Individual Globular Domains and Domain Unfolding Visualized in Overstretched Titin Molecules with Atomic Force Microscopy

Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin’s globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme’s ATP-binding site and hence contributing to the molecule’s mechanosensory function

Transthyretin amiloid fibrillumok nanobiofizikai vizsgálata = Nanobiophysical exploration of transthyretin amyloid fibrils

Kísérleteinkben AFM segítségével vizsgáltuk transthyretin (TTR) amiloid fibrillumok amiloidogén fibrillum képződési mechanizmusait. Protofibrillumokon végzett egyedi molekula erőspektroszkópiai mérések eredményeit a natív TTR szerkezeti paramétereivel hasonlítottuk össze annak érdekében, hogy szerkezeti és dinamikai bepillantást nyerjünk a fibrillumok belső elrendezésébe és az összetartó erők természetébe. Időfüggő AFM felvételek segítségével a protofibrillum képződést követő belső szerkezeti változásokat térképeztük fel. Eredményeink szerint a protofibrillum képződés első lépése amorf aggregátumok kialakulása, amelyek idővel gyűrű alakú szerkezetekké állnak össze. Hasonló gyűrű alakú intermediéreket más amiloid fibrillumok esetében is megfigyeltek. A gyűrűk egymáshoz rendeződve tubuláris strukturákat alakítanak ki, amely a protofibrillum kialakulásának további alapját képezi. Az oldat kicserélésére a struktura szétesik, szétzippzározódási lépéseken keresztül. A nanomechanikai mérések arra utalnak, hogy a TTR egységek ß-szálak mentén tekerednek szét, továbbá érett protofibrillumokban az intermonomerikus kapcsolatok megerősödnek. Megfigyeléseink alapján a TTR fibrillogenezis egy szerkezeti modelljét állítottuk fel. | In this work we used AFM to follow the amyloidogenetic pathway of transthyretin (TTR) by imaging the events leading to the formation of amyloid protofilaments. Single-molecule force spectroscopy (SMFS) of protofilaments was compared to naive TTR in order to probe dynamic and structural differences. We observed that the pathway proceeds through the formation of transient amorphous aggregates, followed by the occurrence of annular oligomers (rings or doughnuts). In other types of amyloidoses similar ring structures have been implicated in cytoxicity, but their properties and involvement in the amyloid pathway are poorly understood. We show that the rings have a tendency to stack, forming tubular protofilaments. These tubular protofilaments precede the appearance of amyloid protofilaments. Their height and pitch resemble those of previous structural models for the TTR amyloid protofilament. Upon solvent exchange we also observed amyloid protofilament dissociation. The dissociation appears to proceed through an unzipping mechanism, revealing structures reminiscent of the TTR annular oligomers. SMFS of protofilaments revealed a time-dependent increase in the length of the manipulated structure, suggesting that associations between monomers stabilize with time. Force spectra of native TTR and protofilaments contained transitions spaced 4 nm apart, indicating that the component ß-strands unfold sequentially. Based on our results a model of TTR protofilament assembly is proposed

Dynamic Strength of Titin's Z-Disk End

Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment

Exclusion-zone dynamics explored with microfluidics and optical tweezers

The exclusion zone (EZ) is a boundary region devoid of macromolecules and microscopic particles formed spontaneously in the vicinity of hydrophilic surfaces. The exact mechanisms behind this remarkable phenomenon are still not fully understood and are debated. We measured the short- and long-time-scale kinetics of EZ formation around a Nafion gel embedded in specially designed microfluidic devices. The time-dependent kinetics of EZ formation follow a power law with an exponent of 0.6 that is strikingly close to the value of 0.5 expected for a diffusion-driven process. By using optical tweezers we show that exclusion forces, which are estimated to fall in the sub-pN regime, persist within the fully-developed EZ, suggesting that EZ formation is not a quasi-static but rather an irreversible process. Accordingly, the EZ-forming capacity of the Nafion gel could be exhausted with time, on a scale of hours in the presence of 1 mM Na2HPO4. EZ formation may thus be a non-equilibrium thermodynamic cross-effect coupled to a diffusion-driven transport process. Such phenomena might be particularly important in the living cell by providing mechanical cues within the complex cytoplasmic environment. © 2014 by the authors; licensee MDPI, Basel, Switzerland

Cardiac Computed Tomography Radiomics: A Comprehensive Review on Radiomic Techniques

Radiologic images are vast three-dimensional data sets in which each voxel of the underlying volume represents distinct physical measurements of a tissue-dependent characteristic. Advances in technology allow radiologists to image pathologies with unforeseen detail, thereby further increasing the amount of information to be processed. Even though the imaging modalities have advanced greatly, our interpretation of the images has remained essentially unchanged for decades. We have arrived in the era of precision medicine where even slight differences in disease manifestation are seen as potential target points for new intervention strategies. There is a pressing need to improve and expand the interpretation of radiologic images if we wish to keep up with the progress in other diagnostic areas. Radiomics is the process of extracting numerous quantitative features from a given region of interest to create large data sets in which each abnormality is described by hundreds of parameters. From these parameters datamining is used to explore and establish new, meaningful correlations between the variables and the clinical data. Predictive models can be built on the basis of the results, which may broaden our knowledge of diseases and assist clinical decision making. Radiomics is a complex subject that involves the interaction of different disciplines; our objective is to explain commonly used radiomic techniques and review current applications in cardiac computed tomography imaging.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/

Microfluidic channels laser-cut in thin double-sided tapes: cost-effective biocompatible fluidics in minutes from design to final integration with optical biochips

A simple, reliable and cost-effective fluidic channel, fabricated by using double-sided pressure-sensitive tapes, is demonstrated here. A laser-cutting method is applied to engrave structures in sheets of the tapes. After peeling off the tape liners, the structures could be easily integrated at room temperature with label-free optical waveguide biochips without further modifications or additional processing steps. It is shown that the well-defined and controllable height of the channels is advantageous for stopped-flow measurements of analyte binding. The easy fabrication of a fully transparent integrated sensor unit – tape cuvette system is also demonstrated for parallel microscopic investigations. The transparent unit was used to on-line monitor the surface adhesion of Salmonella cells on poly-l-lysine-coated biochip surfaces, followed by the straightforward microscopic visualization of the adhered bacterial cells. The material of the double sided tape is stable in aqueous solutions. Furthermore, its material is biocompatible, making it ideal for biological applications. Excellent, stable and reversible bonding of the microstructured tapes to biocompatible plastic and glass is also demonstrated. The simplicity of the fabrication at ambient temperatures makes the developed processes appealing for lab-on-a-chip applications, particularly if the bonded biochips are precious

Reducing data dimension boosts neural network-based stage-specific malaria detection

Although malaria has been known for more than 4 thousand years(1), it still imposes a global burden with approx. 240 million annual cases(2). Improvement in diagnostic techniques is a prerequisite for its global elimination. Despite its main limitations, being time-consuming and subjective, light microscopy on Giemsa-stained blood smears is still the gold-standard diagnostic method used worldwide. Autonomous computer assisted recognition of malaria infected red blood cells (RBCs) using neural networks (NNs) has the potential to overcome these deficiencies, if a fast, high-accuracy detection can be achieved using low computational power and limited sets of microscopy images for training the NN. Here, we report on a novel NN-based scheme that is capable of the high-speed classification of RBCs into four categories—healthy ones and three classes of infected ones according to the parasite age—with an accuracy as high as 98%. Importantly, we observe that a smart reduction of data dimension, using characteristic one-dimensional cross-sections of the RBC images, not only speeds up the classification but also significantly improves its performance with respect to the usual two-dimensional NN schemes. Via comparative studies on RBC images recorded by two additional techniques, fluorescence and atomic force microscopy, we demonstrate that our method is universally applicable for different types of microscopy images. This robustness against imaging platform-specific features is crucial for diagnostic applications. Our approach for the reduction of data dimension could be straightforwardly generalised for the classification of different parasites, cells and other types of objects