Atomic force microscopy: from cell imaging to molecular manipulation

The atomic force microscope (AFM) allows to explore the surface of biological samples bathed in physiological solutions, with vertical and horizontal resolutions ranging from nanometers to angstr?ms. Complex biological structures as well as single molecules can be observed and recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra and intermolecular forces from single molecules are also presented.     

Statocysts in crabs: short-term control of locomotion and long-term monitoring of hydrostatic pressure

Crabs show well-coordinated locomotion. They have proprioceptors similar to those of lobsters, but they differ in terms of their balancing systems and their condensed nervous system, which allows rapid interganglionic conduction. Typically they exhibit dynamically stable locomotion with a highly developed semicircular canal system that codes angular acceleration in each of three orthogonal planes (horizontal and vertical at 45 degrees and 135 degrees to the pitching plane). Left and right interneurons each code one direction of angular acceleration, carrying information between the brain and the thoracic ganglia. Cell A codes head-up vertical plane angular accelerations. Cell B codes rotations in the horizontal plane. Interneurons C and D code headdown vertical plane information, carrying it ipsilaterally and contralaterally respectively. These interneurons have a central role in locomotion. They are activated and have their responsiveness to angular acceleration enhanced before and during locomotion. Such simple activation pathways point to how an angular-acceleration-controlled robot (CRABOT) could be constructed. Hydrostatic pressure information carried by the thread hairs, which also sense angular acceleration, is filtered out from direct pathways onto the interneurons, but spectral analysis shows that it still has an influence via central pathways. Long-term recordings from equilibrium interneurons in free-walking crabs taken from the wild into constant conditions show tidally changing frequencies     

A single-degree-of-freedom dynamic model predicts the range of human responses to impulsive forces produced by power hand tools

The human operator is modelled as a single-degree-of-freedom dynamic mechanical system for predicting the response to impulsive torque reaction forces produced by rotating spindle power hand tools such as nutrunners or screwdrivers. The model uses mass, spring and damping elements to represent the standing operator supporting the tool in the hand. It was hypothesized that these mechanical elements are affected by work location and vary among individuals. These elements were ascertained by measuring the resulting frequency and amplitude of a freely oscillating defined mechanical system when externally loaded using maximal effort to oppose its motion. Twenty-five subjects (13 female, 12 male) participated in the full factorial experiment that measured the effects of gender, vertical and horizontal work location for various tool shapes (in-line, pistol, right angle), and orientations (horizontal and vertical). The mean operator stiffness decreased from 1721 to 1195 N/m when the horizontal work location increased from 30 to 90 cm in front of the ankles for a pistol-grip handle used on a vertical surface. Males had greater mass moment of inertia of (0.0099 kg m²) than females (0.0072 kg m²) for an in-line handle used on a horizontal surface. Internal validation by independently measuring apparatus torque found that the model satisfactorily explained the measured operator dynamics with an average error of 2.86%. Group variance reflects the range of operator capacities to react against power hand tool generated forces for the sample group and therefore it may also be useful for understanding the range of capacities among a group of operators performing similar tasks.     

Variation in petiole and internode length affects plant performance in <Emphasis Type="Italic">Trifolium repens</Emphasis> under opposing selection regimes

We studied the effects of genotypic and plastic variation in vertical and horizontal spacer lengths on plant performance in a stoloniferous herb subjected to opposing selection regimes. We hypothesized that longer vertical structures are beneficial if plants are subjected to competition, but they should negatively affect plant performance if plants are exposed to aboveground disturbance. To test these hypotheses we subjected 34 genotypes of Trifolium repens to competition and disturbance treatments. Competition was imposed by a grass canopy consisting of Lolium perenne, and disturbance was simulated by regularly clipping the target plants and all the surrounding vegetation at 1 cm above soil level. Conform to our hypothesis, genotypes with longer vertical structures (petioles) produced fewer ramets than genotypes with shorter petioles in the disturbance treatment. However, genotypes with longer petioles did not perform better under competition than genotypes with shorter petioles. Genotypes with highly plastic vertical structures tended to produce more shoot mass under competition, and they produced fewer ramets if subjected to disturbance. Unexpectedly, horizontal structures (stolon internodes) expanded in response to competition which, furthermore, was associated with enhanced plant performance. However, producing longer internodes is inherently associated with costs in terms of increased resource allocation to the longer structures, but not to benefits in terms of increased resource capture. Positive correlations among the length and plasticity of vertical and horizontal structures may explain the apparent positive effect of producing longer internodes on plant performance. Our data thus support the notion that trait correlations may weaken selective forces acting on a focal trait in a specific environment if opposing selection pressures act on genetically correlated traits.     

The fine structure of the pupal plastron of simuliid flies

The outer surface of the plastron of the pupal spiracular gills of the Simuliidae was previously thought to be the network formed by the apical branches of the vertical struts. It is now shown that this horizontal network supports a thin laminate outer epicuticle and also a fine meshwork, both of which have been previously overlooked. A simple method is described for quickly producing three-dimensional micrographs of great depth with the transmission electron microscope.     

Comparing the invasibility of experimental "reefs" with field observations of natural reefs and artificial structures

Natural systems are increasingly being modified by the addition of artificial habitats which may facilitate invasion. Where invaders are able to disperse from artificial habitats, their impact may spread to surrounding natural communities and therefore it is important to investigate potential factors that reduce or enhance invasibility. We surveyed the distribution of non-indigenous and native invertebrates and algae between artificial habitats and natural reefs in a marine subtidal system. We also deployed sandstone plates as experimental 'reefs' and manipulated the orientation, starting assemblage and degree of shading. Invertebrates (non-indigenous and native) appeared to be responding to similar environmental factors (e.g. orientation) and occupied most space on artificial structures and to a lesser extent reef walls. Non-indigenous invertebrates are less successful than native invertebrates on horizontal reefs despite functional similarities. Manipulative experiments revealed that even when non-indigenous invertebrates invade vertical "reefs", they are unlikely to gain a foothold and never exceed covers of native invertebrates (regardless of space availability). Community ecology suggests that invertebrates will dominate reef walls and algae horizontal reefs due to functional differences, however our surveys revealed that native algae dominate both vertical and horizontal reefs in shallow estuarine systems. Few non-indigenous algae were sampled in the study, however where invasive algal species are present in a system, they may present a threat to reef communities. Our findings suggest that non-indigenous species are less successful at occupying space on reef compared to artificial structures, and manipulations of biotic and abiotic conditions (primarily orientation and to a lesser extent biotic resistance) on experimental "reefs" explained a large portion of this variation, however they could not fully explain the magnitude of differences.     

Comparison of horizontal and vertical constructed wetland systems for landfill leachate treatment

The main purpose of this study was to treat organic pollution, ammonia and heavy metals present in landfill leachate by the use of constructed wetland systems and to quantify the effect of feeding mode. The effect of different bedding material (gravel and zeolite surface) was also investigated. A pilot-scale study was conducted on subsurface flow constructed wetland systems operated in vertical and horizontal mode. Two vertical systems differed from each other with their bedding material. The systems were planted with cattail (Typha latifolia) and operated identically at a flow rate of 10 l/day and hydraulic retention times of 11.8 and 12.5 day in vertical 1, vertical 2 and horizontal systems, respectively. Concentration based average removal efficiencies for VF1, VF2 and HF were NH₄–N, 62.3%, 48.9% and 38.3%; COD, 27.3%, 30.6% and 35.7%; PO₄–P, 52.6%, 51.9% and 46.7%; Fe(III), 21%, 40% and 17%, respectively. Better NH₄–N removal performance was observed in the vertical system with zeolite layer than that of the vertical 2 and horizontal system. In contrast, horizontal system was more effective in COD removal.     

Insights into the adaptive significance of vertical pupil shape in snakes

Pupil shape in vertebrates ranges from circular to vertical, with multiple phylogenetic shifts in this trait. Our analyses challenge the widely held view that the vertical pupil evolved as an adaptation to enhance night vision. On functional grounds, a variable‐aperture vertical pupil (i) allows a nocturnal species to have a sensitive retina for night vision but avoid dazzle by day by adjusting pupil closure, and (ii) increases visual acuity by day, because a narrow vertical pupil can project a sharper image onto the retina in the horizontal plane. Detection of horizontal movement may be critical for predators that wait in ambush for moving prey, suggesting that foraging mode (ambush predation) as well as polyphasic activity may favour the evolution of vertical pupil shape. Camouflage (disruption of the circular outline of the eye) also may be beneficial for ambush predators. A comparative analysis in snakes reveals significant functional links between pupil shape and foraging mode, as well as between pupil shape and diel timing of activity. Similar associations between ambush predation and vertically slit pupils occur in lizards and mammals also, suggesting that foraging mode has exerted major selective forces on visual systems in vertebrates.     

How do low horizontal forces produce disproportionately high torques in human locomotion?

Although horizontal ground forces are only approximately 15% of vertical forces, they account for 47% and 33% of the metabolic cost in walking and running. To explain these disproportionately high metabolic costs, we hypothesized that low horizontal ground forces generate relatively high torques on body segments during locomotion and this is mediated by long moment arms. We compared external force moment arms and discreet torques applied to the body segments by horizontal and vertical forces during walking and running. Sixteen subjects (21.9+/-1.9 years) walked at 1.5m/s and ten subjects (23.2+/-2.0 years) ran at 3.83 m/s. Segmental torques in the sagittal plane were partitioned into components due to horizontal and vertical forces and quantified by their angular impulses. The mean (+/-S.E.) ratios of horizontal to vertical ground forces (GF ratio) and angular impulses (AI ratio) in walking were 0.131 (+/-0.003, 95% confidence interval (CI) 0.124-0.137) and 0.530 (+/-0.018, CI 0.497-0.569). Results were similar in running. In both gaits the AI ratios were significantly greater than the GF ratios because the respective CI's did not overlap. The horizontal forces produced 53% and 41% as much angular impulse on the body segments, as did the vertical forces in walking and running despite being only 13% as large. In the two movements the moment arms for the horizontal forces averaged across foot, leg, thigh, and trunk body segments were 3.8 fold larger than those for the vertical forces. The data supported the hypothesis and suggest that the relatively low horizontal vs. vertical forces accounted for a disproportionately higher percentage of the angular impulses placed on the body segments and this effect was due to relatively long moment arms for horizontal forces. These results partially explain the relatively large metabolic cost of generating relatively low horizontal forces.     

On Heels and Toes: How Ants Climb with Adhesive Pads and Tarsal Friction Hair Arrays

Ants are able to climb effortlessly on vertical and inverted smooth surfaces. When climbing, their feet touch the substrate not only with their pretarsal adhesive pads but also with dense arrays of fine hairs on the ventral side of the 3^rd and 4^th tarsal segments. To understand what role these different attachment structures play during locomotion, we analysed leg kinematics and recorded single-leg ground reaction forces in Weaver ants (Oecophylla smaragdina) climbing vertically on a smooth glass substrate. We found that the ants engaged different attachment structures depending on whether their feet were above or below their Centre of Mass (CoM). Legs above the CoM pulled and engaged the arolia (‘toes’), whereas legs below the CoM pushed with the 3^rd and 4^th tarsomeres (‘heels’) in surface contact. Legs above the CoM carried a significantly larger proportion of the body weight than legs below the CoM. Force measurements on individual ant tarsi showed that friction increased with normal load as a result of the bending and increasing side contact of the tarsal hairs. On a rough sandpaper substrate, the tarsal hairs generated higher friction forces in the pushing than in the pulling direction, whereas the reverse effect was found on the smooth substrate. When the tarsal hairs were pushed, buckling was observed for forces exceeding the shear forces found in climbing ants. Adhesion forces were small but not negligible, and higher on the smooth substrate. Our results indicate that the dense tarsal hair arrays produce friction forces when pressed against the substrate, and help the ants to push outwards during horizontal and vertical walking.     

Contribution of microbial mats to sedimentary surface structures

Summary This paper summarizes studies of sedimentary surface structures in which microbial mats play a role. Intertidal/supratidal transitions of tidal flats of the North Sea coast, and shallow hypersaline water bodies of salterns (Bretagne, Canary and Balearic Islands), and Gavish Sabkha (Sinai) reveal a multitude of sedimentary surface structures which can be grouped and primary biologically controlled structures. Physically controlled surface structures include shrinkage cracks, erosion marks, deformation structures caused by water friction, gas pressure and mineral encrustation. Shrinkage cracks in microbial mats reveal the following features: (i) horizontally arranged cauliflower pattern that differs from the usually orthogonally regular crack morphology in clay, (ii) rounded edges and pillow-like thickening along the crack edges, caused by the growth of mats into the cracks. Criteria of erosion are pocket-like depressions and ripple marks on the thus exposed non-stabilized sand, and residual stacks of microbial mats. Deformation structures are due to water friction causing flotation of loosely attached microbial mats which fold and tear. Gas migration from deeper layers causes domal upheaval, protuberance structures, folds and “fairy rings”. Protuberance structures are caused by the rupture of gas domes and rapid escape of the enclosed gas. The sudden drop of pressure forces sediment to well up from below through the gas channels and to fill the internal hollow spaces of the domes. “Fairy rings” are horizontal ringshaped structures. Their center is the exit point of gas bubbles which escape from the substrate into the shallow water. The bubbles generate concentric waves which cause displacement of fine muddy sediments at the sediment-water interface Such gradual displacement guides mat-constructing microbes to grow concentrically. The “fairy rings” are crowned by pinnacle structures of bacterial and diatom origin. Pinnacles, “fairy rings” and pillow-like coatings of crack margins are biogenic structures which have to be genetically separated from purely physically controlled structures.     

An in vitro study of implant--tooth-supported connections using a robot test system.

Many unsolved problems in dental implant research concern the interfacial stress distributions between the implant components, as well as between the implant surface and contacting bone. To obtain a mechanical understanding of how vertical and horizontal occlusal forces are distributed in this context, it is crucial to develop in vitro testing systems to measure the force transmission between dental implants and attached prostheses. A new approach to such testing, involving a robotic system, is described in this investigation. The system has been designed to produce simulated mandibular movements and occlusal contact forces so that various implant designs and procedures can be thoroughly tested and evaluated before animal testing or human clinical trials. Two commonly used fixed prosthesis designs used to connect an implant and a tooth, a rigid connection and a nonrigid connection, were fabricated and used for experimental verification. The displacement and force distributions generated during simulated chewing activities were measured in vitro. Force levels, potentially harmful to human bone surrounding the connected dental implant and tooth, were analyzed. These results are useful in the design of prostheses and connecting components that will reduce failures and limit stress transfer to the implant/bone interface.     

Effects of Pendular Waist on Gecko's Climbing: Dynamic Gait,Analytical Model and Bio-inspired Robot

Most quadruped reptiles,such as lizards,salamanders and crocodiles,swing their waists while climbing on horizontal or vertical surfaces.Accompanied by body movement,the centroid trajectory also becomes more of a zigzag path rather than a straight line.Inspired by gecko's gait and posture on a vertical surface,a gecko inspired model with one pendular waist and four active axil legs,which is called GPL model,is proposed.Relationship between the waist position,dynamic gait,and driving forces on supporting feet is analyzed.As for waist trajectory planning,a singular line between the supporting feet is found and its effects on driving forces are discussed.Based on the GPL model,it is found that a sinusoidal waist trajectory,rather than a straight line,makes the driving forces on the supporting legs smaller.Also,a waist close to the pygal can reduce the driving forces compared to the one near middle vertebration,which is in accord with gecko's body bending in the process of climbing.The principles of configuration design and gait planning are proposed based on theoretical analyses.Finally,a bio-inspired robot DracoBot is developed and both of the driving force measurements and climbing experiments reinforce theoretical analysis and the rationality of gecko's dynamic gait.     

Surface organelles assembled by secretion systems of Gram-negative bacteria: diversity in structure and function

Gram-negative bacteria express a wide variety of organelles on their cell surface. These surface structures may be the end products of secretion systems, such as the hair-like fibers assembled by the chaperone/usher (CU) and type IV pilus pathways, which generally function in adhesion to surfaces and bacterial-bacterial and bacterial-host interactions. Alternatively, the surface organelles may be integral components of the secretion machinery itself, such as the needle complex and pilus extensions formed by the type III and type IV secretion systems, which function in the delivery of bacterial effectors inside host cells. Bacterial surface structures perform functions critical for pathogenesis and have evolved to withstand forces exerted by the external environment and cope with defenses mounted by the host immune system. Given their essential roles in pathogenesis and exposed nature, bacterial surface structures also make attractive targets for therapeutic intervention. This review will describe the structure and function of surface organelles assembled by four different Gram-negative bacterial secretion systems: the CU pathway, the type IV pilus pathway, and the type III and type IV secretion systems.     

Cortical bone failure mechanisms during screw pullout

An experimental and computational study of screw pullout from cortical bone has been conducted. A novel modification of standard pullout tests providing real time image capture of damage mechanisms during screw pullout was developed. Pullout forces, measured using the novel test rig, have been validated against standard pullout tests. Pullout tests were conducted, considering osteon alignment, to investigate the effect of osteons aligned parallel to the axis of the orthopaedic screw (longitudinal pullout) as well as the effect of osteons aligned perpendicular to the axis of the screw (transverse pullout). Distinctive alternate failure mechanisms, for longitudinally and transversely orientated cortical bone during screw pullout, were uncovered. Vertical crack propagation, parallel to the axis of the screw, was observed for a longitudinal pullout. Horizontal crack propagation, perpendicular to the axis of the screw, was observed for a transverse pullout. Finite element simulation of screw pullout, incorporating material damage and crack propagation, was also performed. Simulations revealed that a homogenous material model for cortical bone predicts vertical crack propagation patterns for both longitudinal and transverse screw pullout. A bi-layered composite model representing cortical bone microstructure was developed. A unique set of material and damage properties was used for both transverse and longitudinal pullout simulations, with only layer orientations being changed. Simulations predicted: (i) higher pullout forces for transverse pullout; (ii) horizontal crack paths perpendicular to screw axis for transverse pullout, whereas vertical crack paths were computed for longitudinal pullout. Computed results agreed closely with experimental observations in terms of pullout force and crack propagation.     

The role of cutaneous feedback for anticipatory grip force adjustments during object movements and externally imposed variation of the direction of gravity

Grip force adjustments to changes of object loading induced by external changes of the direction of gravity during discrete arm movements with a grasped object were analyzed during normal and anesthetized finger sensibility. Two subjects were seated upright in a rotatable chair and rotated backwards into a horizontal position during discrete movements with a hand-held instrumented object. The movement direction varied from vertical to horizontal inducing corresponding changes in the direction of gravity, but the orientation of the movement in relation to the body remained unaffected. During discrete vertical movements a maximum of load force occurs early in upward and late in downward movements; during horizontal movements two load force peaks result from both acceleratory and deceleratory phases of the movement. During performance with normal finger sensibility grip force was modulated in parallel with fluctuations of load force during vertical and horizontal movements. The grip force profile adopted to the varying load force profile during the transition from the vertical to the horizontal position. The maximum grip force occurred at the same time of maximum load force irrespective of the movement plane. During both subjects' first experience of digital anesthesia the object slipped from the grasp during rotation to the horizontal plane. During the following trials with anesthetized fingers subjects substantially increased their grip forces, resulting in elevated force ratios between maximum grip and load force. However, grip force was still modulated with the movement-induced load fluctuations and maximum grip force coincided with maximum load force during vertical and horizontal movements. This implies that the elevated force ratio between maximum grip and load force does not alter the feedforward system of grip force control. Cutaneous afferent information from the grasping digits seems to be important for the economic scaling of the grip force magnitude according to the actual loading conditions and for reactive grip force adjustments in response to load perturbations. However, it plays a subordinate role for the precise anticipatory temporal coupling between grip and load forces during voluntary object manipulation.     

Coupling between elytra of some beetles: Mechanism, forces and effect of surface texture

Lightweight materials, structures and coupling mechanisms are very important for realizing advanced flight vehicles. Here, we obtained the geometric structures and morphologies of the elytra of beetles and ascertained its coupling zone by using the histological section technique and SEM. We set up a three-dimensional motion observing system to monitor the opening and closing behaviour of elytra in beetles and to determine the motion mechanism. We constructed a force measuring system to measure the coupling forces between elytra. The results show that elytra open and close by rotating about a single axle, where the coupling forces may be as high as 160 times its own bodyweight, the elytra coupling with the tenon and mortise mechanism, surface texture and opening angle between elytra heavily influence the coupling forces. These results may provide insights into the design mechanism and structure for future vehicles of flight.     

Why bones bend but don't break

The musculoskeletal system is adept at dissipating potentially damaging energy that could accelerate fracture consequent to multiple loading cycles. Microstructural damage reduces bone's residual properties, but prevents high stresses within the material by dissipating energy that can lead to eventual failure. Thus skeletal microdamage can be viewed as an adaptive process to prevent bone failure by dissipating energy. Because a damaged bone has reduced strength and stiffness, it must be repaired, so bone has evolved a system of self-repair that relies on microdamage-stimulated signaling mechanisms. When repair cannot occur quickly enough, low energy stress fractures can occur. The regulating effects of muscle also prevent failure by controlling where high stresses occur. Acting synergistically, muscle forces dissipate energy by appropriately regulating accelerations and decelerations of the limbs during movement. When muscles become fatigued, these functions are constrained, larger amounts of energy are imparted to bone, increasing the likelihood of microstructural damage and fracture. Thus, healthy bones are maintained by the ability of the musculoskeletal system to dissipate the energy through synergistic muscular activity and through the maintenance of microstructural and material properties that allow for crack initiation, but also for their repair.     

The interplay between speed,kinetics, and hand postures during primate terrestrial locomotion

Nonprimate terrestrial mammals may use digitigrade postures to help moderate distal limb joint moments and metapodial stresses that may arise during high‐speed locomotion with high‐ground reaction forces (GRF). This study evaluates the relationships between speed, GRFs, and distal forelimb kinematics in order to evaluate if primates also adopt digitigrade hand postures during terrestrial locomotion for these same reasons. Three cercopithecine monkey species (Papio anubis, Macaca mulatta, Erythrocebus patas) were videotaped moving unrestrained along a horizontal runway instrumented with a force platform. Three‐dimensional forelimb kinematics and GRFs were measured when the vertical force component reached its peak. Hand posture was measured as the angle between the metacarpal segment and the ground (MGA). As predicted, digitigrade hand postures (larger MGA) are associated with shorter GRF moment arms and lower wrist joint moments. Contrary to expectations, individuals used more palmigrade‐like (i.e. less digitigrade) hand postures (smaller MGA) when the forelimb was subjected to higher forces (at faster speeds) resulting in potentially larger wrist joint moments. Accordingly, these primates may not use their ability to alter their hand postures to reduce rising joint moments at faster speeds. Digitigrady at slow speeds may improve the mechanical advantage of antigravity muscles crossing the wrist joint. At faster speeds, greater palmigrady is likely caused by joint collapse, but this posture may be suited to distribute higher GRFs over a larger surface area to lower stresses throughout the hand. Thus, a digitigrade hand posture is not a cursorial (i.e. high speed) adaptation in primates and differs from that of other mammals. Am J Phys Anthropol 2010. © 2009 Wiley‐Liss, Inc.     

Maximum allowable force on a safety harness cable to discriminate a successful from a failed balance recovery

A safety harness system is essential to ensure participant safety in experiments at the threshold of balance recovery where avoiding a fall is not always possible. The purpose of this study was to propose a method to determine the maximum allowable force on a safety harness cable to discriminate a successful from a failed balance recovery. Data from 12 younger adults, who participated in experiments to determine the maximum forward lean angles that participants could be suddenly released from and still recover balance using three different limits on the number of steps, were used. For each participant, the coefficients of an asymptotic exponential regression, between the maximum vertical force on the safety harness cable and the initial lean angle at each trial, were evaluated by a least squares method. A proposed threshold for the maximum allowable vertical force of five force constants ensured that the initial lean angle reached 99% of its steady state value with respect to its initial value. It should thus discriminate well a successful (below the threshold) from a failed (above the threshold) balance recovery. Furthermore, although the amplitude of the horizontal forces should not be neglected in safety harness system designs, the contributions of the medial–lateral and anterior–posterior forces can be neglected in experiments at the threshold of balance recovery. Finally, although our five force constants method could be used, the actual value obtained for the maximum allowable vertical force may vary with other safety harness systems and postural perturbations.