Search Results (18)

Single-molecule force spectroscopy (SMFS) experiments can monitor protein refolding by applying a small force of a few piconewtons (pN) and slowing down the folding process. Bell theory predicts that in the narrow force regime where refolding can occur, the folding time should increase exponentially with increased external force. In this work, using coarse-grained molecular dynamics simulations, we compared the refolding pathways of SARS-CoV-1 RBD and SARS-CoV-2 RBD (RBD refers to the receptor binding domain) starting from unfolded conformations with and without a force applied to the protein termini. For SARS-CoV-2 RBD, the number of trajectories that fold is significantly reduced with the application of a 5 pN force, indicating that, qualitatively consistent with Bell theory, refolding is slowed down when a pulling force is applied to the termini. In contrast, the refolding times of SARS-CoV-1 RBD do not change meaningfully when a force of 5 pN is applied. How this lack of a Bell response could arise at the molecular level is unknown. Analysis of the entanglement changes of the folded conformations revealed that in the case of SARS-CoV-1 RBD, an external force minimizes misfolding into kinetically trapped states, thereby promoting efficient folding and offsetting any potential slowdown due to the external force. These misfolded states contain non-native entanglements that do not exist in the native state of either SARS-CoV-1-RBD or SARS-CoV-2-RBD. These results indicate that non-Bell behavior can arise from this class of misfolding and, hence, may be a means of experimentally detecting these elusive, theoretically predicted states. Full article

Scanning force microscopy (SFM) is one of the most widely used techniques in biomaterials research. In addition to imaging the materials of interest, SFM enables the mapping of mechanical properties and biological responses with sub-nanometer resolution and piconewton sensitivity. This review aims to give an overview of using the scanning force microscope (SFM) for investigations on dental materials. In particular, SFM-derived methods such as force–distance curves (scanning force spectroscopy), lateral force spectroscopy, and applications of the FluidFM^® will be presented. In addition to the properties of dental materials, this paper reports the development of the pellicle by the interaction of biopolymers such as proteins and polysaccharides, as well as the interaction of bacteria with dental materials. Full article

Atomic force microscopy (AFM) is a powerful technique to study the nanomechanical properties of a wide range of materials at the piconewton level. AFM force–indentation curves can be fitted with appropriate contact models, enabling the determination of material properties for a given sample. However, the analysis of large datasets comprising thousands of curves using conventional methods presents a time-intensive challenge. As a result, there is an increasing interest in exploring alternative methodologies, such as integrating machine learning (ML) models to streamline and improve the efficiency of this process. In this work, two data-driven regressors were tuned to predict the Young’s modulus and adhesion energy from force–indentation curves of soft samples (Young’s modulus up to 10 kPa). Both models were trained exclusively on synthetic data derived from the contact theories developed by Hertz as well as Johnson, Kendall and Roberts (JKR). The PyTorch library was employed to build and train the models; then, the key hyperparameters were refined by implementing the optimization framework Optuna. The first model was successfully tested with synthetic and experimental curves from AFM nanoindentations, and the second presented promising results on the synthetic data. Our work suggests that experimental data may not be essential for training data-driven models to predict surface properties from AFM nanoindentations. By delivering accurate predictions in a computationally efficient way, our regressors validate the potential of a deep learning approach in exploring AFM nanoindentations and motivate further development of similar strategies to overcome current limitations in AFM postprocessing. Full article

Many phenomena observed in synthetic and biological colloidal suspensions are dominated by the static interaction energies and the hydrodynamic interactions that act both between individual particles and also between colloids and macroscopic interfaces. This calls for methods that allow precise measurements of the corresponding forces. One method used for this purpose is total internal reflection microscopy (TIRM), which has been employed for around three decades to measure in particular the interactions between a single particle suspended in a liquid and a solid surface. However, given the importance of the observable variables, it is crucial to understand the possibilities and limitations of the method. In this paper, we investigate the influence of technically unavoidable noise effects and an inappropriate choice of particle size and sampling time on TIRM measurement results. Our main focus is on the measurement of diffusion coefficients and drift velocities, as the influence of error sources on dynamic properties has not been investigated so far. We find that detector shot noise and prolonged sampling times may cause erroneous results in the steep parts of the interaction potential where forces of the order of pico-Newtons or larger act on the particle, while the effect of background noise is negligible below certain thresholds. Furthermore, noise does not significantly affect dynamic data but we find that lengthy sampling times and/or probe particles with too small a radius will cause issues. Most importantly, we observe that dynamic results are very likely to differ from the standard hydrodynamic predictions for stick boundary conditions due to partial slip. Full article

Atomic force microscopy (AFM) is a method that provides the nanometer-resolution three-dimensional imaging of living cells in their native state in their natural physiological environment. In addition, AFM’s sensitivity to measure interaction forces in the piconewton range enables researchers to probe surface properties, such as elasticity, viscoelasticity, hydrophobicity and adhesion. Despite the growing number of applications of AFM as a method to study biological systems, AFM is not yet an established technique for studying microalgae. Following a brief introduction to the basic principles and operation modes of AFM, this review highlights the major contributions of AFM in the field of microalgae research. A pioneering AFM study on microalgae was performed on diatoms, revealing the fine structural details of diatom frustule, without the need for sample modification. While, to date, diatoms are the most studied class of microalgae using AFM, it has also been used to study microalgae belonging to other classes. Besides using AFM for the morphological characterization of microalgae at the single cell level, AFM has also been used to study the surface properties of microalgal cells, with cell elasticity being most frequently studied one. Here, we also present our preliminary results on the viscoelastic properties of microalgae cell (Dunaliella tertiolecta), as the first microrheological study of microalgae. Overall, the studies presented show that AFM, with its multiparametric characterization, alone or in combination with other complementary techniques, can address many outstanding questions in the field of microalgae. Full article

Myometrium cells are an important reproductive niche in which cyclic mechanical forces of a pico-newton range are produced continuously at millisecond and second intervals. Overproduction and/or underproduction of micro-forces, due to point or epigenetic mutation, aberrant methylation, and abnormal response to hypoxia, may lead to the transformation of fibroid stem cells into fibroid-initiating stem cells. Fibroids are tumors with a high modulus of stiffness disturbing the critical homeostasis of the myometrium and they may cause unfavorable and strong mechanical forces. Micro-mechanical forces and soluble-chemical signals play a critical role in transcriptional and translational processes’ maintenance, by regulating communication between the cell nucleus and its organelles. Signals coming from the external environment can stimulate cells in the format of both soluble biochemical signals and mechanical ones. The shape of the cell and the plasma membrane have a significant character in sensing electro-chemical signals, through specialized receptors and generating responses, accordingly. In order for mechanical signals to be perceived by the cell, they must be converted into biological stimuli, through a process called mechanotransduction. Transmission of fibroid-derived mechanical signals to the endometrium and their effects on receptivity modulators are mediated through a pathway known as solid-state signaling. It is not sufficiently clear which type of receptors and mechanical signals impair endometrial receptivity. However, it is known that biomechanical signals reaching the endometrium affect epithelial sodium channels, lysophosphatidic acid receptors or Rho GTPases, leading to conformational changes in endometrial proteins. Translational changes in receptivity modulators may disrupt the selectivity and receptivity functions of the endometrium, resulting in failed implantation or early pregnancy loss. By hypermethylation of the receptivity genes, micro-forces can also negatively affect decidualization and implantation. The purpose of this narrative review is to summarize the state of the art of the biomechanical forces which can determine fibroid stem cell transformation and, thus, affect the receptivity status of the endometrium with regard to fertilization and pregnancy. Full article

Force-spectroscopy techniques have led to significant progress in studying the physicochemical properties of biomolecules that are not accessible in bulk assays. The application of piconewton forces with laser optical tweezers to single nucleic acids has permitted the characterization of molecular thermodynamics and kinetics with unprecedented accuracy. Some examples are the hybridization reaction between complementary strands in DNA and the folding of secondary, tertiary, and other heterogeneous structures, such as intermediate and misfolded states in RNA. Here we review the results obtained in our lab on deriving the nearest-neighbor free energy parameters in DNA and RNA duplexes from mechanical unzipping experiments. Remarkable nonequilibrium effects are also observed, such as the large irreversibility of RNA unzipping and the formation of non-specific secondary structures in single-stranded DNA. These features originate from forming stem-loop structures along the single strands of the nucleic acid. The recently introduced barrier energy landscape model quantifies kinetic trapping effects due to stem-loops being applicable to both RNA and DNA. The barrier energy landscape model contains the essential features to explain the many behaviors observed in heterogeneous nucleic-acid folding. Full article

We report on the observation of the detachment in situ and in vivo of Dunaliella tertiolecta microalgae cells from a glass surface using a 1064 nm wavelength trapping laser beam. The principal bends of both flagella of Dunaliella were seen self-adhered to either the top or bottom coverslip surfaces of a 50 $\mathsf{\mu }$m thick chamber. When a selected attached Dunaliella was placed in the trapping site, it photoresponded to the laser beam by moving its body and flagellar tips, which eventually resulted in its detachment. The dependence of the time required for detachment on the trapping power was measured. No significant difference was found in the detachment time for cells detached from the top or bottom coverslip, indicating that the induced detachment was not due solely to the optical forces applied to the cells. After detachment, the cells remained within the optical trap. Dunaliella detached from the bottom were seen rotating about their long axis in a counterclockwise direction, while those detached from the top did not rotate. The rotation frequency and the minimal force required to escape from the trap were also measured. The average rotation frequency was found to be independent of the trapping power, and the swimming force of a cell escaping the laser trap ranged from 4 to 10 picoNewtons. Our observations provide insight into the photostimulus produced when a near-infrared trapping beam encounters a Dunaliella. The microalgae frequently absorb more light than they can actually use in photosynthesis, which could cause genetic and molecular changes. Our findings may open new research directions into the study of photomovement in species of Dunaliella and other swimming microorganisms that could eventually help to solve technological problems currently confronting biomass production. In future work, studies of the response to excess light may uncover unrecognized mechanisms of photoprotection and photoacclimation. Full article

With recent advances in scanning probe microscopy (SPM), it is now routine to determine the atomic structure of surfaces and molecules while quantifying the local tip-sample interaction potentials. Such quantitative experiments using noncontact frequency modulation atomic force microscopy is based on the accurate measurement of the resonance frequency shift due to the tip-sample interaction. Here, we experimentally show that the resonance frequency of oscillating probes used for SPM experiments change systematically as a function of oscillation amplitude under typical operating conditions. This change in resonance frequency is not due to tip-sample interactions, but rather due to the cantilever strain or geometric effects and thus the resonance frequency is a function of the oscillation amplitude. Our numerical calculations demonstrate that the amplitude dependence of the resonance frequency is an additional yet overlooked systematic error source that can result in nonnegligible errors in measured interaction potentials and forces. Our experimental results and complementary numerical calculations reveal that the frequency shift due to this amplitude dependence needs to be corrected even for experiments with active oscillation amplitude control to be able to quantify the tip-sample interaction potentials and forces with milli-electron volt and pico-Newton resolutions. Full article

At the molecular level biology is intrinsically noisy. The forces that regulate the myriad of molecular reactions in the cell are tiny, on the order of piconewtons (10⁻¹² Newtons), yet they proceed in concerted action making life possible. Understanding how this is possible is one of the most fundamental questions biophysicists would like to understand. Single molecule experiments offer an opportunity to delve into the fundamental laws that make biological complexity surface in a physical world governed by the second law of thermodynamics. Techniques such as force spectroscopy, fluorescence, microfluidics, molecular sequencing, and computational studies project a view of the biomolecular world ruled by the conspiracy between the disorganizing forces due to thermal motion and the cosmic evolutionary drive. Here we will digress on some of the evidences in support of this view and the role of physical information in biology. Full article

The detection of long-chain biomolecules on mineral surfaces is presented using an atomic force microscope (AFM). This is achieved by using the AFM’s ability to manipulate molecules and measure forces at the pico-newton scale. We show that a highly characteristic force-distance signal is produced when the AFM tip is used to detach long-chain molecules from a surface. This AFM force spectroscopy method is demonstrated on bio-films, spores, fossils and mineral surfaces. The method works with AFM imaging and correlated tip enhanced infrared spectroscopy. The use of AFM force spectroscopy to detect this class of long chain bio-markers has applications in paleontology, life detection and planetary science. Full article

Morphogenesis remains a riddle, wrapped in a mystery, inside an enigma. It remains a formidable problem viewed from many different perspectives of morphology, genetics, and computational modelling. We propose a biochemical reductionist approach that shows how both internal and external physical forces contribute to plant morphogenesis via mechanical stress–strain transduction from the primary cell wall tethered to the plasma membrane by a specific arabinogalactan protein (AGP). The resulting stress vector, with direction defined by Hechtian adhesion sites, has a magnitude of a few piconewtons amplified by a hypothetical Hechtian growth oscillator. This paradigm shift involves stress-activated plasma membrane Ca²⁺ channels and auxin-activated H⁺-ATPase. The proton pump dissociates periplasmic AGP-glycomodules that bind Ca²⁺. Thus, as the immediate source of cytosolic Ca²⁺, an AGP-Ca²⁺ capacitor directs the vectorial exocytosis of cell wall precursors and auxin efflux (PIN) proteins. In toto, these components comprise the Hechtian oscillator and also the gravisensor. Thus, interdependent auxin and Ca²⁺ morphogen gradients account for the predominance of AGPs. The size and location of a cell surface AGP-Ca²⁺ capacitor is essential to differentiation and explains AGP correlation with all stages of morphogenetic patterning from embryogenesis to root and shoot. Finally, the evolutionary origins of the Hechtian oscillator in the unicellular Chlorophycean algae reflect the ubiquitous role of chemiosmotic proton pumps that preceded DNA at the dawn of life. Full article

We report the development of a magnetic tweezers that can be used to micromanipulate single DNA molecules by applying picoNewton (pN)-scale forces in the horizontal plane. The resulting force–extension data from our experiments show high-resolution detection of changes in the DNA tether’s extension: ~0.5 pN in the force and <10 nm change in extension. We calibrate our instrument using multiple orthogonal techniques including the well-characterized DNA overstretching transition. We also quantify the repeatability of force and extension measurements, and present data on the behavior of the overstretching transition under varying salt conditions. The design and experimental protocols are described in detail, which should enable straightforward reproduction of the tweezers. Full article

Recent trends on microbiology point out the urge to develop optical micro-tools with multifunctionalities such as simultaneous manipulation and sensing. Considering that miniaturization has been recognized as one of the most important paradigms of emerging sensing biotechnologies, optical fiber tools, including Optical Fiber Tweezers (OFTs), are suitable candidates for developing multifunctional small sensors for Medicine and Biology. OFTs are flexible and versatile optotools based on fibers with one extremity patterned to form a micro-lens. These are able to focus laser beams and exert forces onto microparticles strong enough (piconewtons) to trap and manipulate them. In this paper, through an exploratory analysis of a 45 features set, including time and frequency-domain parameters of the back-scattered signal of particles trapped by a polymeric lens, we created a novel single feature able to differentiate synthetic particles (PMMA and Polystyrene) from living yeasts cells. This single statistical feature can be useful for the development of label-free hybrid optical fiber sensors with applications in infectious diseases detection or cells sorting. It can also contribute, by revealing the most significant information that can be extracted from the scattered signal, to the development of a simpler method for particles characterization (in terms of composition, heterogeneity degree) than existent technologies. Full article

The advent of atomic force microscopy (AFM) has provided a powerful tool for investigating the behaviors of single native biological molecules under physiological conditions. AFM can not only image the conformational changes of single biological molecules at work with sub-nanometer resolution, but also sense the specific interactions of individual molecular pair with piconewton force sensitivity. In the past decade, the performance of AFM has been greatly improved, which makes it widely used in biology to address diverse biomedical issues. Characterizing the behaviors of single molecules by AFM provides considerable novel insights into the underlying mechanisms guiding life activities, contributing much to cell and molecular biology. In this article, we review the recent developments of AFM studies in single-molecule assay. The related techniques involved in AFM single-molecule assay were firstly presented, and then the progress in several aspects (including molecular imaging, molecular mechanics, molecular recognition, and molecular activities on cell surface) was summarized. The challenges and future directions were also discussed. Full article