2Y1 receptor pathway. Our study identifies for the first time CD86 as a key player in the modulation of microglia phenotype and suggests that CTLA4-Ig-derived compounds might represent new tools to manipulate CNS microglia.

  15. Influence of smectite suspension structure on sheet orientation in dry sediments: XRD and AFM applications.

    PubMed

    Zbik, Marek S; Frost, Ray L

    2010-06-15

    The structure-building phenomena within clay aggregates are governed by forces acting between clay particles. Measurements of such forces are important to understand in order to manipulate the aggregate structure for applications such as dewatering of mineral processing tailings. A parallel particle orientation is required when conducting XRD investigation on the oriented samples and conduct force measurements acting between basal planes of clay mineral platelets using atomic force microscopy (AFM). To investigate how smectite clay platelets were oriented on silicon wafer substrate when dried from suspension range of methods like SEM, XRD and AFM were employed. From these investigations, we conclude that high clay concentrations and larger particle diameters (up to 5 microm) in suspension result in random orientation of platelets in the substrate. The best possible laminar orientation in the clay dry film, represented in the XRD 001/020 intensity ratio of 47 was obtained by drying thin layers from 0.02 wt.% clay suspensions of the natural pH. Conducted AFM investigations show that smectite studied in water based electrolytes show very long-range repulsive forces lower in strength than electrostatic forces from double-layer repulsion. It was suggested that these forces may have structural nature. Smectite surface layers rehydrate in water environment forms surface gel with spongy and cellular texture which cushion approaching AFM probe. This structural effect can be measured in distances larger than 1000 nm from substrate surface and when probe penetrate this gel layer, structural linkages are forming between substrate and clay covered probe. These linkages prevent subsequently smooth detachments of AFM probe on way back when retrieval. This effect of tearing new formed structure apart involves larger adhesion-like forces measured in retrieval. It is also suggested that these effect may be enhanced by the nano-clay particles interaction.

  16. Structural impact of cations on lipid bilayer models: nanomechanical properties by AFM-force spectroscopy.

    PubMed

    Redondo-Morata, Lorena; Giannotti, Marina I; Sanz, Fausto

    2014-02-01

    Atomic Force Microscopy (AFM) has become an invaluable tool for studying the micro- and nanoworlds. As a stand-alone, high-resolution imaging technique and force transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility. The main strength of AFM relies on the possibility to operate in an aqueous environment on a wide variety of biological samples, from single molecules - DNA or proteins - to macromolecular assemblies like biological membranes. Understanding the effect of mechanical stress on membranes is of primary importance in biophysics, since cells are known to perform their function under a complex combination of forces. In the later years, AFM-based Force-Spectroscopy (AFM-FS) has provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Lipid membranes are electrostatically charged entities that physiologically coexist with electrolyte solutions. Thus, specific interactions with ions are a matter of considerable interest. The distribution of ions in the solution and their interaction with the membranes are factors that substantially modify the structure and dynamics of the cell membranes. Furthermore, signaling processes are modified by the membrane capability of retaining ions. Supported Lipid Bilayers (SLBs) are a versatile tool to investigate phospholipid membranes mimicking biological surfaces. In the present contribution, we review selected experiments on the mechanical stability of SLBs as models of lipid membranes by means of AFM-FS, with special focus on the effect of cations and ionic strength in the overall nanomechanical stability. PMID:24341385

  17. SAR650984 directly induces multiple myeloma cell death via lysosomal-associated and apoptotic pathways, which is further enhanced by pomalidomide.

    PubMed

    Jiang, H; Acharya, C; An, G; Zhong, M; Feng, X; Wang, L; Dasilva, N; Song, Z; Yang, G; Adrian, F; Qiu, L; Richardson, P; Munshi, N C; Tai, Y-T; Anderson, K C

    2016-02-01

    The anti-CD38 monoclonal antibody SAR650984 (SAR) is showing promising clinical activity in treatment of relapsed and refractory multiple myeloma (MM). Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define molecular mechanisms of SAR-directed MM cell death and enhanced anti-MM activity triggered by SAR with Pomalidomide (Pom). Without Fc-cross-linking agents or effector cells, SAR specifically induces homotypic aggregation (HA)-associated cell death in MM cells dependent on the level of cell surface CD38 expression, actin cytoskeleton and membrane lipid raft. SAR and its F(ab)'2 fragments trigger caspase 3/7-dependent apoptosis in MM cells highly expressing CD38, even with p53 mutation. Importantly, SAR specifically induces lysosome-dependent cell death (LCD) by enlarging lysosomes and increasing lysosomal membrane permeabilization associated with leakage of cathepsin B and LAMP-1, regardless of the presence of interleukin-6 or bone marrow stromal cells. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR further upregulates reactive oxygen species. Pom enhances SAR-induced direct and indirect killing even in MM cells resistant to Pom/Len. Taken together, SAR is the first therapeutic monoclonal antibody mediating direct cytotoxicity against MM cells via multiple mechanisms of action. Our data show that Pom augments both direct and effector cell-mediated MM cytotoxicity of SAR, providing the framework for combination clinical trials. PMID:26338273

  18. 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.

  19. 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. PMID:21446752

  20. Characterization of single 1.8-nm Au nanoparticle attachments on AFM tips for single sub-4-nm object pickup

    NASA Astrophysics Data System (ADS)

    Cheng, Hui-Wen; Chang, Yuan-Chih; Tang, Song-Nien; Yuan, Chi-Tsu; Tang, Jau; Tseng, Fan-Gang

    2013-11-01

    This paper presents a novel method for the attachment of a 1.8-nm Au nanoparticle (Au-NP) to the tip of an atomic force microscopy (AFM) probe through the application of a current-limited bias voltage. The resulting probe is capable of picking up individual objects at the sub-4-nm scale. We also discuss the mechanisms involved in the attachment of the Au-NP to the very apex of an AFM probe tip. The Au-NP-modified AFM tips were used to pick up individual 4-nm quantum dots (QDs) using a chemically functionalized method. Single QD blinking was reduced considerably on the Au-NP-modified AFM tip. The resulting AFM tips present an excellent platform for the manipulation of single protein molecules in the study of single protein-protein interactions.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. The bile acid chenodeoxycholic acid directly modulates metabolic pathways in white adipose tissue in vitro: insight into how bile acids decrease obesity.

    PubMed

    Teodoro, João Soeiro; Rolo, Anabela Pinto; Jarak, Ivana; Palmeira, Carlos Marques; Carvalho, Rui Albuquerque

    2016-10-01

    Obesity is a worldwide epidemic, and associated pathologies, including type 2 diabetes and cardiovascular alterations, are increasingly escalating morbidity and mortality. Despite intensive study, no effective simple treatment for these conditions exists. As such, the need for go-to drugs is serious. Bile acids (BAs) present the possibility of reversing these problems, as various in vivo studies and clinical trials have shown significant effects with regard to weight and obesity reduction, insulin sensitivity restoration and cardiovascular improvements. However, the mechanism of action of BA-induced metabolic improvement has yet to be fully established. The currently most accepted model involves non-shivering thermogenesis for energy waste, but this is disputed. As such, we propose to determine whether the BA chenodeoxycholic acid (CDCA) can exert anti-obesogenic effects in vitro, independent of thermogenic brown adipose tissue activation. By exposing differentiated 3 T3-L1 adipocytes to high glucose and CDCA, we demonstrate that this BA has anti-obesity effects in vitro. Nuclear magnetic resonance spectroscopic analysis of metabolic pathways clearly indicates an improvement in metabolic status, as these cells become more oxidative rather than glycolytic, which may be associated with an increase in fatty acid oxidation. Our work demonstrates that CDCA-induced metabolic alterations occur in white and brown adipocytes and are not totally dependent on endocrine/nervous system signaling, as thought until now. Furthermore, future exploration of the mechanisms behind these effects will undoubtedly reveal interesting targets for clinical modulation. PMID:27488269

  6. Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

    PubMed Central

    Oehlenschlæger, Christian Berg; Løvgreen, Monika Nøhr; Reinauer, Eva; Lehtinen, Emilia; Pind, Marie-Louise Lindberg; Martinussen, Jan

    2015-01-01

    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. PMID:25746996

  7. Research on the metabolic engineering of the direct oxidation pathway for extraction of phosphate from ore has generated preliminary evidence for PQQ biosynthesis in Escherichia coli as well as a possible role for the highly conserved region of quinoprotein dehydrogenases.

    PubMed

    Goldstein, Alan; Lester, Trevor; Brown, Jacquelyn

    2003-04-11

    The ability of some bacteria to dissolve poorly soluble calcium phosphates (CaPs) has been termed 'mineral phosphate solubilizing' (MPS). Since most microorganisms and plants must assimilate P via membrane transport, biotransformation of CaP into soluble phosphate is considered an essential component of the global P cycle. In many Gram-negative bacteria, strong organic acids produced in the periplasm via the direct oxidation pathway have been shown to dissolve CaP in the adjacent environment. Therefore, the quinoprotein glucose dehydrogenase (PQQGDH) may function in the ecophysiology of many soil bacteria. There is interest in using MPS bacteria for industrial bioprocessing of rock phosphate ore (a substituted fluroapatite) or even for direct inoculation of soils as a 'biofertilizer' analogous to nitrogen fixation. Our laboratory has spent 20 years studying superior MPS bacteria. Screening genomic libraries in the appropriate E. coli genetic background can 'trap' PQQ or GDH genes from these bacteria via functional complementation. In setting the 'trap' for PQQ genes, we have identified DNA fragments that apparently induce PQQGDH activity in E. coli with no sequence homology to known PQQ genes. These data suggest that E. coli may have an alternative, inducible PQQ biosynthesis pathway. Finally, a novel protein engineering strategy to increase the catalytic rate of PQQGDH has emerged and will be discussed.

  8. Identification of ATR-Chk1 pathway inhibitors that selectively target p53-deficient cells without directly suppressing ATR catalytic activity

    PubMed Central

    Kawasumi, Masaoki; Bradner, James E.; Tolliday, Nicola; Thibodeau, Renee; Sloan, Heather; Brummond, Kay M.; Nghiem, Paul

    2014-01-01

    Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells where the G1/S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2/M checkpoints mediated by the ATR and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR-Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs. PMID:25336189

  9. 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.

  10. In Escherichia coli, MreB and FtsZ direct the synthesis of lateral cell wall via independent pathways that require PBP 2.

    PubMed

    Varma, Archana; Young, Kevin D

    2009-06-01

    In Escherichia coli, the cytoplasmic proteins MreB and FtsZ play crucial roles in ensuring that new muropeptide subunits are inserted into the cell wall in a spatially correct way during elongation and division. In particular, to retain a constant diameter and overall shape, new material must be inserted into the wall uniformly around the cell's perimeter. Current thinking is that MreB accomplishes this feat through intermediary proteins that tether peptidoglycan synthases to the outer face of the inner membrane. We tested this idea in E. coli by using a DD-carboxypeptidase mutant that accumulates pentapeptides in its peptidoglycan, allowing us to visualize new muropeptide incorporation. Surprisingly, inhibiting MreB with the antibiotic A22 did not result in uneven insertion of new wall, although the cells bulged and lost their rod shapes. Instead, uneven (clustered) incorporation occurred only if MreB and FtsZ were inactivated simultaneously, providing the first evidence in E. coli that FtsZ can direct murein incorporation into the lateral cell wall independently of MreB. Inhibiting penicillin binding protein 2 (PBP 2) alone produced the same clustered phenotype, implying that MreB and FtsZ tether peptidoglycan synthases via a common mechanism that includes PBP 2. However, cell shape was determined only by the presence or absence of MreB and not by the even distribution of new wall material as directed by FtsZ.

  11. 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

  12. AFM combines functional and morphological analysis of peripheral myelinated and demyelinated nerve fibers.

    PubMed

    Heredia, Alejandro; Bui, Chin Chu; Suter, Ueli; Young, Peter; Schäffer, Tilman E

    2007-10-01

    Demyelination of the myelinated peripheral or central axon is a common pathophysiological step in the clinical manifestation of several human diseases of the peripheral and the central nervous system such as the majority of Charcot-Marie-Tooth syndromes and multiple sclerosis, respectively. The structural degradation of the axon insulating myelin sheath has profound consequences for ionic conduction and nerve function in general, but also affects the micromechanical properties of the nerve fiber. We have for the first time investigated mechanical properties of rehydrated, isolated peripheral nerve fibers from mouse using atomic force microscopy (AFM). We have generated quantitative maps of elastic modulus along myelinated and demyelinated axons, together with quantitative maps of axon topography. This study shows that AFM can combine functional and morphological analysis of neurological tissue at the level of single nerve fibers.

  13. 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.

  14. 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.

  15. Surface electrical properties of stainless steel fibres: An AFM-based study

    NASA Astrophysics Data System (ADS)

    Yin, Jun; D'Haese, Cécile; Nysten, Bernard

    2015-03-01

    Atomic force microscopy (AFM) electrical modes were used to study the surface electrical properties of stainless steel fibres. The surface electrical conductivity was studied by current sensing AFM and I-V spectroscopy. Kelvin probe force microscopy was used to measure the surface contact potential. The oxide film, known as passivation layer, covering the fibre surface gives rise to the observation of an apparently semiconducting behaviour. The passivation layer generally exhibits a p-type semiconducting behaviour, which is attributed to the predominant formation of chromium oxide on the surface of the stainless steel fibres. At the nanoscale, different behaviours are observed from points to points, which may be attributed to local variations of the chemical composition and/or thickness of the passivation layer. I-V curves are well fitted with an electron tunnelling model, indicating that electron tunnelling may be the predominant mechanism for electron transport.

  16. Cell mechanics as a marker for diseases: Biomedical applications of AFM

    NASA Astrophysics Data System (ADS)

    Rianna, Carmela; Radmacher, Manfred

    2016-08-01

    Many diseases are related to changes in cell mechanics. Atomic Force Microscopy (AFM) is one of the most suitable techniques allowing the investigation of both topography and mechanical properties of adherent cells with high spatial resolution under physiological conditions. Over the years the use of this technique in medical and clinical applications has largely increased, resulting in the notion of cell mechanics as a biomarker to discriminate between different physiological and pathological states of cells. Cell mechanics has proven to be a biophysical fingerprint able discerning between cell phenotypes, unraveling processes in aging or diseases, or even detecting and diagnosing cellular pathologies. We will review in this report some of the works on cell mechanics investigated by AFM with clinical and medical relevance in order to clarify the state of research in this field and to highlight the role of cell mechanics in the study of pathologies, focusing on cancer, blood and cardiovascular diseases.

  17. Determination of work of adhesion of biological cell under AFM bead indentation.

    PubMed

    Zhu, Xinyao; Siamantouras, E; Liu, K K; Liu, X

    2016-03-01

    Hertz contact theory has been widely used for the determination of cell elasticity based on AFM indentation experiments. In light of the adhesive contact between AFM tip and cell, this study applied Johnson-Kendall-Roberts (JKR) model to fit the indentation force-displacement (F-D) curves reported previously. A MIN6 cell has been modeled as first a sphere and then a flattened cell with different thicknesses. The results have shown that both basic JKR model and "generalized" JKR model can best describe the unloading force-displacement behaviors of the indentation curves. The Young׳s modulus of the cell and the work of adhesion of the cell-indenter interface are obtained. In comparison to the Hertzian contact model, the JKR model provides obviously better fitting to the experimental results, indicating that the adhesion is significant in the cell interaction. PMID:26688423

  18. Pattern formation and control in polymeric systems: From Minkowski measures to in situ AFM imaging

    NASA Astrophysics Data System (ADS)

    Jacobs, Karin

    2014-03-01

    Thin liquid polymer films are not only of great technical importance, they also exhibit a variety of dynamical instabilities. Some of them may be desired, some rather not. To analyze and finally control pattern formation, modern thin film theories are as vital as techniques to characterize the morphologies and structures in and on the films. Examples for the latter are atomic force microscopy (AFM) as well as scattering techniques. The talk will introduce into the practical applications of Minkowski measures to characterize patterns and explain what thin film properties (e.g. capillary number, solid/liquid boundary condition, glass transition temperature, chain mobility) can further be extracted including new technical possibilities by AFM and scattering techniques.

  19. AFM imaging reveals the tetrameric structure of the TRPM8 channel

    SciTech Connect

    Stewart, Andrew P.; Egressy, Kinga; Lim, Annabel; Edwardson, J. Michael

    2010-04-02

    Several members of the transient receptor potential (TRP) channel superfamily have been shown to assemble as tetramers. Here we have determined the subunit stoichiometry of the transient receptor potential M8 (TRPM8) channel using atomic force microscopy (AFM). TRPM8 channels were isolated from transfected cells, and complexes were formed between the channels and antibodies against a V5 epitope tag present on each subunit. The complexes were then subjected to AFM imaging. A frequency distribution of the molecular volumes of antibody decorated channels had a peak at 1305 nm{sup 3}, close to the expected size of a TRPM8 tetramer. The frequency distribution of angles between pairs of bound antibodies had two peaks, at 93{sup o} and 172{sup o}, confirming that the channel assembles as a tetramer. We suggest that this assembly pattern is common to all members of the TRP channel superfamily.

  20. 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).

  1. Mode coupling in a hanging-fiber AFM used as a rheological probe

    NASA Astrophysics Data System (ADS)

    Devailly, C.; Laurent, J.; Steinberger, A.; Bellon, L.; Ciliberto, S.

    2014-06-01

    We analyze the advantages and drawbacks of a method which measures the viscosity of liquids at microscales, using a thin glass fiber fixed on the tip of a cantilever of an ultra-low-noise Atomic Force Microscope (AFM). When the fiber is dipped into a liquid, the dissipation of the cantilever-fiber system, which is linked to the liquid viscosity, can be computed from the power spectral density of the thermal fluctuations of the cantilever deflection. The high sensitivity of the AFM allows us to show the existence and to develop a model of the coupling between the dynamics of the fiber and that of the cantilever. This model, which accurately fits the experimental data, gives also more insights into the dynamics of coupled microdevices in a viscous environment.

  2. AFM characterization of nanobubble formation and slip condition in oxygenated and electrokinetically altered fluids.

    PubMed

    Bhushan, Bharat; Pan, Yunlu; Daniels, Stephanie

    2013-02-15

    Nanobubbles are gas-filled features that spontaneously form at the interface of hydrophobic surfaces and aqueous solutions. In this study, an atomic force microscope (AFM) was used to characterize the morphology of nanobubbles formed on hydrophobic polystyrene (PS) and octadecyltrichlorosilane (OTS) films immersed in DI water, saline, saline with oxygen and an electrokinetically altered saline solution produced with Taylor-Couette-Poiseuille flow under elevated oxygen pressure. AFM force spectroscopy was used to evaluate hydrodynamic and electrostatic forces and boundary slip condition in various fluids. The effect of solution, electric field and surface charge on shape, size and density of nanobubbles as well as slip length was quantified and the results and underlying mechanisms are presented in this paper.

  3. 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.

  4. 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.

  5. Force-controlled manipulation of single cells: from AFM to FluidFM.

    PubMed

    Guillaume-Gentil, Orane; Potthoff, Eva; Ossola, Dario; Franz, Clemens M; Zambelli, Tomaso; Vorholt, Julia A

    2014-07-01

    The ability to perturb individual cells and to obtain information at the single-cell level is of central importance for addressing numerous biological questions. Atomic force microscopy (AFM) offers great potential for this prospering field. Traditionally used as an imaging tool, more recent developments have extended the variety of cell-manipulation protocols. Fluidic force microscopy (FluidFM) combines AFM with microfluidics via microchanneled cantilevers with nano-sized apertures. The crucial element of the technology is the connection of the hollow cantilevers to a pressure controller, allowing their operation in liquid as force-controlled nanopipettes under optical control. Proof-of-concept studies demonstrated a broad spectrum of single-cell applications including isolation, deposition, adhesion and injection in a range of biological systems. PMID:24856959

  6. High precision attachment of silver nanoparticles on AFM tips by dielectrophoresis.

    PubMed

    Leiterer, Christian; Wünsche, Erik; Singh, Prabha; Albert, Jens; Köhler, Johann M; Deckert, Volker; Fritzsche, Wolfgang

    2016-05-01

    AFM tips are modified with silver nanoparticles using an AC electrical field. The used technique works with sub-micron precision and also does not require chemical modification of the tip. Based on the electrical parameters applied in the process, particle density and particle position on the apex of the tip can be adjusted. The feasibility of the method is proven by subsequent tip-enhanced Raman spectroscopy (TERS) measurements using the fabricated tips as a measurement probe. Since this modification process itself does not require any lithographic processing, the technique can be easily adapted to modify AFM tips with a variety of nanostructures with pre-defined properties, while being parallelizable for a potential commercial application.

  7. Carbon nanotube/carbon nanotube composite AFM probes prepared using ion flux molding

    NASA Astrophysics Data System (ADS)

    Chesmore, Grace; Roque, Carrollyn; Barber, Richard

    The performance of carbon nanotube-carbon nanotube composite (CNT/CNT composite) atomic force microscopy (AFM) probes is compared to that of conventional Si probes in AFM tapping mode. The ion flux molding (IFM) process, aiming an ion beam at the CNT probe, aligns the tip to a desired angle. The result is a relatively rigid tip that is oriented to offset the cantilever angle. Scans using these probes reveal an improvement in image accuracy over conventional tips, while allowing higher aspect ratio imaging of 3D surface features. Furthermore, the lifetimes of CNT-CNT composite tips are observed to be longer than both conventional tips and those claimed for other CNT technologies. Novel applications include the imaging of embiid silk. Supported by the Clare Boothe Luce Research Scholars Award and Carbon Design Innovations.

  8. 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

  9. 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

    2011-10-01

    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.

  10. Characterization and Optimization of Quartz Tuning Fork-Based Force Sensors for Combined STM/AFM

    NASA Astrophysics Data System (ADS)

    Castellanos-Gomez, Andres; Agraït, Nicolás; Rubio-Bollinger, Gabino

    This chapter will be divided in two main parts. In the first one, we will show a detailed analysis of the dynamics of quartz tuning fork resonators which are being increasingly used in scanning probe microscopy as force sensors. We will also show that a coupled harmonic oscillators model, which includes a finite coupling between the prongs, is in remarkable agreement with the observed motion of the tuning forks. Relevant parameters for the tuning fork performance such as the effective spring constant can be obtained from our analysis. In the second one, we will present an implementation of a quartz tuning fork supplemented with optimized tips based on carbon fibers. The remarkable electrical and mechanical properties of carbon fiber make these tips more suitable for combined and/or simultaneous STM and AFM than conventional metallic tips. The fabrication and the characterization of these carbon fiber tips as well as their performance in STM/AFM will be detailed.

  11. Troponate/Aminotroponate Ruthenium-Arene Complexes: Synthesis, Structure, and Ligand-Tuned Mechanistic Pathway for Direct C-H Bond Arylation with Aryl Chlorides in Water.

    PubMed

    Dwivedi, Ambikesh D; Binnani, Chinky; Tyagi, Deepika; Rawat, Kuber S; Li, Pei-Zhou; Zhao, Yanli; Mobin, Shaikh M; Pathak, Biswarup; Singh, Sanjay K

    2016-07-01

    A series of water-soluble troponate/aminotroponate ruthenium(II)-arene complexes were synthesized, where O,O and N,O chelating troponate/aminotroponate ligands stabilized the piano-stool mononuclear ruthenium-arene complexes. Structural identities for two of the representating complexes were also established by single-crystal X-ray diffraction studies. These newly synthesized troponate/aminotroponate ruthenium-arene complexes enable efficient C-H bond arylation of arylpyridine in water. The unique structure-activity relationship in these complexes is the key to achieve efficient direct C-H bond arylation of arylpyridine. Moreover, the steric bulkiness of the carboxylate additives systematically directs the selectivity toward mono- versus diarylation of arylpyridines. Detailed mechanistic studies were performed using mass-spectral studies including identification of several key cyclometalated intermediates. These studies provided strong support for an initial cycloruthenation driven by carbonate-assisted deprotonation of 2-phenylpyridine, where the relative strength of η(6)-arene and the troponate/aminotroponate ligand drives the formation of cyclometalated 2-phenylpyridine Ru-arene species, [(η(6)-arene)Ru(κ(2)-C,N-phenylpyridine) (OH2)](+) by elimination of troponate/aminotroponate ligands and retaining η(6)-arene, while cyclometalated 2-phenylpyridine Ru-troponate/aminotroponate species [(κ (2)-troponate/aminotroponate)Ru(κ(2)-C,N-phenylpyridine)(OH2)2] was generated by decoordination of η(6)-arene ring during initial C-H bond activation of 2-phenylpyridine. Along with the experimental mass-spectral evidence, density functional theory calculation also supports the formation of such species for these complexes. Subsequently, these cycloruthenated products activate aryl chloride by facile oxidative addition to generate C-H arylated products. PMID:27305143

  12. 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.

  13. Troponate/Aminotroponate Ruthenium-Arene Complexes: Synthesis, Structure, and Ligand-Tuned Mechanistic Pathway for Direct C-H Bond Arylation with Aryl Chlorides in Water.

    PubMed

    Dwivedi, Ambikesh D; Binnani, Chinky; Tyagi, Deepika; Rawat, Kuber S; Li, Pei-Zhou; Zhao, Yanli; Mobin, Shaikh M; Pathak, Biswarup; Singh, Sanjay K

    2016-07-01

    A series of water-soluble troponate/aminotroponate ruthenium(II)-arene complexes were synthesized, where O,O and N,O chelating troponate/aminotroponate ligands stabilized the piano-stool mononuclear ruthenium-arene complexes. Structural identities for two of the representating complexes were also established by single-crystal X-ray diffraction studies. These newly synthesized troponate/aminotroponate ruthenium-arene complexes enable efficient C-H bond arylation of arylpyridine in water. The unique structure-activity relationship in these complexes is the key to achieve efficient direct C-H bond arylation of arylpyridine. Moreover, the steric bulkiness of the carboxylate additives systematically directs the selectivity toward mono- versus diarylation of arylpyridines. Detailed mechanistic studies were performed using mass-spectral studies including identification of several key cyclometalated intermediates. These studies provided strong support for an initial cycloruthenation driven by carbonate-assisted deprotonation of 2-phenylpyridine, where the relative strength of η(6)-arene and the troponate/aminotroponate ligand drives the formation of cyclometalated 2-phenylpyridine Ru-arene species, [(η(6)-arene)Ru(κ(2)-C,N-phenylpyridine) (OH2)](+) by elimination of troponate/aminotroponate ligands and retaining η(6)-arene, while cyclometalated 2-phenylpyridine Ru-troponate/aminotroponate species [(κ (2)-troponate/aminotroponate)Ru(κ(2)-C,N-phenylpyridine)(OH2)2] was generated by decoordination of η(6)-arene ring during initial C-H bond activation of 2-phenylpyridine. Along with the experimental mass-spectral evidence, density functional theory calculation also supports the formation of such species for these complexes. Subsequently, these cycloruthenated products activate aryl chloride by facile oxidative addition to generate C-H arylated products.

  14. In vitro study of AFB1 and AFM1 effects on human lymphoblastoid Jurkat T-cell model.

    PubMed

    Luongo, D; Russo, R; Balestrieri, A; Marzocco, S; Bergamo, P; Severino, L

    2014-10-01

    Aflatoxin B(1) (AFB(1)) is a mycotoxin produced by Aspergillus spp. that can occur as a natural contaminant in foods and feeds of vegetable origin. Post-ingestion, AFB(1) can be metabolized in the liver of mammals into hydroxylated aflatoxin M(1) (AFM(1)) that is excreted with milk. Although several studies have been carried out to evaluate effects of AFB(1) on the immune system, studies regarding AFM(1) are moreover lacking. The aim of the current study was to investigate effects of AFB(1) and AFM(1) on immune function using a lymphoblastoid Jurkat T-cell line as an experimental model. Both AFB(1) and AFM(1) produced significant decreases in Jurkat cell proliferation, whereas only minor effects were noted on interleukin (IL)-2 and interferon (IFN)-γ cytokines mRNA expression in stimulated cells that had been pre-incubated with AFB(1) and AFM(1). Particularly, AFB(1), but not AFM(1), at the highest concentration (50 µM) induced a marked increase in IL-8 mRNA expression. The results of the current study suggested the existence of a concentration threshold for AFB(1) and AFM(1) needed to exert biological activity on cell viability and innate immunity.

  15. 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

  16. Condensed-phase thermal decomposition of TATB investigated by atomic force microscopy (AFM) and simultaneous thermogravimetric modulated beam mass spectrometry (STMBMS)

    SciTech Connect

    Land, T.A.; Siekhaus, W.J.; Foltz, M.F.; Behrens, R. Jr.

    1993-05-01

    A combination of techniques has been used to investigate the condensed-phase thermal decomposition of TATB. STMBMS has been used to identify the thermal decomposition products and their temporal correlation`s. These experiments have shown that the condensed-phase decomposition proceeds through several autocatalytic pathways. Both low and high molecular weight decomposition products have been identified. Mono-, di- and tri-furazans products have been identified and, their temporal behaviors are consistent with a stepwise loss of water. AFM has been used to correlate the decomposition chemistry with morphological changes occurring as a function of heating. Patches of small 25-140 nm round holes were observed throughout the lattice of TATB crystals that were heated briefly to 300C. It is likely that these holes show where decomposition reactions have started. Evidence of decomposition products have been seen in TATB that has been held at 250C for one hour.

  17. Liquid solution delivery through the pulled nanopipette combined with QTF-AFM system

    NASA Astrophysics Data System (ADS)

    An, Sangmin; Stambaugh, Corey; Kim, Gunn; Lee, Manhee; Kim, Yonghee; Lee, Kunyoung; Jhe, Wonho

    2012-02-01

    Nanopipette is a versatile fluidic tool for biochemical analysis, controlled liquid delivery in bio-nanotechnology. However, most of the researches have been performed in solution based system, thus it is challenge to study nanofluidic properties of the liquid solution delivery through the nanopipette in ambient conditions. In this work, we demonstrated the liquid ejection, dispersion, and subsequent deposition of the nanoparticles via a 30 nm aperture pipette based on the quartz tuning fork -- atomic force microscope (QTF-AFM) combined nanopipette system.

  18. Morphological analysis of stainless steel scale like surface morphology using STM and AFM

    SciTech Connect

    Vignal, V.; Olive, J.M.; Desjardins, D.; Roux, J.C.; Genton, V.

    1997-12-19

    A combined atomic force microscopy (AFM) and scanning tunneling microscopy (STM) investigation of stainless steel like surface morphology formed either in electropolishing bath or in HNO{sub 3} medium is reported. A new numerical technique using the Nanoscope III software is proposed. The dimension, slope and orientation of scales can be easily determined. Moreover, grain boundaries structure and probable oxides present in the upper part of the film can be deduced.

  19. 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.

  20. Functionalized AFM probes for force spectroscopy: eigenmode shapes and stiffness calibration through thermal noise measurements.

    PubMed

    Laurent, Justine; Steinberger, Audrey; Bellon, Ludovic

    2013-06-01

    The functionalization of an atomic force microscope (AFM) cantilever with a colloidal bead is a widely used technique when the geometry between the probe and the sample must be controlled, particularly in force spectroscopy. But some questions remain: how does a bead glued at the end of a cantilever influence its mechanical response? And more importantly for quantitative measurements, can we still determine the stiffness of the AFM probe with traditional techniques?In this paper, the influence of the colloidal mass loading on the eigenmode shape and resonant frequency is investigated by measuring the thermal noise on rectangular AFM microcantilevers with and without beads attached at their extremities. The experiments are performed with a home-made ultra-sensitive AFM, based on differential interferometry. The focused beam from the interferometer probes the cantilever at different positions and the spatial shapes of the modes are determined up to the fifth resonance, without external excitation. The results clearly demonstrate that the first eigenmode is almost unchanged by mass loading. However the oscillation behavior of higher resonances presents a marked difference: with a particle glued at its extremity, the nodes of the modes are displaced towards the free end of the cantilever. These results are compared to an analytical model taking into account the mass and inertial moment of the load in an Euler-Bernoulli framework, where the normalization of the eigenmodes is explicitly worked out in order to allow a quantitative prediction of the thermal noise amplitude of each mode. A good agreement between the experimental results and the analytical model is demonstrated, allowing a clean calibration of the probe stiffness.

  1. 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.

  2. Analysis of time-resolved interaction force mode AFM imaging using active and passive probes.

    PubMed

    Giray Oral, Hasan; Parlak, Zehra; Levent Degertekin, F

    2012-09-01

    We present an in-depth analysis of time-resolved interaction force (TRIF) mode imaging for atomic force microscopy (AFM). A nonlinear model of an active AFM probe, performing simultaneous topography and material property imaging on samples with varying elasticity and adhesion is implemented in Simulink®. The model is capable of simulating various imaging modes, probe structures, sample material properties, tip-sample interaction force models, and actuation and feedback schemes. For passive AFM cantilevers, the model is verified by comparing results from the literature. As an example of an active probe, the force sensing integrated readout and active tip (FIRAT) probe is used. Simulation results indicate that the active and damped nature of FIRAT provides a significant level of control over the force applied to the sample, minimizing sample indentation and topography error. Active tip control (ATC) preserves constant contact time during force control for stable contact while preventing the loss of material property information such as elasticity and adhesive force. Simulation results are verified by TRIF mode imaging of the samples with both soft and stiff regions. PMID:22813887

  3. Rational fabrication of a gold-coated AFM TERS tip by pulsed electrodeposition.

    PubMed

    Yang, Li-Kun; Huang, Teng-Xiang; Zeng, Zhi-Cong; Li, Mao-Hua; Wang, Xiang; Yang, Fang-Zu; Ren, Bin

    2015-11-21

    Reproducible fabrication of sharp gold- or silver-coated tips has become the bottleneck issue in tip-enhanced Raman spectroscopy, especially for atomic force microscopy (AFM)-based TERS. Herein, we developed a novel method based on pulsed electrodeposition to coat a thin gold layer over atomic force microscopy (AFM) tips to produce plasmonic TERS tips with high reproducibility. We systematically investigated the influence of the deposition potential and step time on the surface roughness and sharpness. This method allows the rational control of the radii of gold-coated TERS tips from a few to hundreds of nanometers, which allows us to systematically study the dependence of the TERS enhancement on the radius of the gold-coated AFM tip. The maximum TERS enhancement was achieved for the tip radius in the range of 60-75 nm in the gap mode. The coated gold layer has a strong adhesion with the silicon tip surface, which is highly stable in water, showing the great potential for application in the aqueous environment. PMID:26482226

  4. Brain Tumor Classification Using AFM in Combination with Data Mining Techniques

    PubMed Central

    Huml, Marlene; Silye, René; Zauner, Gerald

    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. PMID:24062997

  5. 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

  6. 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

    2014-01-01

    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

  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. 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.

  9. 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.

  10. 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. PMID:27454881

  11. Nanoscopic imaging of mechanical properties of metal films with magnetic-force-controlled AFM

    NASA Astrophysics Data System (ADS)

    Yamamoto, S.-I.; Yamada, H.; Matsushige, K.; Ishida, T.; Mizutani, W.; Tokumoto, H.

    1999-08-01

    We have succeeded in measuring the Young's moduli of inorganic films with a diamond tip of 160-200 nm in diameter using magnetic-force-controlled atomic force microscopy (MFC-AFM). The measurement of Young's moduli was carried out by detecting the tip amplitude on the sample with an applied force of 20 nN. This technique was applied successfully to obtain Young's moduli mapping of gold islands with 1 nm thickness and nickel films with 10 nm thickness. We obtained an effective spring constant of 3000 N m -1 on gold and 21 000 N m -1 on nickel using MFC-AFM. We could normalize the effective area of the contact radius between the tip and the sample. A value of 350 GPa was derived for the Young's modulus of the nickel film, indicative of the elasticity of native nickel oxide (NiO 2). This shows that the MFC-AFM technique measures the mechanical properties of a thin layer on top of the sample.

  12. Statistical analysis of AFM topographic images of self-assembled quantum dots

    SciTech Connect

    Sevriuk, V. A.; Brunkov, P. N. Shalnev, I. V.; Gutkin, A. A.; Klimko, G. V.; Gronin, S. V.; Sorokin, S. V.; Konnikov, S. G.

    2013-07-15

    To obtain statistical data on quantum-dot sizes, AFM topographic images of the substrate on which the dots under study are grown are analyzed. Due to the nonideality of the substrate containing height differences on the order of the size of nanoparticles at distances of 1-10 {mu}m and the insufficient resolution of closely arranged dots due to the finite curvature radius of the AFM probe, automation of the statistical analysis of their large dot array requires special techniques for processing topographic images to eliminate the loss of a particle fraction during conventional processing. As such a technique, convolution of the initial matrix of the AFM image with a specially selected matrix is used. This makes it possible to determine the position of each nanoparticle and, using the initial matrix, to measure their geometrical parameters. The results of statistical analysis by this method of self-assembled InAs quantum dots formed on the surface of an AlGaAs epitaxial layer are presented. It is shown that their concentration, average size, and half-width of height distribution depend strongly on the In flow and total amount of deposited InAs which are varied within insignificant limits.

  13. 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.

  14. 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.

  15. 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.

  16. If mechanics of cells can be described by elastic modulus in AFM indentation experiments?

    NASA Astrophysics Data System (ADS)

    Sokolov, Igor; Dokukin, Maxim; Guz, Nataliia; Kalaparthi, Vivekanand

    2014-03-01

    We study the question if cells, being highly heterogeneous objects, can be described with an elastic modulus (the Young's modulus) in a self-consistent way. We analyze the elastic modulus using indentation done with AFM of human cervical epithelial cells. Both sharp (cone) and dull AFM probes were used. The indentation data collected were processed through different elastic models. The cell was considered as a homogeneous elastic medium which had either smooth spherical boundary (Hertz/Sneddon models) or the boundary covered with a layer of glycocalyx and membrane protrusions (``brush'' models). Validity of these approximations was investigated. Specifically, we tested the independence of the elastic modulus of the indentation depth, which is assumed in these models. We demonstrate that only one model shows consistency with treating cells as homogeneous elastic medium, the bush model when processing the indentation data collected with the dull probe. The elastic modulus demonstrates strong depth dependence in all other three models. We conclude that it is possible to describe the elastic properties of the cell body by means of an effective elastic modulus in a self-consistent way when using the brush model to analyze data collected with a dull AFM probe.

  17. 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.

  18. 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.

  19. Nano-scale Topographical Studies on the Growth Cones of Nerve Cells using AFM

    NASA Astrophysics Data System (ADS)

    Durkaya, Goksel; Zhong, Lei; Rehder, Vincent; Dietz, Nikolaus

    2009-11-01

    Nerve cells are the fundamental units which are responsible for intercommunication within the nervous system. The neurites, fibrous cable-like extensions for information delivery, of nerve cells are tipped by highly motile sensory structures known as the growth cones which execute important functions; neural construction, decision making and navigation during development and regeneration of the nervous system. The highly dynamic subcomponents of the growth cones are important in neural activity. Atomic Force Microscopy (AFM) is the most powerful microscopy technique which is capable of imaging without conductivity constraint and in liquid media. AFM providing nano-scale topographical information on biological structures is also informative on the physical properties such as: elasticity, adhesion, and softness. This contribution focuses on AFM analysis of the growth cones of the nerve cells removed from the buccal ganglion of Helisoma trivolvis. The results of nano-scale topography and softness analysis on growth cone central domain, filopodia and overlying lamellopodium (veil) are presented. The subcomponents of the growth cones of different nerve cells are compared to each other. The results of the analysis are linked to the mechanical properties and internal molecular density distribution of the growth cones.

  20. 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.

  1. AFM-porosimetry: density and pore volume measurements of particulate materials.

    PubMed

    Sörensen, Malin H; Valle-Delgado, Juan J; Corkery, Robert W; Rutland, Mark W; Alberius, Peter C

    2008-06-01

    We introduced the novel technique of AFM-porosimetry and applied it to measure the total pore volume of porous particles with a spherical geometry. The methodology is based on using an atomic force microscope as a balance to measure masses of individual particles. Several particles within the same batch were measured, and by plotting particle mass versus particle volume, the bulk density of the sample can be extracted from the slope of the linear fit. The pore volume is then calculated from the densities of the bulk and matrix materials, respectively. In contrast to nitrogen sorption and mercury porosimetry, this method is capable of measuring the total pore volume regardless of pore size distribution and pore connectivity. In this study, three porous samples were investigated by AFM-porosimetry: one ordered mesoporous sample and two disordered foam structures. All samples were based on a matrix of amorphous silica templated by a block copolymer, Pluronic F127, swollen to various degrees with poly(propylene glycol). In addition, the density of silica spheres without a template was measured by two independent techniques: AFM and the Archimedes principle. PMID:18503284

  2. 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.

  3. Catalytic Scanning Probe Nanolithography (cSPL): Control of the AFM Parameters in Order to Achieve Sub-100-nm Spatially Resolved Epoxidation of Alkenes Grafted onto a Surface.

    PubMed

    Mesquita, Vincent; Botton, Julien; Valyaev, Dmitry A; François, Cyril; Patrone, Lionel; Balaban, Teodor Silviu; Abel, Mathieu; Parrain, Jean-Luc; Chuzel, Olivier; Clair, Sylvain

    2016-04-26

    Scanning probe lithography (SPL) appears to be a reliable alternative to the use of masks in traditional lithography techniques as it offers the possibility of directly producing specific chemical functionalities with nanoscale spatial control. We have recently extend the range of applications of catalytic SPL (cSPL) by introducing a homogeneous catalyst immobilized on the apex of a scanning probe. Here we investigate the importance of atomic force microscopy (AFM) physical parameters (applied force, writing speed, and interline distance) on the resultant chemical activity in this cSPL methodology through the direct topographic observation of nanostructured surfaces. Indeed, an alkene-terminated self-assembled monolayer (alkene-SAM) on a silicon wafer was locally epoxidized using a scanning probe tip with a covalently grafted manganese complex bearing the 1,4,7-triazacyclononane macrocycle as the ligand. In a post-transformation process, N-octylpiperazine was covalently grafted to the surface via a selective nucleophilic ring-opening reaction. With this procedure, we could write various patterns on the surface with high spatial control. The catalytic AFM probe thus appears to be very robust because a total area close to 500 μm(2) was patterned without any noticeable loss of catalytic activity. Finally, this methodology allowed us to reach a lower lateral line resolution down to 40 nm, thus being competitive and complementary to the other nanolithographical techniques for the nanostructuration of surfaces.

  4. Directed evolution of the CpcA biosynthetic pathway and optimization of conditions for CpcA production and its properties.

    PubMed

    Dong, Dalu; Pan, Hangtao; Yu, Ping

    2014-06-01

    To improve the production of phycocyanin holo-α-subunit (CpcA) from Spirulina maxima, five genes and their spacer region sequences involved in its biosynthesis were subject to the directed evolution by error-prone PCR using the plasmid pETDuet-6 as the template. Mutants were screened by determining the CpcA yield in 96-well plates directly. A mutant strain CPCA713 with the highest CpcA yield of 17.36 mg/l in 96-well plates was obtained, and this yield was 29.7 % higher than that from the control strain ZJGSU09 containing pETDuet-6 (13.38 mg/l). Sequence alignments indicated that 10 nucleotides and 5 amino acids were mutated. Glycerol and beef extract were found to be the best carbon and nitrogen sources for accumulating CpcA in the screened CPCA713 strain, respectively. The concentrations of the key factors that affected the CpcA yield were optimized by response surface methodology with a Box-Behnken design and were as follows: glycerol, 16.0 g/l; yeast extract, 18.2 g/l; and beef extract, 4.8 g/l. Under the optimal conditions, the CpcA yield was up to 71.21 mg/l in the shake flask. Time-course of the CpcA production before and after optimization were performed and compared. After being purified by a Hi-Trap metal chelating affinity column loaded with 100 mM nickel sulfate, CpcA presented a single protein band with an estimated molecular weight of 29 kDa in the sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The purified CpcA had the maximal absorptive and fluorescent emission wavelengths at 623 and 650.8 nm, respectively, and was stable at temperatures of 40 °C below and pHs of 5.5-8.0, and in the dark or in the dim light. It had also a strong scavenging ability to three free radicals ·OH, ·O2 (-), and di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH). The IC50 values of ·OH, ·O2 (-), and DPPH free radicals by purified CpcA were 0.08, 0.46, and 0.48 mg/ml, respectively. This study lays a good foundation for the industrial

  5. Directed evolution of the CpcA biosynthetic pathway and optimization of conditions for CpcA production and its properties.

    PubMed

    Dong, Dalu; Pan, Hangtao; Yu, Ping

    2014-06-01

    To improve the production of phycocyanin holo-α-subunit (CpcA) from Spirulina maxima, five genes and their spacer region sequences involved in its biosynthesis were subject to the directed evolution by error-prone PCR using the plasmid pETDuet-6 as the template. Mutants were screened by determining the CpcA yield in 96-well plates directly. A mutant strain CPCA713 with the highest CpcA yield of 17.36 mg/l in 96-well plates was obtained, and this yield was 29.7 % higher than that from the control strain ZJGSU09 containing pETDuet-6 (13.38 mg/l). Sequence alignments indicated that 10 nucleotides and 5 amino acids were mutated. Glycerol and beef extract were found to be the best carbon and nitrogen sources for accumulating CpcA in the screened CPCA713 strain, respectively. The concentrations of the key factors that affected the CpcA yield were optimized by response surface methodology with a Box-Behnken design and were as follows: glycerol, 16.0 g/l; yeast extract, 18.2 g/l; and beef extract, 4.8 g/l. Under the optimal conditions, the CpcA yield was up to 71.21 mg/l in the shake flask. Time-course of the CpcA production before and after optimization were performed and compared. After being purified by a Hi-Trap metal chelating affinity column loaded with 100 mM nickel sulfate, CpcA presented a single protein band with an estimated molecular weight of 29 kDa in the sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The purified CpcA had the maximal absorptive and fluorescent emission wavelengths at 623 and 650.8 nm, respectively, and was stable at temperatures of 40 °C below and pHs of 5.5-8.0, and in the dark or in the dim light. It had also a strong scavenging ability to three free radicals ·OH, ·O2 (-), and di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH). The IC50 values of ·OH, ·O2 (-), and DPPH free radicals by purified CpcA were 0.08, 0.46, and 0.48 mg/ml, respectively. This study lays a good foundation for the industrial

  6. Simulation of CNT-AFM tip based on finite element analysis for targeted probe of the biological cell

    NASA Astrophysics Data System (ADS)

    Yousefi, Amin Termeh; Mahmood, Mohamad Rusop; Miyake, Mikio; Ikeda, Shoichiro

    2016-07-01

    Carbon nanotubes (CNTs) are potentially ideal tips for atomic force microscopy (AFM) due to the robust mechanical properties, nano scale diameter and also their ability to be functionalized by chemical and biological components at the tip ends. This contribution develops the idea of using CNTs as an AFM tip in computational analysis of the biological cell's. Finite element analysis employed for each section and displacement of the nodes located in the contact area was monitored by using an output database (ODB). This reliable integration of CNT-AFM tip process provides a new class of high performance nanoprobes for single biological cell analysis.

  7. A Pathway to Ignition-Hydrodynamic-Equivalent Implosions in OMEGA Direct Drive Through the Reduction of Cross-Beam Energy Transfer

    NASA Astrophysics Data System (ADS)

    Froula, D. H.; Fiksel, G.; Goncharov, V. N.; Hu, S. X.; Huang, H.; Igumenshchev, I. V.; Kessler, T. J.; Meyerhofer, D. D.; Michel, D. T.; Sangster, T. C.; Shvydky, A.; Zuegel, J. D.

    2014-10-01

    Cross-beam energy transfer (CBET) in OMEGA cryogenic ignition-hydrodynamic-equivalent designs reduces the ablation pressure from 230 Mbar to 140 Mbar. To maintain an ignition-relevant velocity of 3 . 7 ×107 cm/s, areal density of 300 mg/cm2, and hot-spot pressure greater than 100 Gbar on OMEGA, this reduction in ablation pressure requires that the mass of the shell and the adiabat be reduced by 75% and 50%, respectively. Measurements indicate these implosions are hydrodynamically unstable. To improve the stability, the thickness of the shell (target mass) and the adiabat can be increased while maintaining relevant conditions when reducing CBET. To mitigate CBET, several methods to reduce the diameter of the laser beams while maintaining acceptable drive uniformity are being investigated for OMEGA: (1) direct reduction of the laser spots over the entire laser pulse and (2) reduction of the diameter of the laser spots after a sufficient conduction zone has been generated. This two-state zooming is predicted to maintain low-mode uniformity while mitigating CBET. This material is based upon work supported by the Department of Energy National Nuclear Security Administration under Award Number DE-NA0001944.

  8. Meta-Analysis of Arabidopsis KANADI1 Direct Target Genes Identifies a Basic Growth-Promoting Module Acting Upstream of Hormonal Signaling Pathways1[OPEN

    PubMed Central

    Xie, Yakun; Straub, Daniel; Eguen, Tenai; Brandt, Ronny; Stahl, Mark; Martínez-García, Jaime F.; Wenkel, Stephan

    2015-01-01

    An intricate network of antagonistically acting transcription factors mediates the formation of a flat leaf lamina of Arabidopsis (Arabidopsis thaliana) plants. In this context, members of the class III homeodomain leucine zipper (HD-ZIPIII) transcription factor family specify the adaxial domain (future upper side) of the leaf, while antagonistically acting KANADI transcription factors determine the abaxial domain (future lower side). Here, we used a messenger RNA sequencing approach to identify genes regulated by KANADI1 (KAN1) and subsequently performed a meta-analysis combining our data sets with published genome-wide data sets. Our analysis revealed that KAN1 acts upstream of several genes encoding auxin biosynthetic enzymes. When exposed to shade, we found three YUCCA genes, YUC2, YUC5, and YUC8, to be transcriptionally up-regulated, which correlates with an increase in the levels of free auxin. When ectopically expressed, KAN1 is able to transcriptionally repress these three YUC genes and thereby block shade-induced auxin biosynthesis. Consequently, KAN1 is able to strongly suppress shade-avoidance responses. Taken together, we hypothesize that HD-ZIPIII/KAN form the basis of a basic growth-promoting module. Hypocotyl extension in the shade and outgrowth of new leaves both involve auxin synthesis and signaling, which are under the direct control of HD-ZIPIII/KAN. PMID:26246448

  9. 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;