Mechanical forces induced by the transendothelial migration of human neutrophils

The mechanisms regulating neutrophil transmigration of vascular endothelium are not fully elucidated, but involve neutrophil firm attachment and passage through endothelial cell-cell junctions. The goal of this study was to characterize the tangential forces exerted by neutrophils during transendothelial migration at cell-cell junctions using an in vitro laminar shear flow model in which confluent activated endothelium is grown on a microfabricated pillar substrate. The tangential forces are deduced from the measurement of pillar deflection beneath the endothelial cell-cell junction as neutrophils transmigrate. The force diagram displays an initial force increase, which coincides with neutrophil penetration into the intercellular space and formation of a gap in VE-cadherin staining. This is followed by a rapid and large increase of traction forces exerted by endothelial cells on the substrate in response to the transmigration process and the disruption of cell-cell contacts. The average maximum force exerted by an actively transmigrating neutrophil is three times higher than the force generated by an adherent neutrophil that does not transmigrate. Furthermore, we show that substrate rigidity can modify the mechanical forces induced by the transmigration of a neutrophil through the endothelium. Our data suggest that the force induced by neutrophil transmigration plays a key role in the disruption of endothelial adherens junctions.     

Top-down control analysis of the effect of temperature on ectotherm oxidative phosphorylation

Top-down control and elasticity analysis was conducted on mitochondria isolated from the midgut of the tobacco hornworm (Manduca sexta) to assess how temperature affects oxidative phosphorylation in a eurythermic ectotherm. Oxygen consumption and protonmotive force (measured as membrane potential in the presence of nigericin) were monitored at 15, 25, and 35 degrees C. State 4 respiration displayed a Q(10) of 2.4-2.7 when measured over two temperature ranges (15-25 degrees C and 25-35 degrees C). In state 3, the Q(10)s for respiration were 2.0 and 1.7 for the lower and higher temperature ranges, respectively. The kinetic responses (oxygen consumption) of the substrate oxidation system, proton leak, and phosphorylation system increased as temperature rose, although the proton leak and substrate oxidation system showed the greatest thermal sensitivity. Whereas there were temperature-induced changes in the activities of the oxidative phosphorylation subsystems, there was no change in the state 4 membrane potential and little change in the state 3 membrane potential. Top-down control analysis revealed that control over respiration did not change with temperature. In state 4, control of respiration was shared nearly equally by the proton leak and the substrate oxidation system, whereas in state 3 the substrate oxidation system exerted over 90% of the control over respiration. The proton leak and phosphorylation system account for <10% of the temperature-induced change in the state 3 respiration rate. Therefore, when the temperature is changed, the state 3 respiration rate is altered primarily because of temperature's effect on the substrate oxidation system.     

Metabolic cost of generating horizontal forces during human running.

Previous studies have suggested that generating vertical force on the ground to support body weight (BWt) is the major determinant of the metabolic cost of running. Because horizontal forces exerted on the ground are often an order of magnitude smaller than vertical forces, some have reasoned that they have negligible cost. Using applied horizontal forces (AHF; negative is impeding, positive is aiding) equal to -6, -3, 0, +3, +6, +9, +12, and +15% of BWt, we estimated the cost of generating horizontal forces while subjects were running at 3.3 m/s. We measured rates of oxygen consumption (VO2) for eight subjects. We then used a force-measuring treadmill to measure ground reaction forces from another eight subjects. With an AHF of -6% BWt, VO2 increased 30% compared with normal running, presumably because of the extra work involved. With an AHF of +15% BWt, the subjects exerted approximately 70% less propulsive impulse and exhibited a 33% reduction in VO2. Our data suggest that generating horizontal propulsive forces constitutes more than one-third of the total metabolic cost of normal running.     

Finger force vectors in multi-finger prehension

In a majority of studies on grasp, only normal forces were measured and only when a zero torque was exerted on a hand-held object. This study concerns finger force vectors during the torque production tasks. Subjects (n=8) stabilized a handle with an attachment that allowed for change of external torque from -1.5 to 1.5 Nm. Forces and moments exerted by the digit tips on the object were recorded. At the large (>-0.375 Nm) supination torques the index/middle and ring/little pairs of fingers generated oppositely directed tangential forces. The index and middle finger produced forces in a downward direction and therefore did not support the load. At a zero torque and pronation torques, the middle, ring and little fingers produced forces along nearly the same direction. The vector of the index finger force was always directed differently from the vectors of other finger forces, the angles ranged from 19 degrees 30' to 47 degrees 40'. The points of force application were systematically displaced with the torque, with the exception of the little finger. Tangential finger forces contributed substantially to the total torque exerted on the hand-held object.     

Mechanical force can fine-tune redox potentials of disulfide bonds

Mechanical force applied along a disulfide bond alters its rate of reduction. We here aimed at quantifying the direct effect of force onto the chemical reactivity of a sulfur-sulfur bond in contrast to indirect, e.g., steric or mechanistic, influences. To this end, we evaluated the dependency of a disulfide bond's redox potential on a pulling force applied along the system. Our QM/MM simulations of cystine as a model system take conformational dynamics and explicit solvation into account and show that redox potentials increase over the whole range of forces probed here (30-3320 pN), and thus even in the absence of a significant disulfide bond elongation (<500 pN). Instead, at low forces, dihedrals and angles, as the softer degrees of freedom are stretched, contribute to the destabilization of the oxidized state. We find physiological forces to be likely to tune the disulfide's redox potentials to an extent similar to the tuning within proteins by point mutations.     

Three-Dimensional Balance of Cortical Tension and Axial Contractility Enables Fast Amoeboid Migration

Fast amoeboid migration requires cells to apply mechanical forces on their surroundings via transient adhesions. However, the role these forces play in controlling cell migration speed remains largely unknown. We used three-dimensional force microscopy to measure the three-dimensional forces exerted by chemotaxing Dictyostelium cells, and examined wild-type cells as well as mutants with defects in contractility, internal F-actin crosslinking, and cortical integrity. We showed that cells pull on their substrate adhesions using two distinct, yet interconnected mechanisms: axial actomyosin contractility and cortical tension. We found that the migration speed increases when axial contractility overcomes cortical tension to produce the cell shape changes needed for locomotion. We demonstrated that the three-dimensional pulling forces generated by both mechanisms are internally balanced by an increase in cytoplasmic pressure that allows cells to push on their substrate without adhering to it, and which may be relevant for amoeboid migration in complex three-dimensional environments.     

Hydrodynamic effects in fast AFM single-molecule force measurements

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 m/s, although for a more complete investigation of the kinetic properties higher speeds are desirable. Above 10 m/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 m/s pulling speed.Abbrevations AFM atomic force micrcoscopy - pN piconewton - BR bacteriorhodopsin - DFS dynamic force spectroscopy - Ig27 immunoglobulin 27 - If27-8 immunoglobulin 27 octameric construct - BFP biomembrane force probe     

Orthogonal bone cutting: saw design and operating characteristics

The cutting process of orthopaedic bone saws was considered as orthogonal (two-dimensional) cutting for determination of the horizontal and vertical force components of single edge cutting tools with rake angles of 0 to -30 degrees. The Merchant analysis for orthogonal cutting was used to determine the resultant force and other force and work relationships. The effect of an imposed lateral vibration on the cutting tool was also investigated. The results of the tests indicated a strong interaction between the measured and derived forces with the rake angle and feed velocity. It was concluded that to reduce the cutting forces and work expenditure, a negative rake angle between 0 and -10 degrees, high feed velocity, and an imposed lateral vibration provided the greatest reduction in force and energy expenditure.     

The effects of the antagonist muscle force on intersegmental loading during isokinetic efforts of the knee extensors

Hamstrings activation when acting as antagonists is considered very important for knee joint stability. However, the effect of hamstring antagonist activity on knee joint loading in vivo is not clear. Therefore, the purpose of this study was to examine the differences in antagonistic muscle force and their effect on agonist muscle and intersegmental forces during isokinetic eccentric and concentric efforts of the knee extensors. Ten males performed maximum isokinetic eccentric and concentric efforts of the knee extensors at 30 degrees s(-1). The muscular and tibiofemoral joint forces were then estimated using a two-dimensional model with and without including the antagonist muscle forces. The antagonist moment was predicted using an IEMG-moment model. The predicted antagonist force reached a maximum of 2.55 times body weight (BW) and 1.16 BW under concentric and eccentric conditions respectively. Paired t-tests indicated that these were significantly different (p<0.05). A one-way analysis of variance indicated that when antagonist forces are included in the calculations the patella tendon, compressive and posterior shear joint forces are significantly higher compared to those calculated without including the antagonist forces. The anterior shear force was not affected by antagonist activity. The antagonists produce considerable force throughout the range of motion and affect the joint forces exerted at the knee joint. Further, it appears that the antagonist effect depends on the type of muscle action examined as it is higher during concentric compared to eccentric efforts of the knee extensors.     

Structure and mechanics of the tarsal chain in the hornet, Vespa crabro (Hymenoptera: Vespidae): implications on the attachment mechanism

Two combined mechanisms on the hornet tarsus are adapted to attachment to the substrate: a friction-based (claws and spines) and an adhesion-based one (arolium). There are two ranges of substrate roughness optimal for attachment, either very smooth or very rough. There is an intermediate range of substrate grains of small but non-zero size, where both of these mechanisms fail. The optimal size of substrate grains for hornet grasping was 50-100 microm. Maximal hold to the substrate was achieved when surface irregularities were clamped between the claws of opposite legs. In such a position, the insect could withstand an external force which was almost 25 times larger than its own weight. The tarsal chain is an important part of the entire attachment mechanism. The articulations in the kinematic chain of tibia-tarsus-pretarsus are monocondylar. Three tarsal muscles and one head of the claw retractor muscle originate in the tibia. On pull to the retractor tendon, the tarsus bends in a plane. All elements of the tarsal kinematic chain have one active degree of freedom. The distance between the intertarsomeric articulation point and the tendon of the claw retractor (75-194 microm) corresponds to an efficiency of 1 degrees per 1-3 mircom of pulling distance travelled by the tendon. The claw turns about 1 degrees per 4.3-5.0 microm of pulling distance travelled by the unguitractor. The arolium turns forward and downward simultaneously with flexion of the claws. The kinematic chain of the arolium lacks real condylar joints except the joint at the base of the manubrium. Other components are tied by flexible transmissions of the membranous cuticle. The walking hornet rests on distal tarsomeres of extended tarsi. If the retractor tendon inside the tarsus is fixed, passive extension of the tarsomeres might be replaced by claw flexion. Tarsal chain rigidity, measured with the force tester, increased when the retractor tendon was tightened. Probably, pull to the tendon compresses the tarsomeres, increasing friction within contacting areas of rippled surfaces surrounding condyles within articulations.     

Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog

Increasing temperature (4-22 degrees C) increases the Ca2+ concentration required for activation of mechanically skinned frog muscle fibers. The pCa required for 50% maximal force (pCa50) was inversely proportional to absolute temperature. Assuming that relative force is directly related to fractional occupancy of the Ca2+-binding sites on troponin that regulate force, the shift was consistent with a Gibbs free energy change of binding (delta G) of about -7.8 kcal/mol. This is close to the delta G for Ca2+ binding to the calcium-specific sites on troponin C reported by others. Decreasing Mg2+ from 1 mM to 60 microM shifts the force-pCa curves at either 4 or 22 degrees C to higher pCa, but the shift of pCa50 with temperature over this range (0.4 log units) was the same at low and high Mg2+. Maximal force increased with temperature for the entire range 4-22 degrees C with a Q10 of 1.41, and over the restricted range 4-15 degrees C with a Q10 of 1.20. From the dual effects of temperature on Ca2+ activation and maximal force, one would expect that force would respond differently to temperature change at high or low Ca2+. At high Ca2+, a temperature increase will lead to an increased force. However, at low to intermediate Ca2+ levels (below the intersection of the force-pCa curves for the initial and final temperatures), steady state force should decrease with increasing temperature. The inverse responses should occur with a decrease in temperature. These responses are observed when temperature is changed by rapid solution exchange.     

Patterns of thermal adaptation of Bacillus simplex to the microclimatically contrasting slopes of 'Evolution Canyons' I and II, Israel

Identification of selective forces that drive evolution and speciation of bacteria in natural habitats is a central issue in bacterial ecology and evolution. Exploring the adaptive evolution of Bacillus simplex at 'Evolution Canyons' I and II, Israel, we report here on the impact of high heat stress on the speciation progress of individual evolutionary lineages. These canyons represent similar ecological replicates, separated by 40 km, in which the orientation of the sun yields a strong sun-exposed and hot 'African' south-facing slope (SFS) versus a rather cooler and mesic-lush 'European' north-facing slope (NFS) within a distance of only 50-100 m at the bottom and 400 m at the top. Among 131 strains studied, in Luria-Bertani broth, 'African' strains grow better than 'European' strains at a stressful high temperature (43.25 degrees C). The results suggest that adaptation to the hotter and more stressful SFS is continuously ongoing. The patterns of heat adaptation override the phylogenetic history of individual lineages. A positive correlation of growth rates at 43.25 degrees C and 20 degrees C, more markedly among 'African' strains, reflects probably the broader temperature range on the SFS. Summarizing, the hot temperature stress on the 'African' slope is a major environmental force driving the twin evolutionary processes of adaptation and speciation of B. simplex at 'Evolution Canyon'. Finally, we discuss the data in light of current controversies on species concepts.     

Skeletal muscle fatigue in vitro is temperature dependent

Our purpose was to determine the effect of temperature on the fatigability of isolated soleus and extensor digitorum longus (EDL) muscles from rats during repeated isometric contractions. Muscles (70-90 mg) were studied at 20-40 degrees C in vitro. Fatigability was defined with respect to both the time and number of stimuli required to reach 50% of the force (P) developed at the onset of the fatigue test. Fatigue was studied during stimulation protocols of variable [force approximately 70% of maximum force (Po)] and constant frequency (28 Hz). Results for soleus and EDL muscles were qualitatively similar, but fatigue times were longer for soleus than for EDL muscles. During the variable-frequency protocol, development of approximately 70% of Po required an increase in stimulation frequency as temperature increased. During stimulation at these frequencies, fatigue time shortened as temperature increased. For both fatigue protocols, the relationship between temperature and the number of stimuli required to reach fatigue followed a bell-shaped curve, with maximum values at 25-30 degrees C. The temperature optimum for maximizing the number of isometric contractions to reach fatigue reflects direct effects of temperature on muscle function.     

Myofascial force transmission: muscle relative position and length determine agonist and synergist muscle force.

Equal proximal and distal lengthening of rat extensor digitorum longus (EDL) were studied. Tibialis anterior, extensor hallucis longus, and EDL were active maximally. The connective tissues around these muscle bellies were left intact. Proximal EDL forces differed from distal forces, indicating myofascial force transmission to structures other than the tendons. Higher EDL distal force was exerted (ratio approximately 118%) after distal than after equal proximal lengthening. For proximal force, the reverse occurred (ratio approximately 157%). Passive EDL force exerted at the lengthened end was 7-10 times the force exerted at the nonlengthened end. While kept at constant length, synergists (tibialis anterior + extensor hallucis longus: active muscle force difference approximately -10%) significantly decreased in force by distal EDL lengthening, but not by proximal EDL lengthening. We conclude that force exerted at the tendon at the lengthened end of a muscle is higher because of the extra load imposed by myofascial force transmission on parts of the muscle belly. This is mediated by changes of the relative position of most parts of the lengthened muscle with respect to neighboring muscles and to compartment connective tissues. As a consequence, muscle relative position is a major codeterminant of muscle force for muscle with connectivity of its belly close to in vivo conditions.     

Reactivation of newt lung cilia: evidence for a possible temperature- and MgATP-induced activation mechanism.

Optimal conditions have been developed for the isolation and reactivation of highly coupled, demembranated ciliary axonemes from newt lungs [Hard, Cypher, and Schabtach, 1988, Cell Motil. Cytoskeleton 10:271-284]. In the present study, the motility of these cilia was further characterized by examining the effects of nucleotides, divalent cations, and temperature on beat frequency. When exposed to a reactivating solution containing Mg2+ and ATP, nearly 100% of the axonemes were motile and beat at frequencies of 0-50 Hz, depending on [MgATP] and temperature. Divalent cations were required for movement, with Mg2+ 2-3 times more effective than Ca2+. There was no absolute requirement for Ca2+ for motility. The beat frequencies obtained with fixed ATP and varying Mg2+ concentrations indicate that MgATP serves as the actual substrate. The effects of MgATP on beat frequency depended on the degree of mechanochemical coupling and temperature. When highly coupled preparations were reactivated at 21 degrees C, double reciprocal plots of beat frequency vs. [MgATP] were biphasic with extrapolated Fmax values of 22 and 44.8 Hz. However, when reactivated at 10 degrees C and 30 degrees C, linear plots were generated with Fmax values of 18.3 and 48.9 Hz, respectively. The beat frequencies of cultured cells and reactivated axonemes also varied biphasically with temperature. Our data suggest that newt lung respiratory cilia possess an intra-axonemal activation mechanism involving a temperature- and MgATP-induced transition between two distinct states whose maximum beat frequencies differ by 200-300%.     

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase. Evidence for conformational change

Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tarsal morphology and attachment ability of the codling moth Cydia pomonella L. (Lepidoptera, Tortricidae) to smooth surfaces

Despite several studies on the attachment ability of different insect taxa, little is known about this phenomenon in adult Lepidoptera. In this study we combined morphological and experimental analyses of tarsal adhesive devices and the attachment ability of the codling moth Cydia pomonella (L.) (Lepidoptera, Tortricidae) to smooth surfaces. Pretarsi of C. pomonella attach to smooth substrates by means of their smooth, flexible and well developed arolia. Using the centrifugal force measurement technique, friction forces of males and females were assessed on hydrophobic and hydrophilic glass surfaces. Adults of both sexes generated similar forces in spite of the noticeable difference in their body masses. That is why males showed significantly higher safety factors (attachment force divided by body weight) compared to those of females. Hydrophobicity of the substrate had no considerable effect on friction forces. For females, friction forces (sliding parallel to the substrate plane) were compared with adhesive forces (pulling off perpendicularly from the substrate plane) measured on Plexiglas surfaces. It can be concluded that the attachment system of C. pomonella is rather robust against physico-chemical properties of the substrate and is able to achieve a very good attachment on vertical and horizontal substrata.     

The effects of biting and pulling on the forces generated during feeding in the Komodo dragon (Varanus komodoensis)

In addition to biting, it has been speculated that the forces resulting from pulling on food items may also contribute to feeding success in carnivorous vertebrates. We present an in vivo analysis of both bite and pulling forces in Varanus komodoensis, the Komodo dragon, to determine how they contribute to feeding behavior. Observations of cranial modeling and behavior suggest that V. komodoensis feeds using bite force supplemented by pulling in the caudal/ventrocaudal direction. We tested these observations using force gauges/transducers to measure biting and pulling forces. Maximum bite force correlates with both body mass and total body length, likely due to increased muscle mass. Individuals showed consistent behaviors when biting, including the typical medial-caudal head rotation. Pull force correlates best with total body length, longer limbs and larger postcranial motions. None of these forces correlated well with head dimensions. When pulling, V. komodoensis use neck and limb movements that are associated with increased caudal and ventral oriented force. Measured bite force in Varanus komodoensis is similar to several previous estimations based on 3D models, but is low for its body mass relative to other vertebrates. Pull force, especially in the ventrocaudal direction, would allow individuals to hunt and deflesh with high success without the need of strong jaw adductors. In future studies, pull forces need to be considered for a complete understanding of vertebrate carnivore feeding dynamics.     

Biomechanical properties of the crimp grip position in rock climbers

Rock climbers are often using the unique crimp grip position to hold small ledges. Thereby the proximal interphalangeal (PIP) joints are flexed about 90 degrees and the distal interphalangeal joints are hyperextended maximally. During this position of the finger joints bowstringing of the flexor tendon is applying very high load to the flexor tendon pulleys and can cause injuries and overuse syndromes. The objective of this study was to investigate bowstringing and forces during crimp grip position. Two devices were built to measure the force and the distance of bowstringing and one device to measure forces at the fingertip. All measurements of 16 fingers of four subjects were made in vivo. The largest amount of bowstringing was caused by the flexor digitorum profundus tendon in the crimp grip position being less using slope grip position (PIP joint extended). During a warm-up, the distance of bowstringing over the distal edge of the A2 pulley increased by 0.6mm (30%) and was loaded about 3 times the force applied at the fingertip during crimp grip position. Load up to 116N was measured over the A2 pulley. Increase of force in one finger holds by the quadriga effect was shown using crimp and slope grip position.