Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

Toxicity in amyloidogenic protein misfolding disorders is thought to involve intermediate states of aggregation associated with the formation of amyloid fibrils. Despite their relevance, the heterogeneity and transience of these oligomers have placed great barriers in our understanding of their structural properties. Among amyloid intermediates, annular oligomers or annular protofibrils have raised considerable interest because they may contribute to a mechanism of cellular toxicity via membrane permeation. Here we investigated, by using AFM force spectroscopy, the structural detail of amyloid annular oligomers from transthyretin (TTR), a protein involved in systemic and neurodegenerative amyloidogenic disorders. Manipulation was performed in situ , in the absence of molecular handles and using persistence length‐fit values to select relevant curves. Force curves reveal the presence of dimers in TTR annular oligomers that unfold via a series of structural intermediates. This is in contrast with the manipulation of native TTR that was more often manipulated over length scales compatible with a TTR monomer and without unfolding intermediates. Imaging and force spectroscopy data suggest that dimers are formed by the assembly of monomers in a head‐to‐head orientation with a nonnative interface along their β‐strands. Furthermore, these dimers stack through nonnative contacts that may enhance the stability of the misfolded structure.     

Force and torque modelling of drilling simulation for orthopaedic surgery

The advent of haptic simulation systems for orthopaedic surgery procedures has provided surgeons with an excellent tool for training and preoperative planning purposes. This is especially true for procedures involving the drilling of bone, which require a great amount of adroitness and experience due to difficulties arising from vibration and drill bit breakage. One of the potential difficulties with the drilling of bone is the lack of consistent material evacuation from the drill's flutes as the material tends to clog. This clogging leads to significant increases in force and torque experienced by the surgeon. Clogging was observed for feed rates greater than 0.5 mm/s and spindle speeds less than 2500 rpm. The drilling simulation systems that have been created to date do not address the issue of drill flute clogging. This paper presents force and torque prediction models that account for this phenomenon. The two coefficients of friction required by these models were determined via a set of calibration experiments. The accuracy of both models was evaluated by an additional set of validation experiments resulting in average R² regression correlation values of 0.9546 and 0.9209 for the force and torque prediction models, respectively. The resulting models can be adopted by haptic simulation systems to provide a more realistic tactile output.     

A single-spring model predicts the majority of variance in impact force during a fall onto the outstretched hand

The objective of this study was to validate a single-spring model in predicting measured impact forces during an outstretched arm falling scenario. Using an integrated force plate, impact forces were assessed from 10 young adults (5 males; 5 females), falling from planted knees onto outstretched arms, from a random order of drop heights: 3, 5, 7, 10, 15, 20, and 25 cm. A single-spring model incorporating body mass, drop height plus the estimated linear stiffness of the upper extremity (hand, wrist and arm) was used to predict impact force on the hand. We used an analysis of variance linearity test to test the validity of using a linear stiffness coefficient in the model. We used linear regression to assess variance (R²) in experimental impact force predicted by the single-spring model. We derived optimum linear stiffness coefficients for male, female and sex-combined. Our results indicated that the association between experimental and predicted impact forces was linear (P < 0.05). Explain variance in experimental impact force was R² = 0.82 for sex-combined, R² = 0.88 for males and R² = 0.84 for females. Optimum stiffness coefficients were 7436 N/m for sex-combined, 8989 N/m for males and 4527 N/m for females. In conclusion, a linear spring coefficient used in the single-spring model proved valid for predicting impact forces from fall heights up to 25 cm. Results also suggest the use of sex-specific spring coefficients when estimating impact force using the single-spring model. This model may improve impact force to bone strength ratios (factor-of-risk) and prediction of forearm and wrist fracture.     

Novel instrumented probe for measuring 3D pressure distribution along the vaginal canal

We developed an intravaginal instrumented probe (covered with a 10 × 10 matrix of capacitive sensors) for assessing the three-dimensional (3D) spatiotemporal pressure profile of the vaginal canal. The pressure profile was compared to the pelvic floor (PF) digital assessment, and the reliability of the instrument and repeatability of the protocol was tested. We also tested its ability to characterize and differentiate two tasks: PF maximum contraction and Valsalva maneuver (maximum intra-abdominal effort with downward movement of the PF). Peak pressures were calculated for the total matrix, for three major sub-regions, and for 5 planes and 10 rings throughout the vaginal canal. Intraclass correlation coefficients indicated excellent inter- and intra-rater reliability and intra-trial repeatability for the total and medial areas, with moderate reliability for the cranial and caudal areas. There was a moderate correlation between peak pressure and PF digital palpation [Spearman’s coefficient r = 0.55 (p < 0.001)]. Spatiotemporal profiles were completely different between tasks (2-way ANOVAs for repeated measures) with notably higher pressures (above 30 kPa) for the maximum contraction task compared to Valsalva (below 15 kPa). At maximum contraction, higher pressures occurred in the mid-antero-posterior zone, with earlier peak pressure onsets and more variable along the vaginal depth (from rings 3 to 10-caudal). During Valsalva, the highest pressures were observed in rings 4–6, with peak pressure onsets more synchronized between rings. With this protocol and novel instrument, we obtained a high-resolution and highly reliable innovative 3D pressure distribution map of the PF capable of distinguishing vaginal sub-regions, planes, rings and tasks.     

An automatic device to record the force necessary to compensate plant movements

A device, which automatically and continuously keeps moving plants in fixed positions, has been constructed. The apparatus is based on optical detection of any movements of the plant organ under study. The plant organ is kept in the desired position by means of wires, and the force necessary to apply to the wires to achieve this is recorded. The force reflects any tendency of the plant organ to move. The system is controlled by an Apple II computer. The device can work in one or two dimensions and record compensation forces in a wide range. In the experiments mentioned below, the magnitude of the forces necessary to keep the plants in a fixed position was of the order of 10^-4N.
Circadian leaf movements of Oxalis regnellii Mig. were studied in the device. The leaf rhythm continued, although the leaflets were clamped and the light input on the leaf therefore constant. Circumnutation of hypocotyls of Helianthus annus L. cv. Californicus were drastically reduced in amplitude when the hypocotyls were kept in vertical position by the wires. Since the gravitropic input signal to this system was zeroed by the equipment, the results demonstrated that in the absence of gravitropic inputs the circumnutation reactions drastically diminish. This confirmed that circumnutations of these hypocotyls are influenced by gravity. Finally, the apparatus was used to study phototropic reactions: By clamping phototropically stimulated coleoptiles of Avena sativa L. cv. Seger no gravitational stimulations were involved and the phototropic reaction without interference from gravity could be studied.     

Blistering of supported lipid membranes induced by Phospholipase D, as observed by real-time atomic force microscopy

Phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme known to be activated by the phosphatidic acid (PA)-calcium complexes. Despite the vast body of literature that has accumulated on this enzyme, the exact mechanism of activation remains poorly understood. In this work, we report the first observation of PLDSc activity in real time and at nanometer resolution using atomic force microscopy (AFM). AFM images of continuous and patchy dipalmitoylphosphatidylcholine (DPPC) bilayers were recorded, prior and after incubation with PLDSc. For continuous bilayers, the enzyme induced important morphological alterations; holes corresponding to the bilayer thickness were created, while an additional elevated phase, about 2.5 nm high, was observed. This bilayer blistering is believed to be due to the production of the negatively charged lipid PA that would cause localized repulsions between the bilayer and the underlying mica surface. By contrast, these elevated domains were not seen on patchy bilayers incubated with the enzyme. Instead, the shapes of DPPC patches were strongly deformed by enzyme activity and evolved into melted morphologies. These results point to the importance of lipid packing on PLD activity and illustrate the potential of AFM for visualizing remodeling enzymatic activities.     

Architectural specialization of the intrinsic thoracic limb musculature of the American badger (Taxidea taxus)

Evaluation of the relationships between muscle structure and digging function in fossorial species is limited. Badgers and other fossorial specialists are expected to have massive forelimb muscles with long fascicles capable of substantial shortening for high power and applying high out‐force to the substrate. To explore this hypothesis, we quantified muscle architecture in the thoracic limb of the American badger (Taxidea taxus) and estimated the force, power, and joint torque of its intrinsic musculature in relation to the use of scratch‐digging behavior. Architectural properties measured were muscle mass, belly length, fascicle length, pennation angle, and physiological cross‐sectional area. Badgers possess hypertrophied shoulder flexors/humeral retractors, elbow extensors, and digital flexors. The triceps brachii is particularly massive and has long fascicles with little pennation, muscle architecture consistent with substantial shortening capability, and high power. A unique feature of badgers is that, in addition to elbow joint extension, two biarticular heads (long and medial) of the triceps are capable of applying high torques to the shoulder joint to facilitate retraction of the forelimb throughout the power stroke. The massive and complex digital flexors show relatively greater pennation and shorter fascicle lengths than the triceps brachii, as well as compartmentalization of muscle heads to accentuate both force production and range of shortening during flexion of the carpus and digits. Muscles of most functional groups exhibit some degree of specialization for high force production and are important for stabilizing the shoulder, elbow, and carpal joints against high limb forces generated during powerful digging motions. Overall, our findings support the hypothesis and indicate that forelimb muscle architecture is consistent with specializations for scratch‐digging. Quantified muscle properties in the American badger serve as a comparator to evaluate the range of diversity in muscle structure and contractile function that exists in mammals specialized for fossorial habits. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

A scaffold replacement approach towards new sirtuin 2 inhibitors

Sirtuins (SIRT1–SIRT7) are an evolutionary conserved family of NAD⁺-dependent protein deacylases regulating the acylation state of ε-N-lysine residues of proteins thereby controlling key biological processes. Numerous studies have found association of the aberrant enzymatic activity of SIRTs with various diseases like diabetes, cancer and neurodegenerative disorders. Previously, we have shown that substituted 2-alkyl-chroman-4-one/chromone derivatives can serve as selective inhibitors of SIRT2 possessing an antiproliferative effect in two human cancer cell lines. In this study, we have explored the bioisosteric replacement of the chroman-4-one/chromone core structure with different less lipophilic bicyclic scaffolds to overcome problems associated to poor physiochemical properties due to a highly lipophilic substitution pattern required for achieve a good inhibitory effect. Various new derivatives based on the quinolin-4(1H)-one scaffold, bicyclic secondary sulfonamides or saccharins were synthesized and evaluated for their SIRT inhibitory effect. Among the evaluated scaffolds, the benzothiadiazine-1,1-dioxide-based compounds showed the highest SIRT2 inhibitory activity. Molecular modeling studies gave insight into the binding mode of the new scaffold-replacement analogues.     

Electron Microscopic Evidence in Support of α-Solenoid Models of Proteasomal Subunits Rpn1 and Rpn2

Rpn1 (109 kDa) and Rpn2 (104 kDa) are components of the 19S regulatory complex of the proteasome. The central portions of both proteins are predicted to have toroidal α-solenoid folds composed of 9-11 proteasome/cyclosome repeats, each ∼ 40 residues long and containing two α-helices and turns [A. V. Kajava, J. Biol. Chem. 277, 49791-49798, 2002]. To evaluate this prediction, we examined the full-length yeast proteins and truncated versions thereof consisting only of the repeat-containing regions by gel filtration, CD spectroscopy, and negative-staining electron microscopy (EM). All four proteins are monomeric in solution and highly α-helical, particularly the truncated ones. The EM data were analyzed by image classification and averaging techniques. The preponderant projections, in each case, show near-annular molecules 6-7 nm in diameter. Comparison of the full-length with the truncated proteins showed molecules similar in size and shape, indicating that their terminal regions are flexible and thus smeared to invisibility in the averaged images. We tested the toroidal model further by calculating resolution-limited projections and comparing them with the EM images. The results support the α-solenoid model, except that they indicate that the repeats are organized not as symmetrical circular toroids but in less regular horseshoe-like structures.     

Traction fibres in chromosome movement: the pros and cons

The current discussion on the mechanism of chromosome movement in anaphase is dominated by a new hypothesis assuming active movement of the chromosome along stationary kinetochore microtubules. The aim of this article is to call attention to several older as well as to more recent observations on spindle structural dynamics and microtubule rearrangements which, in the author's opinion, cannot be explained by the new model. In fact, the observations seem to be more consistent with the classical concept of a traction fibre. For this reason, these observations should not be disregarded.