Effect of Supporting Substrates on the Structure of DNA and DNA–Trivaline Complexes Studied by Atomic Force Microscopy

Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA–TV complexes onto this surface. On mica, both purified DNA and DNA–TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA–TV displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.     

Nano-mechanical Compliance of Müller Cells Investigated by Atomic Force Microscopy

It has been known that a single Müller cell displays a large variation in the cytoskeletal compositions along its cell body, suggesting different mechanical properties in different segments. Müller cells are thought to be involved in many retinal diseases such as retinoschisis, which can be facilitated by a mechanical stress. Thus, mapping of mechanical properties on localized nano-domains of Müller cells could provide essential information for understanding their structural functions in the retina and roles in their pathological progresses. Using Atomic Force Microscopy (AFM) - based bio-nano-mechanics, we have investigated the local variations of the mechanical properties of Müller cells in vitro. We have a particular interest in identifying elastic moduli in regions closer to three distinctive segments of the cells - process, endfoot, and soma. Using the modified spherical AFM probes, we were able to accurately determine mechanical properties, i.e., elastic moduli from the obtained force-distance curves. We found that the regions closer to soma were mechanically more compliant than regions closer to endfoot and process of Müller cells. We found that this lateral heterogeneity of the mechanical compliance within a single Müller cell is consistent with reports from other cell types. The local variation in mechanical compliances along a single Müller cell may support their diverse mechanical functions in the retina such as a soft mechanical embedding, mechanosensing, and neurotrophic functions for neurons.     

Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

The stability of aptamer–ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans–cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant K_d ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer–target interactions in this case, the stability of the ligand-free aptamer—containing G-quadruplexes—is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar K_d were identified.     

Nanopore Force Spectroscopy of Aptamer–Ligand Complexes

The stability of aptamer–ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans–cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant K_d ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer–target interactions in this case, the stability of the ligand-free aptamer—containing G-quadruplexes—is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar K_d were identified.     

Stoichiometry and Subunit Arrangement of α1β Glycine Receptors As Determined by Atomic Force Microscopy

The glycine receptor is an anion-permeable member of the Cys-loop ion channel receptor family. Synaptic glycine receptors predominantly comprise pentameric α1β subunit heteromers. To date, attempts to define the subunit stoichiometry and arrangement of these receptors have not yielded consistent results. Here we introduced FLAG and six-His epitopes into α1 and β subunits, respectively, and imaged single antibody-bound α1β receptors using atomic force microscopy. This permitted us to infer the number and relative locations of the respective subunits in functional pentamers. Our results indicate an invariant 2α1:3β stoichiometry with a β-α-β-α-β subunit arrangement.     

The topography of soft,adhesive diatom ‘trails’ as observed by Atomic Force Microscopy

Gliding diatoms foul surfaces by leaving behind ‘trails’ of secreted mucilage. Atomic force microscopy (AFM) used in ‘fluid tapping’ mode enabled the topography of the soft, adhesive trails in the natural hydrated state to be imaged, and without the artefacts resulting from fixation and/or dehydration. Diatom trails consist of a continuous, swollen ridge of material that dominates the trail, as well as a diffuse hydrated mucilage coating observed on either side of the main trail. The main trail material is evenly attached to the coverslip along its entire length, and appears to cure, or become less soft/adhesive, over time. Diatom trails observed with the scanning electron microscope were severely damaged by dehydration, while trails imaged by the AFM in ‘contact’ mode were damaged and/or removed by the action of the cantilever. The AFM used in ‘fluid tapping’ mode is an excellent tool for topographical studies of soft/adhesive biological molecules in the hydrated state, and will have great value for measuring their physical and mechanical properties when operated in ‘force modulation’ mode.     

Microscopy basics and the study of actin–actin-binding protein interactions

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin–ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs.     

Calculation of the Affinity of the λ Repressor-Operator Complex Based on Free Energy Component Analysis

A calculation of the binding free energy of the u repressor-operator complex is described based on free energy component analysis. The calculations are based on a thermodynamic cycle of seven steps decomposed into a total of 24 individual components. The values of these terms are estimated using a combination of empirical potential functions from AMBER, generalized Born - solvent accessibility calculations, elementary statistical mechanics and semiempirical physicochemical properties. Two alternative approaches are compared, one based on the crystal structure of the complex and the other based on the molecular dynamics simulation of the u repressor-operator complex. The calculated affinity is m 19.7 kcal/mol from the crystal structure calculation and m 17.9 kcal/mol from the MD method. The corresponding experimental affinity of the complex is about m 12.6 kcal/mol, indicating reasonable agreement between theory and experiment, considering the approximations involved in the computational methodology. The results are analyzed in terms of contributions from electrostatics, van der Waal interactions, the hydrophobic effect and solvent release. The capabilities and limitations of free energy component methodology are assessed and discussed on the basis of these results.     

Dynamics of Inter-Molecular Interactions Between Single Aβ42 Oligomeric and Aggregate Species by High-Speed Atomic Force Microscopy

Within the amyloid hypothesis in Alzheimer's disease, current focus has shifted to earlier stages of amyloid beta (Aβ) peptide assembly, involving soluble oligomers and smaller aggregates, which are more toxic to cells compared to their morphological distinct fibril forms. Critical to the Aβ field is unlocking the molecular-level kinetic pathways of oligomerization, leading to the culprit subset or specific species of Aβ oligomer populations responsible for the disease etiology. Here, we apply high-speed atomic force microscopy to enable direct visualization of dynamic interactions between single Aβ₄₂ oligomers and aggregate forms, with combined nanometre structural and millisecond temporal resolution in liquid. Analysis of dimensions revealed up to three main Aβ₄₂ species distributions, in addition to the appearance of monomers that showed fast surface diffusion compared to the larger Aβ₄₂ species. Significantly, we devised a new single-molecule analysis based on image contrast in high-speed atomic force microscopy movies to quantify rate determining kinetic constants for interactions between the different Aβ₄₂ species. The findings revealed that smaller Aβ₄₂ species show an exponential decay of lifetime distribution, indicating that all molecules undergo the same process with a single well-defined energy barrier. In contrast, larger aggregates show randomized lifetimes, indicating a distribution of interactions energies/barriers that must be overcome in order to dissociate. We interpret the latter as being due to “permissive” binding, arising from different conformation states of the aggregates, along with a variety of accessible interacting groups. Inevitably, this may lead to the formation of different complexes or alloforms, which is known to contribute to difficulties in identifying Aβ oligomer toxicity and has implications for mechanisms underlying neuronal death accompanying Alzheimer's disease.     

Force–Velocity Curves of Motor Proteins Cooperating In Vivo

Motor proteins convert chemical energy into work, thereby generating persistent motion of cellular and subcellular objects. The velocities of motor proteins as a function of opposing loads have been previously determined in vitro for single motors. These single molecule “force–velocity curves” have been useful for elucidating motor kinetics and for estimating motor performance under physiological loads due to, for example, the cytoplasmic drag force on transported organelles. Here we report force–velocity curves for single and multiple motors measured in vivo. Using motion enhanced differential interference contrast (MEDIC) movies of living NT2 (neuron-committed teratocarcinoma) cells at 37°C, three parameters were measured—velocity (v), radius (a), and effective cytoplasmic viscosity (η′)—as they applied to moving vesicles. These parameters were combined in Stokes’ equation, F = 6πaη′v, to determine the force, F, required to transport a single intracellular particle at velocity, v. In addition, the number of active motors was inferred from the multimodal pattern seen in a normalized velocity histogram. Using this inference, the resulting in vivo force–velocity curve for a single motor agrees with previously reported in vitro single motor force–velocity curves. Interestingly, however, the curves for two and three motors lie significantly higher in both measured velocity and computed force, which suggests that motors can work cooperatively to attain higher transport forces and velocities. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Projection of the Mexican National Labor Force, 1980–2005

Abstract

Mexico has a large and rapidly growing labor force. This paper projects the Mexican national labor force from 1980 to 2005, with varying assumptions of vital rates, economic activity, and international migration. Projections are also made for the urban and rural components of the Mexican population, assuming inter‐component migration flows. Results indicate that the Mexican labor force will grow over the projection period at an average annual rate of 907,000 to 1,183,000 workers; will age slightly; and will have a much higher proportion female. Implications are discussed in terms of Mexican‐U.S. migration, possible agreements on free trade, and global trends in workforce.     

Study on Calceolarioside A Anti-Aβ25–35-Induced Damage in SH-SY5Y cells by Atomic Force Microscopy

Calceolarioside A is a phenylethyl glycoside compound, originally isolated from the bark of Fraxinus mandshurica Rupr. In this work, the protective effect of Calceolarioside A on beta Amyloid protein induced toxicity in SH-SY5Y cells was studied. DPPH experiment, MTT assay, SEM, Atomic force microscopy and Colony formation were used to study the activity of Calceolarioside A. The results in this article show that the survival rate of the cells with Calceolarioside A (20–40 mg/mL) was significantly higher than that of the model cells without Calceolarioside A. Calceolarioside A could protect SH-SY5Y cells by improving some parameters in cells, such as the cell height, Young's modulus, adhesion and branch. In summary, Calceolarioside A can reduce Aβ_25–35-induced damage in SH-SY5Y cells. It can be a potential medicine to treatment with AD.     

Form–meaning links in the development of visual word recognition

Learning to read takes time and it requires explicit instruction. Three decades of research has taught us a good deal about how children learn about the links between orthography and phonology during word reading development. However, we have learned less about the links that children build between orthographic form and meaning. This is surprising given that the goal of reading development must be for children to develop an orthographic system that allows meanings to be accessed quickly, reliably and efficiently from orthography. This review considers whether meaning-related information is used when children read words aloud, and asks what we know about how and when children make connections between form and meaning during the course of reading development.     

Force–length relation of skeletal muscles: from sarcomeres to myofibril

Sarcomeres are building blocks of skeletal muscles. Given force–length relations of sarcomeres serially connected in a myofibril, the myofibril force–length relation can be uniquely determined. Necessary and sufficient conditions are derived for capability of fully lengthening or completely shortening a myofibril under isometric, eccentric or concentric contraction, and for the myofibril force–length relation to be a continuous single-valued function. Intriguing phenomena such as sarcomere force–length hysteresis and myofibril regularity are investigated and their important roles in determining myofibril force–length relations are explored. The theoretical analysis leads to experimentally verifiable predictions on myofibril force–length relations. For illustration, simulated force–length relations of a myofibril portion consisting of a sarcomere pair are presented.     

Aggregation of A?(25-35) on DOPC and DOPC/DHA Bilayers: An Atomic Force Microscopy Study

β amyloid peptide plays an important role in both the manifestation and progression of Alzheimer disease. It has a tendency to aggregate, forming low-molecular weight soluble oligomers, higher-molecular weight protofibrillar oligomers and insoluble fibrils. The relative importance of these single oligomeric-polymeric species, in relation to the morbidity of the disease, is currently being debated. Here we present an Atomic Force Microscopy (AFM) study of Aβ(25–35) aggregation on hydrophobic dioleoylphosphatidylcholine (DOPC) and DOPC/docosahexaenoic 22∶6 acid (DHA) lipid bilayers. Aβ(25–35) is the smallest fragment retaining the biological activity of the full-length peptide, whereas DOPC and DOPC/DHA lipid bilayers were selected as models of cell-membrane environments characterized by different fluidity. Our results provide evidence that in hydrophobic DOPC and DOPC/DHA lipid bilayers, Aβ(25-35) forms layered aggregates composed of mainly annular structures. The mutual interaction between annular structures and lipid surfaces end-results into a membrane solubilization. The presence of DHA as a membrane-fluidizing agent is essential to protect the membrane from damage caused by interactions with peptide aggregates; to reduces the bilayer defects where the delipidation process starts.     

Adrenomedullin Improves the Blood–Brain Barrier Function Through the Expression of Claudin-5

Summary 1. Aims: Brain vascular endothelial cells secret Adrenomedullin (AM) has multifunctional biological properties. AM affects cerebral blood flow and blood–brain barrier (BBB) function. We studied the role of AM on the permeability and tight junction proteins of brain microvascular endothelial cells (BMEC).2. Methods: BMEC were isolated from rats and a BBB in vitro model was generated. The barrier functions were studied by measuring the transendothelial electrical resistance (TEER) and the permeability of sodium fluorescein and Evans’ blue albumin. The expressions of tight junction proteins were analyzed using immunocytochemistry and immunoblotting.3. Results: AM increased TEER of BMEC monolayer dose-dependently. Immunocytochemistry revealed that AM enhanced the claudin-5 expression at a cell–cell contact site in a dose-dependent manner. Immunoblotting also showed an overexpression of claudin-5 in AM exposure.4.Conclusions: AM therefore inhibits the paracellular transport in a BBB in vitro model through claudin-5 overexpression.     

Epidemiology of HIV among US Air Force Military Personnel, 1996–2011

ObjectiveThe objectives of this study were to describe the epidemiology of HIV in the United States Air Force (USAF) from 1996 through 2011 and to assess whether socio-demographic characteristics and service-related mobility, including military deployments, were associated with HIV infection.MethodsWe conducted a retrospective cohort analysis of USAF personnel who were HIV-infected during the study period January 1, 1996 through December 31, 2011 and a matched case-control study. Cases were USAF personnel newly-diagnosed with HIV during the study period. Five randomly-selected HIV-uninfected controls were matched to each case by age, length of service, sex, race, service, component, and HIV test collection date. Socio-demographic and service-related mobility factors and HIV diagnosis were assessed using conditional logistic regression.ResultsDuring the study period, the USAF had 541 newly diagnosed HIV-infected cases. HIV incidence rate (per 100,000 person-years) among 473 active duty members was highest in 2007 (16.78), among black/ African-American USAF members (26.60) and those aged 25 to 29 years (10.84). In unadjusted analysis restricted to personnel on active duty, 10 characteristics were identified and considered for final multivariate analysis. Of these single (adjusted odds ratio [aOR], 8.15, 95% confidence interval [CI] 5.71–11.6) or other marital status (aOR 4.60, 95% CI 2.72–7.75), communications/ intelligence (aOR 2.57, 95% CI 1.84–3.60) or healthcare (aOR 2.07, 95% CI 1.28–3.35) occupations, and having no deployment in the past 2 years before diagnosis (aOR 2.02, 95% CI 1.47–2.78) conferred higher odds of HIV infection in adjusted analysis.ConclusionThe highest risk of HIV infection in the USAF was among young unmarried deployment-naïve males, especially those in higher risk occupation groups. In an era when worldwide military operations have increased, these analyses identified potential areas where targeted HIV prevention efforts may be beneficial in reducing HIV incidence in the USAF military population.     

Association of Renal Na,K-ATPase α-Subunit with the β- and γ-Subunits Based on Cryoelectron Microscopy

Na,K-ATPase transports Na(+) and K(+) across cell membranes and consists of alpha- and beta-subunits. Na,K-ATPase also associates with small FXYD proteins that regulate the activity of the pump. We have used cryoelectron microscopy of two-dimensional crystals including data to 8 A resolution to determine the three-dimensional (3-D) structure of renal Na,K-ATPase containing FXYD2, the gamma-subunit. A homology model for the alpha-subunit was calculated from a Ca(2+)-ATPase structure and used to locate the additional beta- and gamma-subunits present in the 3-D map of Na,K-ATPase. Based on the 3-D map, the beta-subunit is located close to transmembrane helices M8 and M10 and the gamma-subunit is adjacent to helices M2 and M9 of the alpha-subunit.     

Accuracy and precision of protein–ligand interaction kinetics determined from chemical shift titrations

NMR-monitored chemical shift titrations for the study of weak protein?Cligand interactions represent a rich source of information regarding thermodynamic parameters such as dissociation constants (K _D ) in the micro- to millimolar range, populations for the free and ligand-bound states, and the kinetics of interconversion between states, which are typically within the fast exchange regime on the NMR timescale. We recently developed two chemical shift titration methods wherein co-variation of the total protein and ligand concentrations gives increased precision for the K _D value of a 1:1 protein?Cligand interaction (Markin and Spyracopoulos in J Biomol NMR 53: 125?C138, 2012). In this study, we demonstrate that classical line shape analysis applied to a single set of ¹H?C¹⁵N 2D HSQC NMR spectra acquired using precise protein?Cligand chemical shift titration methods we developed, produces accurate and precise kinetic parameters such as the off-rate (k _off ). For experimentally determined kinetics in the fast exchange regime on the NMR timescale, k _off ?~?3,000?s^?1 in this work, the accuracy of classical line shape analysis was determined to be better than 5?% by conducting quantum mechanical NMR simulations of the chemical shift titration methods with the magnetic resonance toolkit GAMMA. Using Monte Carlo simulations, the experimental precision for k _off from line shape analysis of NMR spectra was determined to be 13?%, in agreement with the theoretical precision of 12?% from line shape analysis of the GAMMA simulations in the presence of noise and protein concentration errors. In addition, GAMMA simulations were employed to demonstrate that line shape analysis has the potential to provide reasonably accurate and precise k _off values over a wide range, from 100 to 15,000?s^?1. The validity of line shape analysis for k _off values approaching intermediate exchange (~100?s^?1), may be facilitated by more accurate K _D measurements from NMR-monitored chemical shift titrations, for which the dependence of K _D on the chemical shift difference (????) between free and bound states is extrapolated to ?????=?0. The demonstrated accuracy and precision for k _off will be valuable for the interpretation of biological kinetics in weakly interacting protein?Cprotein networks, where a small change in the magnitude of the underlying kinetics of a given pathway may lead to large changes in the associated downstream signaling cascade.