Machine learning method for the classification of the state of living organisms' oscillations.

The World Health Organization highlights the urgent need to address the global threat posed by antibiotic-resistant bacteria. Efficient and rapid detection of bacterial response to antibiotics and their virulence state is crucial for the effective treatment of bacterial infections. However, current methods for investigating bacterial antibiotic response and metabolic state are time-consuming and lack accuracy. To address these limitations, we propose a novel method for classifying bacterial virulence based on statistical analysis of nanomotion recordings. We demonstrated the method by classifying living Bordetella pertussis bacteria in the virulent or avirulence phase, and dead bacteria, based on their cellular nanomotion signal. Our method offers significant advantages over current approaches, as it is faster and more accurate. Additionally, its versatility allows for the analysis of cellular nanomotion in various applications beyond bacterial virulence classification.

A simplified version of rapid susceptibility testing of bacteria and yeasts using optical nanomotion detection.

We present a novel optical nanomotion-based rapid antibiotic and antifungal susceptibility test. The technique consisted of studying the effects of antibiotics or antifungals on the nanometric scale displacements of bacteria or yeasts to assess their sensitivity or resistance to drugs. The technique relies on a traditional optical microscope, a video camera, and custom-made image analysis software. It provides reliable results in a time frame of 2-4 h and can be applied to motile, non-motile, fast, and slowly growing microorganisms. Due to its extreme simplicity and low cost, the technique can be easily implemented in laboratories and medical centers in developing countries.

Accurate and rapid antibiotic susceptibility testing using a machine learning-assisted nanomotion technology platform.

Antimicrobial resistance (AMR) is a major public health threat, reducing treatment options for infected patients. AMR is promoted by a lack of access to rapid antibiotic susceptibility tests (ASTs). Accelerated ASTs can identify effective antibiotics for treatment in a timely and informed manner. We describe a rapid growth-independent phenotypic AST that uses a nanomotion technology platform to measure bacterial vibrations. Machine learning techniques are applied to analyze a large dataset encompassing 2762 individual nanomotion recordings from 1180 spiked positive blood culture samples covering 364 Escherichia coli and Klebsiella pneumoniae isolates exposed to cephalosporins and fluoroquinolones. The training performances of the different classification models achieve between 90.5 and 100% accuracy. Independent testing of the AST on 223 strains, including in clinical setting, correctly predict susceptibility and resistance with accuracies between 89.5% and 98.9%. The study shows the potential of this nanomotion platform for future bacterial phenotype delineation.

An Optical Fiber-Based Nanomotion Sensor for Rapid Antibiotic and Antifungal Susceptibility Tests.

The emergence of antibiotic and antifungal resistant microorganisms represents nowadays a major public health issue that might push humanity into a post-antibiotic/antifungal era. One of the approaches to avoid such a catastrophe is to advance rapid antibiotic and antifungal susceptibility tests. In this study, we present a compact, optical fiber-based nanomotion sensor to achieve this goal by monitoring the dynamic nanoscale oscillation of a cantilever related to microorganism viability. High detection sensitivity was achieved that was attributed to the flexible two-photon polymerized cantilever with a spring constant of 0.3 N/m. This nanomotion device showed an excellent performance in the susceptibility tests of Escherichia coli and Candida albicans with a fast response in a time frame of minutes. As a proof-of-concept, with the simplicity of use and the potential of parallelization, our innovative sensor is anticipated to be an interesting candidate for future rapid antibiotic and antifungal susceptibility tests and other biomedical applications.

Mechanical Properties and Nanomotion of BT-20 and ZR-75 Breast Cancer Cells Studied by Atomic Force Microscopy and Optical Nanomotion Detection Method.

Cells of two molecular genetic types of breast cancer-hormone-dependent breast cancer (ZR-75 cell line) and triple-negative breast cancer (BT-20 cell line)-were studied using atomic force microscopy and an optical nanomotion detection method. Using the Peak Force QNM and Force Volume AFM modes, we revealed the unique patterns of the dependence of Young's modulus on the indentation depth for two cancer cell lines that correlate with the features of the spatial organization of the actin cytoskeleton. Within a 200-300 nm layer just under the cell membrane, BT-20 cells are stiffer than ZR-75 cells, whereas in deeper cell regions, Young's modulus of ZR-75 cells exceeds that of BT-20 cells. Two cancer cell lines also displayed a difference in cell nanomotion dynamics upon exposure to cytochalasin D, a potent actin polymerization inhibitor. The drug strongly modified the nanomotion pattern of BT-20 cells, whereas it had almost no effect on the ZR-75 cells. We are confident that nanomotion monitoring and measurement of the stiffness of cancer cells at various indentation depths deserve further studies to obtain effective predictive parameters for use in clinical practice.

Mitochondrial nanomotion measured by optical microscopy.

Nanometric scale size oscillations seem to be a fundamental feature of all living organisms on Earth. Their detection usually requires complex and very sensitive devices. However, some recent studies demonstrated that very simple optical microscopes and dedicated image processing software can also fulfill this task. This novel technique, termed as optical nanomotion detection (ONMD), was recently successfully used on yeast cells to conduct rapid antifungal sensitivity tests. In this study, we demonstrate that the ONMD method can monitor motile sub-cellular organelles, such as mitochondria. Here, mitochondrial isolates (from HEK 293 T and Jurkat cells) undergo predictable motility when viewed by ONMD and triggered by mitochondrial toxins, citric acid intermediates, and dietary and bacterial fermentation products (short-chain fatty acids) at various doses and durations. The technique has superior advantages compared to classical methods since it is rapid, possesses a single organelle sensitivity, and is label- and attachment-free.

Modulation of the nanoscale motion rate of Candida albicans by X-rays.

Introduction: Patients undergoing cancer treatment by radiation therapy commonly develop Candida albicans infections (candidiasis). Such infections are generally treated by antifungals that unfortunately also induce numerous secondary effects in the patient. Additional to the effect on the immune system, ionizing radiation influences the vital activity of C. albicans cells themselves; however, the reaction of C. albicans to ionizing radiation acting simultaneously with antifungals is much less well documented. In this study, we explored the effects of ionizing radiation and an antifungal drug and their combined effect on C. albicans. Methods: The study essentially relied on a novel technique, referred to as optical nanomotion detection (ONMD) that monitors the viability and metabolic activity of the yeast cells in a label and attachment-free manner. Results and discussion: Our findings demonstrate that after exposure to X-ray radiation alone or in combination with fluconazole, low-frequency nanoscale oscillations of whole cells are suppressed and the nanomotion rate depends on the phase of the cell cycle, absorbed dose, fluconazole concentration, and post-irradiation period. In a further development, the ONMD method can help in rapidly determining the sensitivity of C. albicans to antifungals and the individual concentration of antifungals in cancer patients undergoing radiation therapy.

Simple optical nanomotion method for single-bacterium viability and antibiotic response testing.

Antibiotic resistance is nowadays a major public health issue. Rapid antimicrobial susceptibility tests (AST) are one of the options to fight this deadly threat. Performing AST with single-cell sensitivity that is rapid, cheap, and widely accessible, is challenging. Recent studies demonstrated that monitoring bacterial nanomotion by using atomic force microscopy (AFM) upon exposure to antibiotics constitutes a rapid and highly efficient AST. Here, we present a nanomotion detection method based on optical microscopy for testing bacterial viability. This novel technique only requires a very basic microfluidic analysis chamber, and an optical microscope equipped with a camera or a mobile phone. No attachment of the microorganisms is needed, nor are specific bacterial stains or markers. This single-cell technique was successfully tested to obtain AST for motile, nonmotile, gram-positive, and gram-negative bacteria. The simplicity and efficiency of the method make it a game-changer in the field of rapid AST.

Fast Self-Assembly Dynamics of a ß-Sheet Peptide Soft Material.

Peptide-based hydrogels are promising biocompatible materials for wound healing, drug delivery, and tissue engineering applications. The physical properties of these nanostructured materials depend strongly on the morphology of the gel network. However, the self-assembly mechanism of the peptides that leads to a distinct network morphology is still a subject of ongoing debate, since complete assembly pathways have not yet been resolved. To unravel the dynamics of the hierarchical self-assembly process of the model ß-sheet forming peptide KFE8 (Ac-FKFEFKFE-NH2 ), high-speed atomic force microscopy (HS-AFM) in liquid is used. It is demonstrated that a fast-growing network, based on small fibrillar aggregates, is formed at a solid-liquid interface, while in bulk solution, a distinct, more prolonged nanotube network emerges from intermediate helical ribbons. Moreover, the transformation between these morphologies has been visualized. It is expected that this new in situ and in real-time methodology will set the path for the in-depth unravelling of the dynamics of other peptide-based self-assembled soft materials, as well as gaining advanced insights into the formation of fibers involved in protein misfolding diseases.

High-Speed Atomic Force Microscopy Visualization of Protein-DNA Interactions Using DNA Origami Frames.

Direct, live imaging of protein-DNA interactions under physiological conditions is invaluable for understanding the mechanism and kinetics of binding and understanding the topological changes of the DNA strand. The DNA origami technology allows for precise placement of target molecules in a designed nanostructure. Here, we describe a protocol for the self-assembly of DNA origami frames with 2 stretched DNA sequences containing the binding site of a transcription factor, i.e., the Protein FadR, which is a TetR-family tanscription factor regulator for fatty acid metabolism in the archaeal organism Sulfolobus acidocaldarius. These frames can be used to study the dynamics of transcription factor binding using high-speed AFM and obtain mechanistic insights into the mechanism of action of transcription factors.

Nanomotion Spectroscopy as a New Approach to Characterize Bacterial Virulence.

Atomic force microscopy (AFM)-based nanomotion detection is a label-free technique that has been used to monitor the response of microorganisms to antibiotics in a time frame of minutes. The method consists of attaching living organisms onto an AFM cantilever and in monitoring its nanometric scale oscillations as a function of different physical-chemical stimuli. Up to now, we only used the cantilever oscillations variance signal to assess the viability of the attached organisms. In this contribution, we demonstrate that a more precise analysis of the motion pattern of the cantilever can unveil relevant medical information about bacterial phenotype. We used B. pertussis as the model organism, it is a slowly growing Gram-negative bacteria which is the agent of whooping cough. It was previously demonstrated that B. pertussis can expresses different phenotypes as a function of the physical-chemical properties of the environment. In this contribution, we highlight that B. pertussis generates a cantilever movement pattern that depends on its phenotype. More precisely, we noticed that nanometric scale oscillations of B. pertussis can be correlated with the virulence state of the bacteria. The results indicate a correlation between metabolic/virulent bacterial states and bacterial nanomotion pattern and paves the way to novel rapid and label-free pathogenic microorganism detection assays.

Nano-Motion Analysis for Rapid and Label Free Assessing of Cancer Cell Sensitivity to Chemotherapeutics.

Background and Objectives: Optimization of chemotherapy is crucial for cancer patients. Timely and costly efficient treatments are emerging due to the increasing incidence of cancer worldwide. Here, we present a methodology of nano-motion analysis that could be developed to serve as a screening tool able to determine the best chemotherapy option for a particular patient within hours. Materials and Methods: Three different human cancer cell lines and their multidrug resistant (MDR) counterparts were analyzed with an atomic force microscope (AFM) using tipless cantilevers to adhere the cells and monitor their nano-motions. Results: The cells exposed to doxorubicin (DOX) differentially responded due to their sensitivity to this chemotherapeutic. The death of sensitive cells corresponding to the drop in signal variance occurred in less than 2 h after DOX application, while MDR cells continued to move, even showing an increase in signal variance. Conclusions: Nano-motion sensing can be developed as a screening tool that will allow simple, inexpensive and quick testing of different chemotherapeutics for each cancer patient. Further investigations on patient-derived tumor cells should confirm the method's applicability.

Nanomotion Detection-Based Rapid Antibiotic Susceptibility Testing.

Rapid antibiotic susceptibility testing (AST) could play a major role in fighting multidrug-resistant bacteria. Recently, it was discovered that all living organisms oscillate in the range of nanometers and that these oscillations, referred to as nanomotion, stop as soon the organism dies. This finding led to the development of rapid AST techniques based on the monitoring of these oscillations upon exposure to antibiotics. In this review, we explain the working principle of this novel technique, compare the method with current ASTs, explore its application and give some advice about its implementation. As an illustrative example, we present the application of the technique to the slowly growing and pathogenic Bordetella pertussis bacteria.

Environmental Control of Amyloid Polymorphism by Modulation of Hydrodynamic Stress.

The phenomenon of amyloid polymorphism is a key feature of protein aggregation. Unravelling this phenomenon is of great significance for understanding the underlying molecular mechanisms associated with neurodegenerative diseases and for the development of amyloid-based functional biomaterials. However, the understanding of the molecular origins and the physicochemical factors modulating amyloid polymorphs remains challenging. Herein, we demonstrate an association between amyloid polymorphism and environmental stress in solution, induced by an air/water interface in motion. Our results reveal that low-stress environments produce heterogeneous amyloid polymorphs, including twisted, helical, and rod-like fibrils, whereas high-stress conditions generate only homogeneous rod-like fibrils. Moreover, high environmental stress converts twisted fibrils into rod-like fibrils both in-pathway and after the completion of mature amyloid formation. These results enrich our understanding of the environmental origin of polymorphism of pathological amyloids and shed light on the potential of environmentally controlled fabrication of homogeneous amyloid biomaterials for biotechnological applications.

Single yeast cell nanomotions correlate with cellular activity.

Living single yeast cells show a specific cellular motion at the nanometer scale with a magnitude that is proportional to the cellular activity of the cell. We characterized this cellular nanomotion pattern of nonattached single yeast cells using classical optical microscopy. The distribution of the cellular displacements over a short time period is distinct from random motion. The range and shape of such nanomotion displacement distributions change substantially according to the metabolic state of the cell. The analysis of the nanomotion frequency pattern demonstrated that single living yeast cells oscillate at relatively low frequencies of around 2 hertz. The simplicity of the technique should open the way to numerous applications among which antifungal susceptibility tests seem the most straightforward.

Fiber-Tip Polymer Microcantilever for Fast and Highly Sensitive Hydrogen Measurement.

Hydrogen as an antioxidant gas has been widely used in the medical and biological fields for preventing cancer or treating inflammation. However, controlling the hydrogen concentration is crucial for practical use due to its explosive property when its volume concentration in air reaches the explosive limit (4%). In this work, a polymer-based microcantilever (µ-cantilever) hydrogen sensor located at the end of a fiber tip is proposed to detect the hydrogen concentration in medical and biological applications. The proposed sensor was developed using femtosecond laser-induced two-photon polymerization (TPP) to print the polymer µ-cantilever and magnetron sputtering to coat a palladium (Pd) film on the upper surface of the µ-cantilever. Such a device exhibits a high sensitivity, roughly -2 nm %-1 when the hydrogen concentration rises from 0% to 4.5% (v/v) and a short response time, around 13.5 s at 4% (v/v), making it suitable for medical and environmental applications. In addition to providing an ultracompact optical solution for fast and highly sensitive hydrogen measurement, the polymer µ-cantilever fiber sensor can be used for diverse medical and biological sensing applications by replacing Pd with other functional materials.

A perspective view on the nanomotion detection of living organisms and its features.

The insurgence of newly arising, rapidly developing health threats, such as drug-resistant bacteria and cancers, is one of the most urgent public-health issues of modern times. This menace calls for the development of sensitive and reliable diagnostic tools to monitor the response of single cells to chemical or pharmaceutical stimuli. Recently, it has been demonstrated that all living organisms oscillate at a nanometric scale and that these oscillations stop as soon as the organisms die. These nanometric scale oscillations can be detected by depositing living cells onto a micro-fabricated cantilever and by monitoring its displacements with an atomic force microscope-based electronics. Such devices, named nanomotion sensors, have been employed to determine the resistance profiles of life-threatening bacteria within minutes, to evaluate, among others, the effect of chemicals on yeast, neurons, and cancer cells. The data obtained so far demonstrate the advantages of nanomotion sensing devices in rapidly characterizing microorganism susceptibility to pharmaceutical agents. Here, we review the key aspects of this technique, presenting its major applications. and detailing its working protocols.

Silver Nanomaterials in Contemporary Molecular Physiology Research.

Silver nanoparticles have numerous potential applications in engineering, industry, biology and medicine. Because of their unique chemical properties, they have become the focus of many research teams all over the world. Silver nanoparticles may exhibit significant antimicrobial and anticancer effects, and they may be a valuable part of various bioassays and biosensors. However, the research on biological and medical uses of AgNPs is related with numerous potential problems and challenges that need to be overcome in the years ahead. Possible toxic effects of silver nanoparticles on living organisms represent a great concern, both in clinical medicine and public health. Nevertheless, in the future, it may be expected that all metallic nanomaterials, including the ones made from silver will greatly benefit almost all natural scientific fields. In this short review, we focus on the recent research on silver nanoparticles in experimental physiology, as well as other areas of fundamental and clinical medicine.