Terahertz s-SNOM Imaging of a Single Cell with Nanoscale Resolution.

Terahertz scattering scanning near-field optical microscopy is a robust spectral detection technique with a nanoscale resolution. However, there are still major challenges in investigating the heterogeneity of cell membrane components in individual cells. Here, we present a novel and comprehensive analytical approach for detecting and investigating heterogeneity in cell membrane components at the single-cell level. In comparison to the resolution of the topographical atomic force microscopy image, the spatial resolution of the terahertz near-field amplitude image is 3 times that of the former. This ultrafine resolution enables the compositional distribution in the cell membrane, such as the distribution of extracellular vesicles, to be finely characterized. Furthermore, via extraction of the near-field absorption images at specific frequencies, the visualization and compositional difference analysis of cell membrane components can be presented in detail. These findings have significant implications for the intuitive and visual analysis of cell development and disease evolutionary pathways.

Nanomechanical collective vibration of SARS-CoV-2 spike proteins.

The development of effective therapeutics against COVID-19 requires a thorough understanding of the receptor recognition mechanism of the SARS-CoV-2 spike (S) protein. Here the multidomain collective dynamics on the trimer of the spike protein has been analyzed using normal mode analysis (NMA). A common nanomechanical profile was identified in the spike proteins of SARS-CoV-2 and its variants. The profile involves collective vibrations of the receptor-binding domain (RBD) and the N-terminal domain (NTD), which may mediate the physical interaction process. Quantitative analysis of the collective modes suggests a nanomechanical property involving large-scale conformational changes, which explains the difference in receptor binding affinity among different variants. These results support the use of intrinsic global dynamics as a valuable perspective for studying the allosteric and functional mechanisms of the S protein. This approach also provides a low-cost theoretical toolkit for screening potential pathogenic mutations and drug targets.

Ultrafine FeF3·0.33H2O Nanocrystal-Doped Graphene Aerogel Cathode Materials for Advanced Lithium-Ion Batteries.

FeF3 has been extensively studied as an alternative positive material owing to its superior specific capacity and low cost, but the low conductivity, large volume variation, and slow kinetics seriously hinder its commercialization. Here, we propose the in situ growth of ultrafine FeF3·0.33H2O NPs on a three-dimensional reduced graphene oxide (3D RGO) aerogel with abundant pores by a facile freeze drying process followed by thermal annealing and fluorination. Within the FeF3·0.33H2O/RGO composites, the three-dimensional (3D) RGO aerogel and hierarchical porous structure ensure rapid diffusion of electrons/ions within the cathode, enabling good reversibility of FeF3. Benefiting from these advantages, a superior cycle behavior of 232 mAh g-1 under 0.1C over 100 cycles as well as outstanding rate performance is achieved. These results provide a promising approach for advanced cathode materials for Li-ion batteries.

Oxygen therapy accelerates apoptosis induced by selenium compounds via regulating Nrf2/MAPK signaling pathway in hepatocellular carcinoma.

Selenium has good antitumor effects in vitro, but the hypoxic microenvironment in solid tumors makes its clinical efficacy unsatisfactory. We hypothesized that the combination with oxygen therapy might improve the treatment efficacy of selenium in hypoxic tumors through the changes of redox environment. In this work, two selenium compounds, Na2SeO3 and CysSeSeCys, were selected to interrogate their therapeutic effects on hepatocellular carcinoma (HCC) under different oxygen levels. In tumor-bearing mice, both selenium compounds significantly inhibited the tumor growth, and combined with oxygen therapy further reduced the tumor volume about 50 %. In vitro HepG2 cell experiments, selenium induced autophagy and delayed apoptosis under hypoxia (1 % O2), while inhibited autophagy and accelerated apoptosis under hyperoxia (60 % O2). We found that, in contrast to hypoxia, the hyperoxic environment facilitated the H2Se, produced by the selenium metabolism in cells, to be rapidly oxidized to generate H2O2, leading to inhibit the expression level of Nrf2 and to increase that of phosphorylation of p38 and MKK4, resulting in inhibiting autophagy and accelerating apoptosis. Once the Nrf2 gene was knocked down, selenium compounds combined with hyperoxia treatment would further activate the MAPK signaling pathway and further increase apoptosis. These findings highlight oxygen can significantly enhance the anti-HCC effect of selenium compounds through regulating the Nrf2 and MAPK signaling pathways, thus providing novel therapeutic strategy for the hypoxic tumors and pave the way for the application of selenium in clinical treatment.

Hydrogel microenvironment contributes to chemical-induced differentiation of mesenchymal stem cells: single-cell infrared microspectroscopy characterization.

Stem cell microenvironment plays vital roles in directing cell proliferation and differentiation. Due to the tiny biochemical changes in the early stage of stem cell development, technical challenges to characterize the potential effects of environmental signals remain. In this work, we have introduced synchrotron radiation-based Fourier transform infrared microspectroscopy to evaluate the synergistic effects of physical and chemical factors on stem cell differentiation at the single-cell level. By using principal component analysis and cell-cell Euclidean distance calculation, the phenotypic heterogeneity changes during stem cell osteogenesis induced by lithium chloride or Wnt5a protein loaded in the polyvinyl alcohol (PVA) hydrogel were characterized in detail. The results demonstrated that PVA hydrogel could lead to the distinct effects between low-concentration lithium and wnt5a on human mesenchymal stem cells, suggesting a vital role of niche signals in Wnt pathway. These findings highlight the importance of microenvironment to the chemical-induced effects on stem cell differentiation and also provide a label-free, noninvasive method to sensitively identify the niche function in stem cell biology.

Label-free analysis of biofilm phenotypes by infrared micro- and correlation spectroscopy.

The methodology development for deeply describing the complex biofilm phenotypes is an urgent demand for understanding their basic biology and the central clinic relevance. Here, we developed an infrared microspectroscopy-based method for the quantitative evaluation and description of biofilm phenotypic characteristics by calculating the spectral similarity of the infrared data. Using this approach, we revealed the phenotypic variation during the biofilm formation process and biofilm heterogeneity between two E. coli strains. Two-dimensional correlation spectroscopy was further combined to deeply investigate the biochemical component evolution sequences during E. coli biofilm formation and revealed the first-order of the polysaccharide molecules change, expanding new opportunities for infrared microspectroscopy in revealing molecule evolution in the biofilm formation. This novel development offers a label-free optical toolkit for the bioanalytical analysis of biofilm phenotypes but also paves the way for screening the drugs to modulate the structure and ecology of biofilm microbiome.

Low-frequency collective motion of DNA-binding domain defines p53 function.

Most mutations in the DNA-binding domain (DBD) of p53 inactivate or rescue the protein function interacting with the minor groove of DNA. However, how the conformation changes propagating from the mutation sites result in distinct molecular recognition is still not well understood. As the protein mobility is an intrinsic property encrypted in its primary structure, we examined if different structures of wild-type and mutant p53 core domains display any unique patterns of intrinsic mobility. Normal mode calculation was employed to characterize the collective dynamics of DBD in p53 monomer and tetramer as well as their mutants. Intriguingly, the low-frequency collective motions of DBD show similar patterns between the wild-type protein and the rescued mutants. The analysis on atomic backbone fluctuations and low-frequency vibration mode statistics does further support the correlation between the intrinsic collective motion of DBD and the p53 protein function. The mutations in the DBD influence the low-frequency vibration of the p53 tetramer via the change of the collective motions among its four monomers. These findings thus provide new insights for understanding the physical mechanism of p53 protein structure-function relationship and help find the small molecule drug to modulate protein dynamic for disease therapy.

Membrane phosphatidylserine allosterically regulates the cytosolic phospholipase A2 activity via an electrostatic-switch mechanism.

Phospholipase A2 (PLA2) is a peripheral membrane protein that plays an essential role in many inflammatory responses. However, the activation mechanisms of PLA2 on the membrane surface have not been fully understood. Herein, we have combined experimental techniques and theoretical approaches to investigate the activation and association of the PLA2 protein on an artificial phospholipid membrane. Using a phosphatidylserine (PS) nanodomain containing membrane to mimic the inflammatory conditions, we found that the activity of cytosolic PLA2s (cPLA2s) increases with higher ratios of PS in the membrane. Molecular dynamics simulations reveal that significant changes in the protein structure are related to negatively charged membranes. In particular, the alteration of negatively charged residues in the C2 domain brings about an opened binding pocket and the catalytic site access to the substrate phospholipid. Meanwhile, the negative residues in the loop 650-665 facilitate the optimal interfacial orientation of the protein with a closed binding pocket on the membrane surface. These results lead us to suggest an electrostatic-switch allosteric mechanism for cPLA2 activation on the cell membrane surface under the inflammatory state.

Synchrotron Infrared Microspectroscopy for Stem Cell Research.

Stem cells have shown great potential functions for tissue regeneration and repair because of their unlimited self-renewal and differentiation. Stem cells reside in their niches, making them a hotspot for the development and diagnosis of diseases. Complex interactions between niches and stem cells create the balance between differentiation, self-renewal, maturation, and proliferation. However, the multi-facet applications of stem cells have been challenged since the complicated responses of stem cells to biological processes were explored along with the limitations of current systems or methods. Emerging evidence highlights that synchrotron infrared microspectroscopy, known as synchrotron radiation-based Fourier transform infrared microspectroscopy, has been investigated as a potentially attractive technology with its non-invasive and non-biological probes in stem cell research. With their unique vibration bands, the quantitative mapping of the content and distribution of biomolecules can be detected and characterized in cells or tissues. In this review, we focus on the potential applications of synchrotron infrared microspectroscopy for investigating the differentiation and fate determination of stem cells.

Single-Cell Infrared Microspectroscopy Quantifies Dynamic Heterogeneity of Mesenchymal Stem Cells during Adipogenic Differentiation.

The central relevance of cellular heterogeneity to biological phenomena raises the rational needs for analytical techniques with single-cell resolution. Here, we developed a single-cell FTIR microspectroscopy-based method for the quantitative evaluation of cellular heterogeneity by calculating the cell-to-cell similarity distance of the infrared spectral data. Based on this method, we revealed the infrared phenotypes might reflect the dynamic heterogeneity changes in the cell population during the adipogenic differentiation of the human mesenchymal stem cells. These findings provide an alternative label-free optical approach for quantifying the cellular heterogeneity, and the combination with other single-cell analysis tools will be very helpful for understanding the genotype-to-phenotype relationship in cellular populations.

What factors predict physicians' utilization behavior of contrast-enhanced ultrasound? Evidence from the integration of the Theory of Planned Behavior and Technology Acceptance Model using a structural equation modeling approach.

BACKGROUND: The promotion of early diagnosis is undoubtedly effective in reducing the burden of disease. Contrast-enhanced ultrasound (CEUS) is a diagnostic technology for liver cancer, but its implementation faces some challenges. Understanding the influencing factors of CEUS utilization is crucial for its successful implementation. However, such research is rare. The aims of this study were to investigate the status of CEUS utilization and its predictors in China. METHODS: Through multistage random sampling, a cross-sectional study design was conducted among physicians in charge of direct use of CEUS working at liver disease-related departments of sampled health institutions. To access the potential influencing factors of physicians' CEUS utilization, a structured questionnaire was developed based on the theoretical model, which was developed by integration of the Theory of Planned Behavior (TPB) and Technology Acceptance Model (TAM). Structural equation modeling was used to verify the proposed hypotheses, and analyze the relationship and mechanism between the factors. RESULTS: A total of 309 physicians were enrolled. The mean score of utilization behavior was 2.04 (SD = 1.07), and 37.22% above the mean. The favorable fitting results demonstrated that the integration of TAM and TPB was an acceptable model. SEM results also identified physicians' intentions to use CEUS was directly associated with utilization behavior (ß = 0.287, P < 0.001). Attitude (ß = 0.272, P < 0.001), subjective norm (ß = 0.172, P = 0.013), perceived behavioral control (ß = 0.491, P < 0.001) and perceived usefulness (ß = 0.108, P = 0.027) significantly influenced physicians' intentions. Besides, subjective norm (ß = 0.065, P = 0.021), perceived behavioral control (ß = 0.141, P = 0.003), and perceived ease of use (ß = 0.022, P = 0.033) indirectly affected physicians' CEUS utilization. CONCLUSIONS: The findings provide a reference for understanding the factors associated with physicians' utilization of CEUS. Additionally, the proposed measures such as building innovative and incentive environment, providing high quality and adequate training, etc., will help promote the utilization of CEUS, thereby increasing the detection rate of liver cancer, and improving the survival rate and the quality of life for liver cancer patients.

Single-molecule nanomechanical spectroscopy shows calcium ions contribute to chain association and structural flexibility of blood clotting factor VIII.

Blood coagulation factor VIII (FVIII) can bind calcium ions and ion-protein interactions appear central importance for both their structure and function in coagulation cascade. However, the mechanism and details of how calcium dependent structure change of proteins to fulfill their function remain to be fully defined. In this work, PeakForce Quantitative Nanomechanics (PF-QNM) mode atomic force microscopy (AFM) was used to map the topography and mechanical properties of FVIII with single protein resolution under different calcium concentrations. The obtained nanomechanical spectroscopy showed that calcium ions play dual roles in the chain association and structural flexibility of FVIII. Low concentration of calcium ions prefer to bind isolated chains and increase their mechanical properties, whereas they link the heavy and light chains to keep the protein re-association under higher ions concentration. Our results provide a novel insight into the mechanistic details of the metal ions on the stability and function of blood clotting proteins.

Unconventional Atomic Structure of Graphene Sheets on Solid Substrates.

The atomic structure of free-standing graphene comprises flat hexagonal rings with a 2.5 Å period, which is conventionally considered the only atomic period and determines the unique properties of graphene. Here, an unexpected highly ordered orthorhombic structure of graphene is directly observed with a lattice constant of ≈5 Å, spontaneously formed on various substrates. First-principles computations show that this unconventional structure can be attributed to the dipole between the graphene surface and substrates, which produces an interfacial electric field and induces atomic rearrangement on the graphene surface. Further, the formation of the orthorhombic structure can be controlled by an artificially generated interfacial electric field. Importantly, the 5 Å crystal can be manipulated and transformed in a continuous and reversible manner. Notably, the orthorhombic lattice can control the epitaxial self-assembly of amyloids. The findings reveal new insights about the atomic structure of graphene, and open up new avenues to manipulate graphene lattices.

Evaluating the biochemical changes of LPS-stimulated endothelial cells by synchrotron FTIR microspectroscopy at a single-cell level.

Endothelial damage is a major manifestation in many forms of heart and lung injuries induced by lipopolysaccharide (LPS), but the biochemical responses and activation mechanisms of endothelial cells have not been fully explicit. In this study, the biochemical changes to endothelial cells exposed to LPS were investigated by synchrotron FTIR microspectroscopy at a single-cell level. We found that the whole infrared spectrum of endothelial cells shifted after LPS treatment, indicating chemical component changes within cells. Principal component analysis (PCA) and t tests on subspectra (fatty acid region, protein region, and nucleic acid-sugar region, respectively) further showed that sugar components as well as fatty acids changed dramatically while proteins had no significant variation following LPS exposure. These results suggested that the glycocalyx layer structure on endothelial cell membrane may be mainly influenced by LPS and also proved that synchrotron FTIR microspectroscopy was a useful technique to evaluate the biochemical changes of endothelial damage at the single-cell level. Graphical abstract.

Synchrotron FTIR microspectroscopy reveals early adipogenic differentiation of human mesenchymal stem cells at single-cell level.

Human mesenchymal stem cells (hMSCs) have been used as an ideal in vitro model to study human adipogenesis. However, little knowledge of the early stage differentiation greatly hinders our understanding on the mechanism of the adipogenesis processes. In this study, synchrotron radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy was applied to track the global structural and compositional changes of lipids, proteins and nucleic acids inside individual hMSCs along the time course. The multivariate analysis of the SR-FTIR spectra distinguished the dynamic and significant changes of the lipids and nucleic acid at early differentiation stage. Importantly, changes of lipid structure during early days (Day 1-3) of differentiation might serve as a potential biomarker in identifying the state in early differentiation at single cell level. These results proved that SR-FTIR is a powerful tool to study the stem cell fate determination and early lipogenesis events.

Size-Dependent Stiffness of Nanodroplets: A Quantitative Analysis of the Interaction between an AFM Probe and Nanodroplets.

The interfacial properties of nanodroplets are very significant for the exploration of the basic law governing the fluid behavior at the nanoscale and also the applications in some important processes in novel materials fabrication by forming a special and local reaction environment. However, many basic factors such as the interfacial tension or stiffness of nanodroplets are still lacking, partially because of the difficulty of making quantitative measurements of the interfacial interactions at the nanometer scale. Here, we used a novel atomic force microscopy (AFM) mode, PeakForce mode, to control the interaction between an AFM probe and nanodroplets, by which we could obtain the morphology and stiffness of nanodroplets simultaneously. The change in the stiffness with the size of the nanodroplets was observed where the smaller nanodroplets usually had a larger stiffness. To explain this phenomenon, we then established a theoretical model based on the Young-Laplace equation in which the deformation and size-dependent stiffness could be described quantitatively and the experimental observations could be explained with our numerical calculations very well. The general methodology presented here could also be extended to analyze the relevant behavior of nanobubbles and other wetting phenomena at the nanoscale.

Stiffness and evolution of interfacial micropancakes revealed by AFM quantitative nanomechanical imaging.

Micropancakes are quasi-two-dimensional micron-sized domains on crystalline substrates (e.g. highly oriented pyrolytic graphite (HOPG)) immersed in water. They are only a few nanometers thick, and are suspected to come from the accumulation of dissolved air at the solid-water interface. However, the exact chemical nature and basic physical properties of micropancakes have been under debate ever since their first observation, primarily due to the lack of a suitable characterization technique. In this study, the stiffness of micropancakes at the interface between HOPG and ethanol-water solutions was investigated by using PeakForce Quantitative NanoMechanics (PF-QNM) mode Atomic Force Microscopy (AFM). Our measurements showed that micropancakes were stiffer than nanobubbles, and for bilayer micropancakes, the bottom layer in contact with the substrate was stiffer than the top one. Interestingly, the micropancakes became smaller and softer with an increase in the ethanol concentration in the solution, and were undetectable by AFM above a critical concentration of ethanol. But they re-appeared after the ethanol concentration in the solution was reduced. Clearly the evolution and stiffness of the micropancakes were dependent on the chemical composition in the solution, which could be attributed to the correlation of the mechanical properties of the micropancakes with the surface tension of the liquid phase. Based on the "go-and-come" behaviors of micropancakes with the ethanol concentration, we found that the micropancakes could actually tolerate the ethanol concentration much higher than 5%, a value reported in the literature. The results from this work may be helpful in alluding the chemical nature of micropancakes.

The origin of the "snap-in" in the force curve between AFM probe and the water/gas interface of nanobubbles.

The long-range attractive force or "snap-in" is an important phenomenon usually occurring when a solid particle interacts with a water/gas interface. By using PeakForce quantitative nanomechanics the origin of snap-in in the force curve between the atomic force microscopy (AFM) probe and the water/gas interface of nanobubbles has been investigated. The snap-in frequently happened when the probe was preserved for a certain time or after being used for imaging solid surfaces under atmospheric conditions. In contrast, imaging in liquids rarely induced a snap-in. After a series of control experiments, it was found that the snap-in can be attributed to hydrophobic interactions between the water/gas interface and the AFM probe, which was either modified or contaminated with hydrophobic material. The hydrophobic contamination could be efficiently removed by a conventional plasma-cleaning treatment, which prevents the occurring of the snap-in. In addition, the adsorption of sodium dodecyl sulfate onto the nanobubble surface changed the water/gas interface into hydrophilic, which also eliminated the snap-in phenomenon.

Unraveling the Drug Response Heterogeneity with Single-Cell Vibrational Phenomics.

Drug responses heterogeneity is often highlighted to justify the need for precision medicine. However, due to the highly complex nature of cell phenotypes in many diseases, one of key challenges is how to obtain the high content features in a cellular population. Here we present a single-cell vibrational phenomics approach, integrating synchrotron infrared microspectroscopy and multivariate calculation, for quantitatively evaluating the cellular responses to drug perturbation with single cell resolution. In a human hepatocellular carcinoma HepG2 cell model, the phenotypic changes induced by two types of drugs, taxol (TAX) and protopanaxadiol (PPD), were analyzed and revealed the response heterogeneity in drug concentration and chemical components. These findings not only provide a label-free strategy for determining the drug response at the single cell level, but also demonstrate the great potential of vibrational phenomics as a drug discovery platform.

Vibrational phenomics decoding of the stem cell stepwise aging process at single-cell resolution.

We introduce vibrational spectroscopy to quantitatively measure the phenotypic heterogeneity of senescent stem cells in the aging process at the single cell level. Using an aging model of serially passaged human mesenchymal stem cells (MSCs), we characterized the phenotypic changes of MSCs during different aged stages and discovered a stepwise aging process with several distinct subtypes.