[The Structural Features of Skeletal Muscle Titin Aggregates].

Titin is a multidomain protein of striated and smooth muscles of vertebrates. The protein consists of repeating immunoglobulin-like (Ig) and fibronectin-like (FnIII) domains, which are ß-sandwiches with a predominant ß-structure, and also contains disordered regions. In this work, the methods of atomic force microscopy (AFM), X-ray diffraction, and Fourier transform infrared spectroscopy were used to study the morphology and structure of aggregates of rabbit skeletal muscle titin obtained in two different solutions: 0.15 M glycine-KOH, pH 7.0 and 200 mM KCl, 10 mM imidazole, pH 7.0. According to AFM data, skeletal muscle titin formed amorphous aggregates of different morphologies in the above two solutions. Amorphous aggregates of titin formed in a solution containing glycine consisted of much larger particles than aggregates of this protein formed in a solution containing KCl. The "KCl-aggregates" according to AFM data had the form of a "sponge"-like structure, while amorphous "glycine-aggregates" of titin formed "branching" structures. Spectrofluorometry revealed the ability of "glycine-aggregates" of titin to bind to the dye thioflavin T (TT), and X-ray diffraction revealed the presence of one of the elements of the amyloid cross ß-structure, a reflection of ~4.6 Å, in these aggregates. These data indicate that "glycine-aggregates" of titin are amyloid or amyloid-like. No similar structural features were found in "KCl-aggregates" of titin; they also did not show the ability to bind to thioflavin T, indicating the non-amyloid nature of these titin aggregates. Fourier transform infrared spectroscopy revealed differences in the secondary structure of the two types of titin aggregates. The data we obtained demonstrate the features of structural changes during the formation of intermolecular bonds between molecules of the giant titin protein during its aggregation. The data expand the understanding of the process of amyloid protein aggregation.

Hevea brasiliensis rubber particles' fluid interfaces reveal size impact on early coagulation steps.

Natural rubber originates from the coagulation of rubber particles (RP) from Hevea brasiliensis latex. The size distribution of Hevea RP is bimodal with the presence of small rubber particles (SRP) and large rubber particles (LRP). This study aims at getting a better understanding of the early coagulation steps of Hevea RP taking into account the particle size. SRP and LRP were obtained by centrifugation of freshly tapped ammonia-free latex from RRIM600 clone. Size and zeta potential measurements showed that both RP fractions were efficiently separated and stable in basic buffer. SRP and LRP dispersions were placed in a Langmuir trough and RP were let to adsorb at the air-liquid interface to form interfacial films. Surface tension and ellipsometry indicate that the formation kinetics and the stabilization of the film at the air-liquid interface are faster for SRP than LRP. Moreover, the arrangement of RP at the interface differs between SRP and LRP, as shown by Brewster angle microscopy, atomic force microscopy and confocal laser scanning microscopy. First, the RP membrane and cis-1,4-polyisoprene core spread at the air-liquid interface before clustering. Then, while the SRP fuse, the LRP keep their structure in individual particles in floating aggregate. The role of the non-isoprene molecules on the different organization of SRP and LRP films is discussed, the one of the two major RP proteins, SRPP1 (Small Rubber Particle Protein) and Rubber Elongation Factor (REF1) in the early coagulation steps.

Investigating the role of extracellular polymeric substances produced by Parachlorella kessleri in Zn(II) bioremediation using atomic force microscopy.

Microalgae, such as Parachlorella kessleri, have significant potential for environmental remediation, especially in removing heavy metals like zinc from water. This study investigates how P. kessleri, isolated from a polluted river in Argentina, can remediate zinc. Using atomic force microscopy (AFM), the research examined the interactions between Zn particles and cells grown with different nitrogen sources-nitrate or ammonium. The results showed that cells grown with nitrate produced extracellular polymeric substances (EPS), while those grown with ammonium did not. Raman spectroscopy revealed distinct metabolic responses based on the nitrogen source, with nitrate-grown cells showing altered profiles after zinc exposure. Zinc exposure also changed the surface roughness and nanomechanical properties of the cells, particularly in those producing EPS. AFM force spectroscopy experiments then confirmed strong Zn binding to EPS in nitrate-grown cells, while interactions were weaker in ammonium-grown cells that lacked EPS. Overall, our results elucidate the critical role of EPS in Zn removal by P. kessleri cells and show that Zn remediation is mediated by EPS adsorption. This study underscores the significance of regulating nitrogen sources to stimulate EPS production, offering insights that are essential for subsequent bioremediation applications.

Facile fabrication of ZnO nanoparticles via non-thermal plasma technique and their anti-corrosive effects on X60 API 5L steel in 1M HCl solution.

This work aims to explore the efficiency of ZnO nanoparticles synthesized via the non-thermal gliding arc discharge-assisted plasma (NT-GAD) technique for inhibiting the corrosion of X60 API 5L steel in a 1M HCl environment. The XRD pattern revealed that the ZnO nanoparticles exhibit hexagonal wurtzite structure with average particle size of ∼24 nm. UV-visible spectroscopy analysis revealed an absorption peak centering at 365 nm, corresponding to an energy band gap of 3.29 eV. SEM and TEM analysis revealed that the nanoparticles exhibit an agglomerated and irregular morphology. The corrosion inhibition of ZnO NPs was investigated via the electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization tests (PDP), while varying both concentration and temperature. The results revealed that the increase in inhibitor concentration resulted in a higher activity at ambient temperature, with an optimal efficiency of 93 % at a concentration of 100 mg/L. However, the increase in temperature remarkably reduced the inhibition efficiency, suggesting a physisorption behavior of ZnO NPs onto the steel surface. AFM and FE-SEM analysis confirmed the formation of a protective layer on the X60 API 5L steel surface. This study emphasizes the significant potential of ZnO NPs synthesized via the NT-GAD assisted plasma technique as corrosion inhibitor for X60 API 5L carbon steel in 1M HCl corrosive media.

Unveiling the Dynamic Mechanism of SARS-CoV-2 Entry Host Cells at the Single-Particle Level.

Understanding the dynamic features of severe acute respiratory coronavirus 2 (SARS-CoV-2) binding to the cell membrane and entry cells is crucial for comprehending viral pathogenesis and transmission and facilitating the development of effective drugs against COVID-19. Herein, we employed atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) to study the binding dynamics between the virus and cell membrane. Our findings revealed that the Omicron variant of SARS-CoV-2 virus-like particles (VLPs) exhibited a slightly stronger affinity for the angiotensin-converting enzyme-2 (ACE2) receptor compared with the Delta variant and was significantly higher than the wild-type (WT). Using a real-time force-tracing technique, we quantified the dynamic parameters for a single SARS-CoV-2 VLP entry into cells, showing that approximately 200 ms and 60 pN are required. The parameters aligned with the analysis obtained from coarse-grained molecular dynamics (CGMD) simulations. Additionally, the Omicron variant invades cells at a higher entry cell speed, smaller force, and higher probability. Furthermore, single-particle fluorescence tracking visually demonstrated clathrin-dependent endocytosis for SARS-CoV-2 entry into A549 cells. The dynamic features of endocytosis provide valuable insights into the SARS-CoV-2 entry mechanism and possible intervention strategies targeting the viral infection process.

Complement-mediated killing of Escherichia coli by mechanical destabilization of the cell envelope.

Complement proteins eliminate Gram-negative bacteria in the blood via the formation of membrane attack complex (MAC) pores in the outer membrane. However, it remains unclear how outer membrane poration leads to inner membrane permeation and cell lysis. Using atomic force microscopy (AFM) on living Escherichia coli (E. coli), we probed MAC-induced changes in the cell envelope and correlated these with subsequent cell death. Initially, bacteria survived despite the formation of hundreds of MACs that were randomly distributed over the cell surface. This was followed by larger-scale disruption of the outer membrane, including propagating defects and fractures, and by an overall swelling and stiffening of the bacterial surface, which precede inner membrane permeation. We conclude that bacterial cell lysis is only an indirect effect of MAC formation; outer membrane poration leads to mechanical destabilization of the cell envelope, reducing its ability to contain the turgor pressure, leading to inner membrane permeation and cell death.

Atomically Precise Control of Topological State Hybridization in Conjugated Polymers.

Realization of topological quantum states in carbon nanostructures has recently emerged as a promising platform for hosting highly coherent and controllable quantum dot spin qubits. However, their adjustable manipulation remains elusive. Here, we report the atomically accurate control of the hybridization level of topologically protected quantum edge states emerging from topological interfaces in bottom-up-fabricated π-conjugated polymers. Our investigation employed a combination of low-temperature scanning tunneling microscopy and spectroscopy, along with high-resolution atomic force microscopy, to effectively modify the hybridization level of neighboring edge states by the selective dehydrogenation reaction of molecular units in a pentacene-based polymer and demonstrate their reversible character. Density functional theory, tight binding, and complete active space calculations for the Hubbard model were employed to support our findings, revealing that the extent of orbital overlap between the topological edge states can be finely tuned based on the geometry and electronic bandgap of the interconnecting region. These results demonstrate the utility of topological edge states as components for designing complex quantum arrangements for advanced electronic devices.

Ex vivo SIM-AFM measurements reveal the spatial correlation of stiffness and molecular distributions in 3D living tissue.

Living tissues each exhibit a distinct stiffness, which provides cells with key environmental cues that regulate their behaviors. Despite this significance, our understanding of the spatiotemporal dynamics and the biological roles of stiffness in three-dimensional tissues is currently limited due to a lack of appropriate measurement techniques. To address this issue, we propose a new method combining upright structured illumination microscopy (USIM) and atomic force microscopy (AFM) to obtain precisely coordinated stiffness maps and biomolecular fluorescence images of thick living tissue slices. Using mouse embryonic and adult skin as a representative tissue with mechanically heterogeneous structures inside, we validate the measurement principle of USIM-AFM. Live measurement of tissue stiffness distributions revealed the highly heterogeneous mechanical nature of skin, including nucleated/enucleated epithelium, mesenchyme, and hair follicle, as well as the role of collagens in maintaining its integrity. Furthermore, quantitative analysis comparing stiffness distributions in live tissue samples with those in preserved tissues, including formalin-fixed and cryopreserved tissue samples, unveiled the distinct impacts of preservation processes on tissue stiffness patterns. This series of experiments highlights the importance of live mechanical testing of tissue-scale samples to accurately capture the true spatiotemporal variations in mechanical properties. Our USIM-AFM technique provides a new methodology to reveal the dynamic nature of tissue stiffness and its correlation with biomolecular distributions in live tissues and thus could serve as a technical basis for exploring tissue-scale mechanobiology. STATEMENT OF SIGNIFICANCE: Stiffness, a simple mechanical parameter, has drawn attention in understanding the mechanobiological principles underlying the homeostasis and pathology of living tissues. To explore tissue-scale mechanobiology, we propose a technique integrating an upright structured illumination microscope and an atomic force microscope. This technique enables live measurements of stiffness distribution and fluorescent observation of thick living tissue slices. Experiments revealed the highly heterogeneous mechanical nature of mouse embryonic and adult skin in three dimensions and the previously unnoticed influences of preservation techniques on the mechanical properties of tissue at microscopic resolution. This study provides a new technical platform for live stiffness measurement and biomolecular observation of tissue-scale samples with micron-scale resolution, thus contributing to future studies of tissue- and organ-scale mechanobiology.

From Proteomics to the Analysis of Single Protein Molecules.

Limit of detection (LoD) is a term that is used to characterize the sensitivity of an analytical method. The existing limitation of the sensitivity of analysis using modern mass spectrometry methods has been experimentally shown to be a limiting factor in the application of proteomic technologies in medicine. This article proposes a concept of a new technology that will set a new vector of development in the development of systems for solving problems of medical diagnostics and deals with theoretical and practical aspects of creating a new technology for the detection of single biomacromolecules (in particular, proteins) in biological samples. Such technology should be based on the principle of signal registration similar to that used in a Geiger counter (also known as a Geiger-Müller counter or G-M counter), a device that automatically counts the number of ionizing particles that hit it. This counter is free from probabilistic components; it registers a signal if there is at least one target molecule in the analysis chamber. Predictive medical diagnostics require technology based on methods where sensitivity allows for the detection of single marker molecules in a biological sample volume of 1-10 µL, the smallest volume of biomaterial used in laboratory diagnostics. Creation of a detector with a sensitivity of 10-18 M would allow for the detection of one molecule in 1 µL of the sample, which fundamentally makes this approach analogous to a G-M counter for solutions. To date, bioanalytical methods are limited to a sensitivity of 10-12 M (which is approximately 1 million molecules per 1 µL), which is insufficient to capture the early stages of pathological processes.

Mica Lattice Orientation of Epitaxially Grown Amyloid ß25-35 Fibrils.

ß-amyloid (Aß) peptides form self-organizing fibrils in Alzheimer's disease. The biologically active, toxic Aß25-35 fragment of the full-length Aß-peptide forms a stable, oriented filament network on the mica surface with an epitaxial mechanism at the timescale of seconds. While many of the structural and dynamic features of the oriented Aß25-35 fibrils have been investigated before, the ß-strand arrangement of the fibrils and their exact orientation with respect to the mica lattice remained unknown. By using high-resolution atomic force microscopy, here, we show that the Aß25-35 fibrils are oriented along the long diagonal of the oxygen hexagon of mica. To test the structure and stability of the oriented fibrils further, we carried out molecular dynamics simulations on model ß-sheets. The models included the mica surface and a single fibril motif built from ß-strands. We show that a sheet with parallel ß-strands binds to the mica surface with its positively charged groups, but the C-terminals of the strands orient upward. In contrast, the model with antiparallel strands preserves its parallel orientation with the surface in the molecular dynamics simulation, suggesting that this model describes the first ß-sheet layer of the mica-bound Aß25-35 fibrils well. These results pave the way toward nanotechnological construction and applications for the designed amyloid peptides.

Single-Molecule-Level Quantification Based on Atomic Force Microscopy Data Reveals the Interaction between Melittin and Lipopolysaccharide in Gram-Negative Bacteria.

The interaction forces and mechanical properties of the interaction between melittin (Mel) and lipopolysaccharide (LPS) are considered to be crucial driving forces for Mel when killing Gram-negative bacteria (GNB). However, how their interaction forces perform at the single-molecule level and the dissociation kinetic characteristics of the Mel/LPS complex remain poorly understood. In this study, the single-molecule-level interaction forces between Mel and LPSs from E. coli K-12, O55:B5, O111:B4, and O128:B12 were explored using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS). AFM-based dynamic force spectroscopy (DFS) and an advanced analytical model were employed to investigate the kinetic characteristics of the Mel/LPS complex dissociation. The results indicated that Mel could interact with both rough (R)-form LPS (E. coli K-12) and smooth (S)-form LPSs (E. coli O55:B5, O111:B4, and O128:B12). The S-form LPS showed a more robust interaction with Mel than the R-form LPS, and a slight difference existed in the interaction forces between Mel and the diverse S-form LPS. Mel interactions with the S-form LPSs showed greater specific and non-specific interaction forces than the R-form LPS (p < 0.05), as determined by AFM-based SMFS. However, there was no significant difference in the specific and non-specific interaction forces among the three samples of S-form LPSs (p > 0.05), indicating that the variability in the O-antigen did not affect the interaction between Mel and LPSs. The DFS result showed that the Mel/S-form LPS complexes had a lower dissociation rate constant, a shorter energy barrier width, a longer bond lifetime, and a higher energy barrier height, demonstrating that Mel interacted with S-form LPS to form more stable complexes. This research enhances the existing knowledge of the interaction micromechanics and kinetic characteristics of Mel and LPS at the single-molecule level. Our research may help with the design and evaluation of new anti-GNB drugs.

Morphological changes and luminescence of Escherichia coli in contact with Mn2O3 and Co3O4 ultrafine particles as components of a mineral feed additive.

Background and Aim: The spread of antibiotic resistance and mineral depletion in soils encourages an intensive search for highly effective and environmentally safe bactericidal agents and sources of macro- and micro-elements. The most profitable solution would combine both the described tasks. Ultrafine particles (UFPs) have this functionality. Thus, this study aimed to analyze the bioluminescence and external morphological changes of Escherichia coli cells after contact with M2O3 and Co3O4 UFPs at effective concentrations (ECs). Materials and Methods: The antibiotic properties of the studied samples were determined on a multifunctional microplate analyzer TECAN Infinite F200 (Tecan Austria GmbH, Austria) by fixing the luminescence value of the bacterial strain E. coli K12 TG11 (Ecolum, NVO Immunotech Closed Joint Stock Company, Russia). Morphological changes in the cell structure were evaluated using a Certus Standard EG-5000 atomic force microscope equipped with NSPEC software (Nano Scan Technology LLC, Russia). Results: The obtained results indicate high bactericidal properties of Co3O4 and Mn2O3 UFPs (EC50 at 3.1 × 10-5 and 1.9 × 10-3 mol/L, respectively) due to the degradation of the cell wall, pathological increase in size, disruption of septic processes, and loss of cytoplasmic contents. Conclusion: The prospects for the environmentally safe use of ultrafine materials are outlined. The limits of the dosages of Co3O4 and Mn2O3 UFPs recommended for further study in vitro and in vivo in feeding farm animals are established (no more than 4.9 × 10-4 mol/L for Mn2O3 UFPs and 1.5 × 10-5 mol/L for Co3O4 UFPs). The limitation of the work is the lack of experiments to determine the mechanisms of the toxic effect of UFP on bacteria, protein structures, and DNA and oxidative stress, which is planned to be performed in the future together with in situ and in vivo studies on animals.

AFM for Nanomechanical Assessment of Polymer Overcoatings on Nanoparticle-Decorated Biomaterials.

Nanoparticle adhesion to polymer and similar substrates may be prone to low nano-Newton forces, disrupting the surface bonds and patterning, potentially reducing the functionality of complex surface patterns. Testing this, a functionalised surface reported for biological and medical applications, consisting of a thin plasma-derived oxazoline-based film with 68 nm diameter covalently bound colloidal gold nanoparticles attached within an aqueous solution, underwent nanomechanical analysis. Atomic Force Microscopy nanomechanical analysis was used to quantify the limits of various adaptations to these nanoparticle-featured substrates. Regular and laterally applied forces in the nano-Newton range were shown to de-adhere surface-bound gold nanoparticles. Applying a nanometre-thick overcoating anchored the nanoparticles to the surface and protected the underlying base substrate in a one-step process to improve the overall stability of the functionalised substrate against lower-range forces. The thickness of the oxazoline-based overcoating displayed protection from forces at different rates. Testing overcoating thickness ranging from 5 to 20 nm in 5 nm increments revealed a significant improvement in stability using a 20 nm-thick overcoating. This approach underscores the importance of optimising overcoating thickness to enhance nanoparticle-based surface modifications' durability and functional integrity.

Automated High-Throughput Atomic Force Microscopy Single-Cell Nanomechanical Assay Enabled by Deep Learning-Based Optical Image Recognition.

Mechanical forces are essential for life activities, and the mechanical phenotypes of single cells are increasingly gaining attention. Atomic force microscopy (AFM) has been a standard method for single-cell nanomechanical assays, but its efficiency is limited due to its reliance on manual operation. Here, we present a study of deep learning image recognition-assisted AFM that enables automated high-throughput single-cell nanomechanical measurements. On the basis of the label-free identification of the cell structures and the AFM probe in optical bright-field images as well as the consequent automated movement of the sample stage and AFM probe, the AFM probe tip could be accurately and sequentially moved onto the specific parts of individual living cells to perform a single-cell indentation assay or single-cell force spectroscopy in a time-efficient manner. The study illustrates a promising method based on deep learning for achieving operator-independent high-throughput AFM single-cell nanomechanics, which will benefit the application of AFM in mechanobiology.

Structure and Defect Identification at Self-Assembled Islands of CO2 Using Scanning Probe Microscopy.

Understanding how carbon dioxide (CO2) behaves and interacts with surfaces is paramount for the development of sensors and materials to attempt CO2 mitigation and catalysis. Here, we combine simultaneous atomic force microscopy (AFM) and scanning tunneling microscopy (STM) using CO-functionalized probes with density functional theory (DFT)-based simulations to gain fundamental insight into the behavior of physisorbed CO2 molecules on a gold(111) surface that also contains one-dimensional metal-organic chains formed by 1,4-phenylene diisocyanide (PDI) bridged by gold (Au) adatoms. We resolve the structure of self-assembled CO2 islands, both confined between the PDI-Au chains as well as free-standing on the surface and reveal a chiral arrangement of CO2 molecules in a windmill-like structure that encloses a standing-up CO2 molecule and other foreign species existing at the surface. We identify these species by the comparison of height-dependent AFM and STM imaging with DFT-calculated images and clarify the origin of the kagome tiling exhibited by this surface system. Our results show the complementarity of AFM and STM using functionalized probes and their potential, when combined with DFT, to explore greenhouse gas molecules at surface-supported model systems.

Sensing Biomolecules Associated with Cells' Radiosusceptibility by Advanced Micro- and Nanospectroscopy Techniques.

Radiotherapy is one of the most common approaches for cancer treatment, especially in the case of peripheral nervous system tumors. As it requires exposure to high doses of ionizing radiation, it is important to look for substances that support efficient reduction of the tumor volume with simultaneous prevention of the surrounding noncancerous cells. Cannabidiol (CBD), which exhibits both anticancer and neuroprotective properties, was applied as a potential modulator of radiological response; however, its influence on cells undergoing irradiation remains elusive. Here, we have applied high-resolution optical spectroscopy techniques to capture biomolecules associated with CBD shielding of normal and damaging cancerous cells upon X-ray exposure. Conventional Raman (RS) and Fourier transformed infrared (FT-IR) spectroscopies provided semiquantitative information mainly about changes in the concentration of total lipids, DNA, cholesteryl esters, and phospholipids in cells. A through assessment of the single cells by atomic force microscopy coupled with infrared spectroscopy (AFM-IR) allowed us to determine not only the alterations in DNA content but also in its conformation due to cell treatment. Pronounced nanoscale changes in cholesteryl ester metabolites, associated with CBD treatment and radiation, were also observed. AFM-IR chemoselective maps of the single cells indicate the modified distribution of cholesteryl esters with 40 nm spatial resolution. Based on the obtained results, we propose a label-free and fast analytical method engaging optical spectroscopy to assess the mechanism of normal and cancerous cell susceptibility to ionizing radiation when pretreated with CBD.

Morphological and Biophysical Study of S100A9 Protein Fibrils by Atomic Force Microscopy Imaging and Nanomechanical Analysis.

Atomic force microscopy (AFM) imaging enables the visualization of protein molecules with high resolution, providing insights into their shape, size, and surface topography. Here, we use AFM to study the aggregation process of protein S100A9 in physiological conditions, in the presence of calcium at a molar ratio 4Ca2+:S100A9. We find that S100A9 readily assembles into a worm-like fibril, with a period dimension along the fibril axis of 11.5 nm. The fibril's chain length extends up to 136 periods after an incubation time of 144 h. At room temperature, the fibril's bending stiffness was found to be 2.95×10-28 Nm2, indicating that the fibrils are relatively flexible. Additionally, the values obtained for the Young's modulus (Ex=6.96×105 Pa and Ey=3.37×105 Pa) are four orders of magnitude lower than those typically reported for canonical amyloid fibrils. Our findings suggest that, under the investigated conditions, a distinct aggregation mechanism may be in place in the presence of calcium. Therefore, the findings reported here could have implications for the field of biomedicine, particularly with regard to Alzheimer's disease.

Membrane Activity and Viroporin Assembly for the SARS-CoV-2 E Protein Are Regulated by Cholesterol.

The SARS-CoV-2 E protein is an enigmatic viral structural protein with reported viroporin activity associated with the acute respiratory symptoms of COVID-19, as well as the ability to deform cell membranes for viral budding. Like many viroporins, the E protein is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the structure of the protein complex have yielded inconclusive results, suggesting several possible oligomers, ranging from dimers to pentamers. Here, we combined patch-clamp, confocal fluorescence microscopy on giant unilamellar vesicles, and atomic force microscopy to show that E protein can exhibit two modes of membrane activity depending on membrane lipid composition. In the absence or the presence of a low content of cholesterol, the protein forms short-living transient pores, which are seen as semi-transmembrane defects in a membrane by atomic force microscopy. Approximately 30 mol% cholesterol is a threshold for the transition to the second mode of conductance, which could be a stable pentameric channel penetrating the entire lipid bilayer. Therefore, the E-protein has at least two different types of activity on membrane permeabilization, which are regulated by the amount of cholesterol in the membrane lipid composition and could be associated with different types of protein oligomers.

Cytoarchitecture of Breast Cancer Cells under Diabetic Conditions: Role of Regulatory Kinases-Rho Kinase and Focal Adhesion Kinase.

Diabetes greatly reduces the survival rates in breast cancer patients due to chemoresistance and metastasis. Reorganization of the cytoskeleton is crucial to cell migration and metastasis. Regulatory cytoskeletal protein kinases such as the Rho kinase (ROCK) and focal adhesion kinase (FAK) play a key role in cell mobility and have been shown to be affected in cancer. It is hypothesized that diabetes/high-glucose conditions alter the cytoskeletal structure and, thus, the elasticity of breast cancer cells through the ROCK and FAK pathway, which can cause rapid metastasis of cancer. The aim of the study was to investigate the role of potential mediators that affect the morphology of cancer cells in diabetes, thus leading to aggressive cancer. Breast cancer cells (MDA-MB-231 and MCF-7) were treated with 5 mM glucose (low glucose) or 25 mM glucose (high glucose) in the presence of Rho kinase inhibitor (Y-27632, 10 mM) or FAK inhibitor (10 mM). Cell morphology and elasticity were monitored using atomic force microscopy (AFM), and actin staining was performed by fluorescence microscopy. For comparative study, normal mammary breast epithelial cells (MCF-10A) were used. It was observed that high-glucose treatments modified the cytoskeleton of the cells, as observed through AFM and fluorescence microscopy, and significantly reduced the elasticity of the cells. Blocking the ROCK or FAK pathway diminished the high-glucose effects. These changes were more evident in the breast cancer cells as compared to the normal cells. This study improves the knowledge on the cytoarchitecture properties of diabetic breast cancer cells and provides potential pathways that can be targeted to prevent such effects.

Applying the Techniques of Materials Science towards an Understanding of the Process of Canine Intervertebral Disc Degeneration.

Intervertebral disc degeneration in dogs occurs in an accelerated way and involves calcification, which is associated with disc herniation or extrusion. The degenerative process is complex and involves the transformation of collagen fibres, loss of proteoglycans and notochord cells and a reduction in water content; however, how these processes are linked to future disc extrusion remains unknown. We have employed techniques including Fourier Transform Infra-red Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Uniaxial Compression Loading and Atomic Force Microscopy (AFM) in an attempt to gain a greater understanding of the degenerative process and its consequences on the physical properties of the disc. FTIR verified by TEM demonstrated that calcium phosphate exists in an amorphous state within the disc and that the formation of crystalline particles of hydroxyapatite occurs prior to disc extrusion. AFM identified crystalline agglomerates consistent with hydroxyapatite as well as individual collagen fibres. SEM enabled the identification of regions rich in calcium, phosphorous and oxygen and allowed the visualization of the topographical landscape of the disc. Compression testing generated stress/strain curves which will facilitate investigation into disc stiffness. Ongoing work is aimed at identifying potential areas of intervention in the degenerative process as well as further characterizing the role of calcification in disc extrusion.