Complex Stability of Single Proteins Explored by Forced Unfolding Experiments

In the last decade atomic force microscopy has been used to measure the mechanical stability of single proteins. These force spectroscopy experiments have shown that many water-soluble and membrane proteins unfold via one or more intermediates. Recently, Li and co-workers found a linear correlation between the unfolding force of the native state and the intermediate in fibronectin, which they suggested indicated the presence of a molecular memory or multiple unfolding pathways (1). Here, we apply two independent methods in combination with Monte Carlo simulations to analyze the unfolding of α-helices E and D of bacteriorhodopsin (BR). We show that correlation analysis of unfolding forces is very sensitive to errors in force calibration of the instrument. In contrast, a comparison of relative forces provides a robust measure for the stability of unfolding intermediates. The proposed approach detects three energetically different states of α-helices E and D in trimeric BR. These states are not observed for monomeric BR and indicate that substantial information is hidden in forced unfolding experiments of single proteins.     

Habitat cues synergize to elicit chemically mediated landing behavior in a specialist phytophagous insect,the grape berry moth

Many phytophagous insects locate their host plant using mixtures of volatile compounds produced by the plant. A key behavior in the host location process that has been the focus of decades of behavioral research is optomotor anemotaxis. Another key step in host location is landing on (or near) the odor source. In previous work, rubber septa emitting a synthetic blend of volatiles extracted from young shoots of grape plants, Vitus spp. (Vitaceae), elicited equivalent levels of oriented upwind flight by female grape berry moths (GBM), Paralobesia viteana (Clemens) (Lepidoptera: Tortricidae), as did actual (control) grape shoots. However, in contrast to the shoots, females did not land on the odor source. In this study, we used flight tunnel assays to investigate the landing response of GBM females with respect to chemical and visual stimuli, as well as differences in relative humidity. When stimuli were presented individually, only the synthetic blend of host plant volatiles elicited equivalent levels of oriented upwind flight compared to the plants. Interestingly, wet cotton strips elicited low but consistent levels of upwind flight. In paired assays, only the synthetic blend paired with wet cotton strips elicited landing, although at significantly lower levels than that elicited by grape shoots. To achieve landing rates equivalent to live grape shoots, grape berry moth females required all three stimuli we tested: host odor cues, moisture, and visual cues simulating a grape shoot. These results suggest the cues have a synergistic effect, and that landing behavior requires complex sensory processing using multiple sensory inputs. Furthermore, these results suggest that moisture plays an important role in the host plant location process.     

Complex stability of single proteins explored by forced unfolding experiments

In the last decade atomic force microscopy has been used to measure the mechanical stability of single proteins. These force spectroscopy experiments have shown that many water-soluble and membrane proteins unfold via one or more intermediates. Recently, Li and co-workers found a linear correlation between the unfolding force of the native state and the intermediate in fibronectin, which they suggested indicated the presence of a molecular memory or multiple unfolding pathways (1). Here, we apply two independent methods in combination with Monte Carlo simulations to analyze the unfolding of alpha-helices E and D of bacteriorhodopsin (BR). We show that correlation analysis of unfolding forces is very sensitive to errors in force calibration of the instrument. In contrast, a comparison of relative forces provides a robust measure for the stability of unfolding intermediates. The proposed approach detects three energetically different states of alpha-helices E and D in trimeric BR. These states are not observed for monomeric BR and indicate that substantial information is hidden in forced unfolding experiments of single proteins.     

Evoked potentials in the brain of adolescents in norm and those with attention deficit during recognition of short-term acoustic stimuli

The effect of stimulus duration on auditory event-related potentials and performance of oddball task was studied in normal children and those with attention-deficit symptoms. Mismatch negativity was absent on presentation of short-term (11 ms) stimuli and present with longer stimuli (50 ms). The adolescents with deficit of attention performed much worse (errors of omission) with the short stimuli. The RT was significantly larger in subjects with attention-deficit with all types of tested stimulus duration. They also manifested a smaller P3b amplitude in response to task-relevant deviant stimuli and larger N2b peaks in response to the standard stimuli. It was possible to differentiate between the MMN and the N2b components owing to the fact that the MMN was absent with shorter stimuli. The findings suggest that there is a deficit in processing of sensory information at the cortical level in subjects with the attention-deficit symptoms.     

The effects of muscle stretching and shortening on isometric forces on the descending limb of the force-length relationship

The purpose of this study was to examine the effects of stretching and shortening on the isometric forces at different lengths on the descending limb of the force-length relationship. Cat soleus (N = 10) was stretched and shortened by various amounts on the descending limb of the force-length relationship, and the steady-state forces following these dynamic contractions were compared to the isometric forces at the corresponding muscle lengths. We found a shift of the force-length relationship to greater force values following muscle stretching, and to smaller force values following muscle shortening. Shifts in both directions critically depended on the magnitude of stretching/shortening and the final muscle length. We confirm recent findings that the steady-state isometric force following some stretch conditions clearly exceeded the maximal isometric forces at optimum muscle length, and that force enhancement was associated with an increase in the passive force, i.e., a passive force enhancement. When the passive force enhancement was subtracted from the total force enhancement, forces following stretch were always equal to or smaller than the isometric force at optimum muscle length. Together, these findings led to the conclusions: (a). that force enhancement is composed of an "active and a "passive" component; (b). that the "passive" component of force enhancement allows for forces greater than the maximal isometric forces at the muscle's optimum length; and (c). that force enhancement and force depression are critically affected by muscle length and stretch/shortening amplitude.     

Quantification of the Contact Area at the Head-Stem Taper Interface of Modular Hip Prostheses

Corrosion of modular taper junctions of hip implants may be associated with clinical failure. Taper design parameters, as well as the intraoperatively applied assembly forces, have been proposed to affect corrosion. Fretting corrosion is related to relative interface shear motion and fluid ingress, which may vary with contact force and area. It was hypothesised in this study that assembly forces modify the extent and distribution of the surface contact area at the taper interface between a cobalt chrome head and titanium stem taper with a standard threaded surface profile. Local abrasion of a thin gold coating applied to the stem taper prior to assembly was used to determine the contact area after disassembly. Profilometry was then used to assess permanent deformation of the stem taper surface profile. With increasing assembly force (500 N, 2000 N, 4000 N and 8000 N) the number of stem taper surface profile ridges in contact with the head taper was found to increase (9.2±9.3%, 65.4±10.8%, 92.8±6.0% and 100%) and the overall taper area in contact was also found to increase (0.6±0.7%, 5.5±1.0%, 9.9±1.1% and 16.1±0.9%). Contact was inconsistently distributed over the length of the taper. An increase in plastic radial deformation of the surface ridges (-0.05±0.14 μm, 0.1±0.14 μm, 0.21±0.22 μm and 0.96±0.25 μm) was also observed with increasing assembly force. The limited contact of the taper surface ridges at lower assembly forces may influence corrosion rates, suggesting that the magnitude of the assembly force may affect clinical outcome. The method presented provides a simple and practical assessment of the contact area at the taper interface.     

Mechanobiological dysregulation of the epidermis and dermis in skin disorders and in degeneration

During growth and development, the skin expands to cover the growing skeleton and soft tissues by constantly responding to the intrinsic forces of underlying skeletal growth as well as to the extrinsic mechanical forces from body movements and external supports. Mechanical forces can be perceived by two types of skin receptors: (1) cellular mechanoreceptors/mechanosensors, such as the cytoskeleton, cell adhesion molecules and mechanosensitive (MS) ion channels, and (2) sensory nerve fibres that produce the somatic sensation of mechanical force. Skin disorders in which there is an abnormality of collagen [e.g. Ehlers–Danlos syndrome (EDS)] or elastic (e.g. cutis laxa) fibres or a malfunction of cutaneous nerve fibres (e.g. neurofibroma, leprosy and diabetes mellitus) are also characterized to some extent by deficiencies in mechanobiological processes. Recent studies have shown that mechanotransduction is crucial for skin development, especially hemidesmosome maturation, which implies that the pathogenesis of skin disorders such as bullous pemphigoid is related to skin mechanobiology. Similarly, autoimmune diseases, including scleroderma and mixed connective tissue disease, and pathological scarring in the form of keloids and hypertrophic scars would seem to be clearly associated with the mechanobiological dysfunction of the skin. Finally, skin ageing can also be considered as a degenerative process associated with mechanobiological dysfunction. Clinically, a therapeutic strategy involving mechanoreceptors or MS nociceptor inhibition or acceleration together with a reduction or augmentation in the relevant mechanical forces is likely to be successful. The development of novel approaches such as these will allow the treatment of a broad range of cutaneous diseases.     

Development and design of a novel loading device for the investigation of bone adaptation around immediately loaded dental implants using the reindeer antler as implant bed

The assessment of the behavior of immediately loaded dental implants using biomechanical methods is of particular importance. The primary goal of this investigation is to optimize the function of the implants to serve for immediate loading. Animal experiments on reindeer antlers as a novel animal model will serve for investigation of the bone remodeling processes in the implant bed. The main interest is directed towards the time and loading-dependant behavior of the antler tissue around the implants. The aim and scope of this work was to design an autonomous loading device that has the ability to load an inserted implant in the antler with predefined occlusal forces for predetermined time protocols. The mechanical part of the device can be attached to the antler and is capable of cyclically loading the implant with forces of up to 100 N. For the calibration and testing of the loading device a biomechanical measuring system has been used. The calibration curve shows a logarithmic relationship between force and motor current and is used to control the force on the implant. A first test on a cast reindeer antler was performed successfully.     

Biomechanical model study of pelvic belt influence on muscle and ligament forces

Many patients with low back and/or pelvic girdle pain feel relief after application of a pelvic belt. External compression might unload painful ligaments and joints, but the exact mechanical effect on pelvic structures, especially in (active) upright position, is still unknown. In the present study, a static three-dimensional (3-D) pelvic model was used to simulate compression at the level of anterior superior iliac spine and the greater trochanter. The model optimised forces in 100 muscles, 8 ligaments and 8 joints in upright trunk, pelvis and upper legs using a criterion of minimising maximum muscle stress. Initially, abdominal muscles, sacrotuberal ligaments and vertical sacroiliac joints (SIJ) shear forces mainly balanced a trunk weight of 500N in upright position. Application of 50N medial compression force at the anterior superior iliac spine (equivalent to 25N belt tension force) deactivated some dorsal hip muscles and reduced the maximum muscle stress by 37%. Increasing the compression up to 100N reduced the vertical SIJ shear force by 10% and increased SIJ compression force with 52%. Shifting the medial compression force of 100N in steps of 10N to the greater trochanter did not change the muscle activation pattern but further increased SIJ compression force by 40% compared to coxal compression. Moreover, the passive ligament forces were distributed over the sacrotuberal, the sacrospinal and the posterior ligaments. The findings support the cause-related designing of new pelvic belts to unload painful pelvic ligaments or muscles in upright posture.     

Directional Coordination of Thumb and Finger Forces during Precision Pinch

The human opposable thumb enables the hand to perform dexterous manipulation of objects, which requires well-coordinated digit force vectors. This study investigated the directional coordination of force vectors generated by the thumb and index finger during precision pinch. Fourteen right-handed, healthy subjects were instructed to exert pinch force on an externally stabilized apparatus with the pulps of the thumb and index finger. Subjects applied forces to follow a force-ramp profile that linearly increased from 0 to 12 N and then decreased to 0 N, at a rate of ±3 N/s. Directional relationships between the thumb and index finger force vectors were quantified using the coordination angle (CA) between the force vectors. Individual force vectors were further analyzed according to their projection angles (PAs) with respect to the pinch surface planes and the shear angles (SAs) within those planes. Results demonstrated that fingertip force directions were dependent on pinch force magnitude, especially at forces below 2 N. Hysteresis was observed in the force-CA relationship for increasing and decreasing forces and fitted with exponential models. The fitted asymptotic values were 156.0±6.6° and 150.8±9.3° for increasing and decreasing force ramps, respectively. The PA of the thumb force vector deviated further from the direction perpendicular to the pinching surface planes than that of the index finger. The SA showed that the index finger force vector deviated in the ulnar-proximal direction, whereas the thumb switched its force between the ulnar-proximal and radial-proximal directions. The findings shed light on the effects of anatomical composition, biomechanical function, and neuromuscular control in coordinating digit forces during precision pinch, and provided insight into the magnitude-dependent force directional control which potentially affects a range of dexterous manipulations.     

Mechanical Properties of Mesh Materials Used for Hernia Repair and Soft Tissue Augmentation

Background

Hernia repair is the most common surgical procedure in the world. Augmentation with synthetic meshes has gained importance in recent decades. Most of the published work about hernia meshes focuses on the surgical technique, outcome in terms of mortality and morbidity and the recurrence rate. Appropriate biomechanical and engineering terminology is frequently absent. Meshes are under continuous development but there is little knowledge in the public domain about their mechanical properties. In the presented experimental study we investigated the mechanical properties of several widely available meshes according to German Industrial Standards (DIN ISO).

Methodology/Principal Findings

Six different meshes were assessed considering longitudinal and transverse direction in a uni-axial tensile test. Based on the force/displacement curve, the maximum force, breaking strain, and stiffness were computed. According to the maximum force the values were assigned to the groups weak and strong to determine a base for comparison. We discovered differences in the maximum force (11.1±6.4 to 100.9±9.4 N/cm), stiffness (0.3±0.1 to 4.6±0.5 N/mm), and breaking strain (150±6% to 340±20%) considering the direction of tension.

Conclusions/Significance

The measured stiffness and breaking strength vary widely among available mesh materials for hernia repair, and most of the materials show significant anisotropy in their mechanical behavior. Considering the forces present in the abdominal wall, our results suggest that some meshes should be implanted in an appropriate orientation, and that information regarding the directionality of their mechanical properties should be provided by the manufacturers.     

Forces acting on the patella during maximal voluntary contraction of the quadriceps femoris muscle at different knee flexion/extension angles

From knee extension moments measured with a dynamometer, the quadriceps muscle force, the patellar ligament force and the reaction force in the patellofemoral joint at various knee angles (0-90 degrees) were estimated. The information needed to calculate the combined effect of both patellofemoral and tibiofemoral joint on the mechanical advantage of the muscle was obtained from lateral-view radiographs of autopsy knees. The results show that the smallest quadriceps force (2,000 N) is exerted at maximal extension, and the largest force (8,000 N) at about 75 degrees of flexion. The patellar ligament force reaches a maximum (5,000 N) at 60 degrees. The reaction force in the patellofemoral joint is the smallest (1,000 N) at extension and is of the same values as the muscle force in a range from 75 to 90 degrees. Especially at large flexion angles, the value of the estimated forces is considerably larger (by 100%) than reported in the literature. This difference is attributed to the influence of the patellofemoral joint on the mechanical advantage of the muscle, which has not been taken into account in other studies.     

Mechanosensitive enteric neurons in the myenteric plexus of the mouse intestine

Background

Within the gut the autonomous enteric nervous system (ENS) is able to sense mechanical stimuli and to trigger gut reflex behaviour. We previously proposed a novel sensory circuit in the ENS which consists of multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the guinea pig. The aim of this study was to validate this concept by studying its applicability to other species or gut regions.

Methodology/Principal Findings

We deformed myenteric ganglia in the mouse small and large intestine and recorded spike discharge using voltage sensitive dye imaging. We also analysed expression of markers hitherto proposed to label mouse sensory myenteric neurons in the ileum (NF145kD) or colon (calretinin). RAMEN constituted 22% and 15% of myenteric neurons per ganglion in the ileum and colon, respectively. They encoded dynamic rather than sustained deformation. In the colon, 7% of mechanosensitive neurons fired throughout the sustained deformation, a behaviour typical for slowly adapting echanosensitive neurons (SAMEN). RAMEN and SAMEN responded directly to mechanical deformation as their response remained unchanged after synaptic blockade in low Ca⁺⁺/high Mg⁺⁺. Activity levels of RAMEN increased with the degree of ganglion deformation. Recruitment of more RAMEN with stronger stimuli may suggest low and high threshold RAMEN. The majority of RAMEN were cholinergic but most lacked expression of NF145kD or calretinin.

Conclusions/Significance

We showed for the first time that fundamental properties of mechanosensitive enteric neurons, such as firing pattern, encoding of dynamic deformation, cholinergic phenotype and their proportion, are conserved across species and regions. We conclude that RAMEN are important for mechanotransduction in the ENS. They directly encode dynamic changes in force as their firing frequency is proportional to the degree of deformation of the ganglion they reside in. The additional existence of SAMEN in the colon is likely an adaptation to colonic motor patterns which consist of phasic and tonic contractions.     

The effect of disconnecting the hippocampus from the septum on the activity of septal neurons]

Extracellular recording of neuronal activity was performed in the medial and lateral septal nuclei (MS and LS) in unanaesthetized rabbits after coagulation of septo-hippocampal connections. The MS neuronal activity had many pathological features. The LS activity was normal in every respect. Spontaneous activity, reactivity to sensory stimuli and main characteristics of responses to sensory stimuli were preserved in LS (and in a part of MS neurones). Sensory effects were augmented in intensity and duration, the number of neurones in LS with theta-bursts increased twofold, theta-bursts were more regular, than in control animals. These effects may be explained by an increase of ascending RF influences, which is supported by the fact of outstanding similarity between sensory and reticular effects in septal neurones after hippocampal disconnection. The number of units with inhibition of activity in response to sensory stimuli decreased, habituation of responses was absent. That means that hippocampal influences are necessary for the organization of inhibitory phenomena in the septum, and, above all, for processes of gradual habituation.     

Nitrogen cycling and water pulses in semiarid grasslands: are microbial and plant processes temporally asynchronous?

Precipitation pulses in arid ecosystems can lead to temporal asynchrony in microbial and plant processing of nitrogen (N) during drying/wetting cycles causing increased N loss. In contrast, more consistent availability of soil moisture in mesic ecosystems can synchronize microbial and plant processes during the growing season, thus minimizing N loss. We tested whether microbial N cycling is asynchronous with plant N uptake in a semiarid grassland. Using ¹⁵N tracers, we compared rates of N cycling by microbes and N uptake by plants after water pulses of 1 and 2?cm to rates in control plots without a water pulse. Microbial N immobilization, gross N mineralization, and nitrification dramatically increased 1?C3?days after the water pulses, with greatest responses after the 2-cm pulse. In contrast, plant N uptake increased more after the 1-cm than after the 2-cm pulse. Both microbial and plant responses reverted to control levels within 10?days, indicating that both microbial and plant responses were short lived. Thus, microbial and plant processes were temporally synchronous following a water pulse in this semiarid grassland, but the magnitude of the pulse substantially influenced whether plants or microbes were more effective in acquiring N. Furthermore, N loss increased after both small and large water pulses (as shown by a decrease in total ¹⁵N recovery), indicating that changes in precipitation event sizes with future climate change could exacerbate N losses from semiarid ecosystems.     

Drag-Based ‘Hovering’ in Ducks: The Hydrodynamics and Energetic Cost of Bottom Feeding

Diving ducks use their webbed feet to provide the propulsive force that moves them underwater. To hold position near the bottom while feeding, ducks paddle constantly to resist the buoyant force of the body. Using video sequences from two orthogonal cameras we reconstructed the 3-dimensional motion of the feet through water and estimated the forces involved with a quasi-steady blade-element model. We found that during station holding, near the bottom, ducks use drag based propulsion with the webbed area of the foot moving perpendicular to the trajectory of the foot. The body was pitched at 76±3.47° below the horizon and the propulsive force was directed 26±1.9° ventral to the body so that 98% of the propulsive force in the sagittal plane of the duck worked to oppose buoyancy. The mechanical work done by moving both feet through a paddling cycle was 1.1±0.2 J which was equivalent to an energy expenditure of 3.7±0.5 W to hold position while feeding at 1.5 m depth. We conclude that in shallow water the high energetic cost of feeding in ducks is due to the need to paddle constantly against buoyancy even after reaching the bottom. The mechanical energy spent on holding position near the bottom, while feeding, is approximately 2 fold higher than previous estimates that were made for similar bottom depths but based on the presumed motion of the body instead of motion of the feet.     

Acute and chronic tactile sensory testing after spinal cord injury in rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia(3). Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.     

A treadmill ergometer for three-dimensional ground reaction forces measurement during walking.

This report describes new treadmill ergometer designed to measure the vertical and horizontal ground reaction forces produced by the left and right legs during walking. It was validated by static and dynamic tests. Non-linearity was from 0.2% (left vertical force) to 1.4% (right antero-posterior force). The resonance frequency was from 219 (right vertical direction) to 58 Hz (left medio-lateral direction). A calibration "leg", an air jack in series with a strain gauge, was developed and used to produce force signals comparable to those obtained during human locomotion. The mean differences between the force measured by the calibration leg and treadmill ergometer at 5 km h(-1) were 3.7 N (0.7%) for the left side and 6.5 N (1.2%) for the right. Measurements obtained during human walking showed that the treadmill ergometer has considerable potential for analysing human gait.     

The bite force of the largest fossil rodent (Hystricognathi,Caviomorpha, Dinomyidae)

Blanco R.E., Rinderknecht, A. & Lecuona, G. 2011: The bite force of the largest fossil rodent (Hystricognathi, Caviomorpha, Dinomyidae). Lethaia, Vol. 45, pp. 157–163. An exceptionally well‐preserved skull of the largest fossil rodent Josephoartigasia monesi allows the first analysis of the bite mechanics of this group of South American giant rodents. In this study, we reconstructed the main anatomical features of the skull of this Pliocene rodent, relating them to the bite force at incisors. Bite force was estimated using three different techniques. Two methods suggest that bite forces at incisors of around 1000 N were possible for these mammals. However, the incisors seem to be stronger than expected for this bite force implying that the bite forces may have been greater than 3000 N. We consider three hypotheses: allometric effects, teeth digging or defence against predators, to explain our results. □Bite force, Dinomyidae, incisors, largest rodent, Pliocene.     

A force transducer to measure individual finger loads during activities of daily living.

A new six-degree-of-freedom force transducer has been manufactured, with the sensitivity to measure forces in the range +/-100 N and moments of up to +/-5 Nm. The transducer incorporates two mechanical components: shear forces and bending moments are measured via a strain-gauged tubular section whilst axial forces are transmitted to a cantilevered load cell. Both components are instrumented with 350 ohms strain gauge full bridge circuits and are temperature compensated. After calibration, measurement errors are less than +/-0.3 N for direct forces and +/-0.03 Nm for applied moments. In order to measure sub-maximal finger loads during activities of daily living, the transducer has been incorporated into several housings representing objects in domestic use: a jar, a tap, a key in a lock and a jug kettle.