Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens.

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.

Cardio PyMEA: A user-friendly, open-source Python application for cardiomyocyte microelectrode array analysis.

Open source analytical software for the analysis of electrophysiological cardiomyocyte data offers a variety of new functionalities to complement closed-source, proprietary tools. Here, we present the Cardio PyMEA application, a free, modifiable, and open source program for the analysis of microelectrode array (MEA) data obtained from cardiomyocyte cultures. Major software capabilities include: beat detection; pacemaker origin estimation; beat amplitude and interval; local activation time, upstroke velocity, and conduction velocity; analysis of cardiomyocyte property-distance relationships; and robust power law analysis of pacemaker spatiotemporal instability. Cardio PyMEA was written entirely in Python 3 to provide an accessible, integrated workflow that possesses a user-friendly graphical user interface (GUI) written in PyQt5 to allow for performant, cross-platform utilization. This application makes use of object-oriented programming (OOP) principles to facilitate the relatively straightforward incorporation of custom functionalities, e.g. power law analysis, that suit the needs of the user. Cardio PyMEA is available as an open source application under the terms of the GNU General Public License (GPL). The source code for Cardio PyMEA can be downloaded from Github at the following repository: https://github.com/csdunhamUC/cardio_pymea.

Pacemaker translocations and power laws in 2D stem cell-derived cardiomyocyte cultures.

Power laws are of interest to several scientific disciplines because they can provide important information about the underlying dynamics (e.g. scale invariance and self-similarity) of a given system. Because power laws are of increasing interest to the cardiac sciences as potential indicators of cardiac dysfunction, it is essential that rigorous, standardized analytical methods are employed in the evaluation of power laws. This study compares the methods currently used in the fields of condensed matter physics, geoscience, neuroscience, and cardiology in order to provide a robust analytical framework for evaluating power laws in stem cell-derived cardiomyocyte cultures. One potential power law-obeying phenomenon observed in these cultures is pacemaker translocations, or the spatial and temporal instability of the pacemaker region, in a 2D cell culture. Power law analysis of translocation data was performed using increasingly rigorous methods in order to illustrate how differences in analytical robustness can result in misleading power law interpretations. Non-robust methods concluded that pacemaker translocations adhere to a power law while robust methods convincingly demonstrated that they obey a doubly truncated power law. The results of this study highlight the importance of employing comprehensive methods during power law analysis of cardiomyocyte cultures.

Cancer cell mechanobiology: a new frontier for cancer research.

The study of physical and mechanical features of cancer cells, or cancer cell mechanobiology, is a new frontier in cancer research. Such studies may enhance our understanding of the disease process, especially mechanisms associated with cancer cell invasion and metastasis, and may help the effort of developing diagnostic biomarkers and therapeutic drug targets. Cancer cell mechanobiological changes are associated with the complex interplay of activation/inactivation of multiple signaling pathways, which can occur at both the genetic and epigenetic levels, and the interactions with the cancer microenvironment. It has been shown that metastatic tumor cells are more compliant than morphologically similar benign cells in actual human samples. Subsequent studies from us and others further demonstrated that cell mechanical properties are strongly associated with cancer cell invasive and metastatic potential, and thus may serve as a diagnostic marker of detecting cancer cells in human body fluid samples. In this review, we provide a brief narrative of the molecular mechanisms underlying cancer cell mechanobiology, the technological platforms utilized to study cancer cell mechanobiology, the status of cancer cell mechanobiological studies in various cancer types, and the potential clinical applications of cancer cell mechanobiological study in cancer early detection, diagnosis, and treatment.

Single Cell Mechanotype and Associated Molecular Changes in Urothelial Cell Transformation and Progression.

Cancer cell mechanotype changes are newly recognized cancer phenotypic events, whereas metastatic cancer cells show decreased cell stiffness and increased deformability relative to normal cells. To further examine how cell mechanotype changes in early stages of cancer transformation and progression, an in vitro multi-step human urothelial cell carcinogenic model was used to measure cellular Young's modulus, deformability, and transit time using single-cell atomic force microscopy, microfluidic-based deformability cytometry, and quantitative deformability cytometry, respectively. Measurable cell mechanotype changes of stiffness, deformability, and cell transit time occur early in the transformation process. As cells progress from normal, to preinvasive, to invasive cells, Young's modulus of stiffness decreases and deformability increases gradually. These changes were confirmed in three-dimensional cultured microtumor masses and urine exfoliated cells directly from patients. Using gene screening and proteomics approaches, we found that the main molecular pathway implicated in cell mechanotype changes appears to be epithelial to mesenchymal transition.

Monomolecular covalent honeycomb nanosheets produced by surface-mediated polycondensation between 1,3,5-triamino benzene and benzene-1,3,5-tricarbox aldehyde on Au(111).

Fabrication of a two-dimensional covalent network of honeycomb nanosheets comprising small 1,3,5-triamino benzene and benzene-1,3,5-tricarboxaldehyde aromatic building blocks was conducted on Au(111) in a pH-controlled aqueous solution. In situ scanning tunneling microscopy revealed a large defect-free and homogeneous honeycomb π-conjugated nanosheet at the Au(111)/liquid interface. An electrochemical potential dependence indicated that the nanosheets were the result of thermodynamic self-assembly based not only on the reaction equilibrium but also on the adsorption (partition) equilibrium, which was controlled by the building block surface coverage as a function of electrode potential.

Emergent dynamics of neuromorphic nanowire networks.

Neuromorphic networks are formed by random self-assembly of silver nanowires. Silver nanowires are coated with a polymer layer after synthesis in which junctions between two nanowires act as resistive switches, often compared with neurosynapses. We analyze the role of single junction switching in the dynamical properties of the neuromorphic network. Network transitions to a high-conductance state under the application of a voltage bias higher than a threshold value. The stability and permanence of this state is studied by shifting the voltage bias in order to activate or deactivate the network. A model of the electrical network with atomic switches reproduces the relation between individual nanowire junctions switching events with current pathway formation or destruction. This relation is further manifested in changes in 1/f power-law scaling of the spectral distribution of current. The current fluctuations involved in this scaling shift are considered to arise from an essential equilibrium between formation, stochastic-mediated breakdown of individual nanowire-nanowire junctions and the onset of different current pathways that optimize power dissipation. This emergent dynamics shown by polymer-coated Ag nanowire networks places this system in the class of optimal transport networks, from which new fundamental parallels with neural dynamics and natural computing problem-solving can be drawn.

Piezoelectric needle sensor reveals mechanical heterogeneity in human thyroid tissue lesions.

Palpable thyroid lesions are common, and although mostly benign, lethal malignant nodules do occur and may be difficult to differentiate. Here, we introduce the use of a piezoelectric system called Smart-touch fine needle (or STFN) mounted directly onto conventional biopsy needles, to evaluate abnormal tissues, through quantitative real-time measurements of variations in tissue stiffness as the needle penetrates tissue. Using well-characterized biomaterials of known stiffness and explanted animal tissue models, we first established experimental protocols for STFN measures on biological tissues, as well as optimized device design for high signal-to-noise ratio. Freshly excised patient thyroids with varying fibrotic and malignant potential revealed discrete variations in STFN based tissue stiffness/stiffness heterogeneity and correlated well with final histopathology. Our piezoelectric needle sensor reveals mechanical heterogeneity in thyroid tissue lesions and provides a foundation for the design of hand-held tools for the rapid, mechano-profiling of malignant lesions in vivo while performing fine needle aspiration (FNA).

Potential role of MRN-100, an iron-based compound, in upregulating production of cytokine IL-10 in human dendritic cells to promote an anti-inflammatory response in vitro.

The hydroferrate fluid MRN-100, an iron-based compound with potent antioxidant characteristics, was examined to identify its possible anti-inflammatory effects on human dendritic cells (DCs) in vitro. Human monocyte-derived DCs were treated with MRN-100 at two concentrations (50 and 100 µL/mL) for 24 h and then stimulated with or without lipopolysaccharides (LPS). The expression of DC maturation markers was assessed by flow cytometry and the production of cytokines was determined by enzyme-linked immunosorbent assay (ELISA). Functional assay was performed by co-culturing MRN-100-treated and untreated DCs with allogeneic naïve CD4+ T cells and assaying the T cells' cytokine production. Results show that treatment with MRN-100 significantly upregulated the co-stimulatory molecules CD80 and CD86 and increased human leukocyte antigen-DR (HLA-DR) though not significantly. MRN-100 treatment also significantly increased the production of the anti-inflammatory cytokine interleukin (IL)-10. On the other hand, MRN-100 significantly induced the secretion of pro-inflammatory cytokines such as IL-6 only at high concentrations. Furthermore, DCs pretreated with MRN-100 and either stimulated or not with LPS were able to prime CD4+ T cells to secrete significant amounts of IL-10 while inhibiting the secretion of pro-inflammatory cytokine tumor necrosis factor (TNF)-α. These results indicate that MRN-100 is a powerful anti-inflammatory agent that promotes the generation of an anti-inflammatory immune response in vitro. MRN-100 could be beneficial for treating patients with inflammatory diseases, including arthritis and type 1 diabetes, and its potential benefits should be examined in clinical trials.

Marina crystal minerals (MCM) activate human dendritic cells to induce CD4+ and CD8+ T cell responses in vitro.

Marina crystal minerals (MCM) are a mixture that contains crystallized minerals along with trace elements extracted from seawater. It is a nutritional supplement that is capable of enhancing natural killer (NK) cell activity and increasing T and B cell proliferation in humans post ingestion. However, its effect on dendritic cells (DCs), the cells that bridge innate and adaptive immunity, is not yet known. In this study, we examine the stimulatory effects of MCM on DCs' maturation and function in vitro. Human monocyte-derived DCs were treated with MCM at two different concentrations (10 and 20 µg/mL) for 24 h. Results showed that MCM treatment activated DCs in a dose-dependent fashion. It caused the upregulation of costimulatory molecules CD80, CD86, and HLA-DR, and prompted the production of DC cytokines, including interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and IL-1ß, and chemokines (monocyte chemotactic protein-1 (MCP-1)) and interferon-gamma-inducible protein-10 (IP-10). In addition, activated DCs primed CD4+ T cells to secrete significant amounts of interferon gamma (IFN-γ), and they also stimulated CD8+ T cells to express higher amounts of CD107a. These results indicate that MCM is a potentially powerful adjuvant, from natural materials, that activates human DCs in vitro and therefore may suggest its possible use in immune-based therapies against cancer and viral infections.

Inhibition of TRPV1 Channel Activity in Human CD4⁺ T Cells by Nanodiamond and Nanoplatinum Liquid, DPV576.

Background: Transient receptor potential vanilloid (TRPV) channels act as sensors of pain, temperature, and other external stimuli. We have recently shown that DPV576, an aqueous mixture of nanodiamond (ND) and nanoplatinum (NP), can modulate the activity of TRPV on human primary keratinocytes, suggesting their potential as a possible pain modulator. Here, we sought to examine the effect of DPV576 in modulating the functions of human CD4⁺ T lymphocytes and whether the modulation is mediated via TRPV channels. Materials and methods: Human primary CD4⁺ T cells were activated with anti CD3/CD28 with and without DPV576 at 1:10 and 1:100 dilutions for 24 h in vitro. TRPV receptor expression (TRPV1 and TRPV4) on CD4⁺ T cells were examined by flow cytometry. The capacity of DPV576 to modulate the activity of TRPV1 agonist capsaicin in CD4⁺ T cells was also determined. Activation of CD4⁺ T cells was determined by production of cytokines TNF-α, IFN-γ, and IL-10 using specific ELISA kits. Results: DPV576 treatment of CD4⁺ T cells that were activated with anti CD3/CD28 resulted in decreased expression of the TRPV1 channel, but had no effect on TRPV4. This was accompanied by decreased secretion of IFN-γ and reduced expression of TRPV1 in capsaicin activated CD4⁺ T cells. In addition, DPV576 inhibited the capsaicin, induced the production of IFN-γ, and enhanced the secretion of IL-10. Conclusion: We conclude that short term exposure to DPV576 inhibits the activity of TRPV1 channels in CD4⁺ T lymphocytes, which may suggest its possible beneficial use for pain management.

Glucose inhibits cardiac muscle maturation through nucleotide biosynthesis.

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is associated with congenital heart disease. However, little is known about how blood glucose impacts heart formation. Using a chemically defined human pluripotent stem-cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic, electrophysiological, and biomechanical levels by promoting nucleotide biosynthesis through the pentose phosphate pathway. Blood glucose level in embryos is stable in utero during normal pregnancy, but glucose uptake by fetal cardiac tissue is drastically reduced in late gestational stages. In a murine model of diabetic pregnancy, fetal hearts showed cardiomyopathy with increased mitotic activity and decreased maturity. These data suggest that high glucose suppresses cardiac maturation, providing a possible mechanistic basis for congenital heart disease in diabetic pregnancy.

DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle.

Progress in whole-genome sequencing using short-read (e.g., <150 bp), next-generation sequencing technologies has reinvigorated interest in high-resolution physical mapping to fill technical gaps that are not well addressed by sequencing. Here, we report two technical advances in DNA nanotechnology and single-molecule genomics: (1) we describe a labeling technique (CRISPR-Cas9 nanoparticles) for high-speed AFM-based physical mapping of DNA and (2) the first successful demonstration of using DVD optics to image DNA molecules with high-speed AFM. As a proof of principle, we used this new "nanomapping" method to detect and map precisely BCL2-IGH translocations present in lymph node biopsies of follicular lymphoma patents. This HS-AFM "nanomapping" technique can be complementary to both sequencing and other physical mapping approaches.

Identification of a Human Airway Epithelial Cell Subpopulation with Altered Biophysical, Molecular, and Metastatic Properties.

Lung cancers are documented to have remarkable intratumoral genetic heterogeneity. However, little is known about the heterogeneity of biophysical properties, such as cell motility, and its relationship to early disease pathogenesis and micrometastatic dissemination. In this study, we identified and selected a subpopulation of highly migratory premalignant airway epithelial cells that were observed to migrate through microscale constrictions at up to 100-fold the rate of the unselected immortalized epithelial cell lines. This enhanced migratory capacity was found to be Rac1-dependent and heritable, as evidenced by maintenance of the phenotype through multiple cell divisions continuing more than 8 weeks after selection. The morphology of this lung epithelial subpopulation was characterized by increased cell protrusion intensity. In a murine model of micrometastatic seeding and pulmonary colonization, the motility-selected premalignant cells exhibit both enhanced survival in short-term assays and enhanced outgrowth of premalignant lesions in longer-term assays, thus overcoming important aspects of "metastatic inefficiency." Overall, our findings indicate that among immortalized premalignant airway epithelial cell lines, subpopulations with heritable motility-related biophysical properties exist, and these may explain micrometastatic seeding occurring early in the pathogenesis of lung cancer. Understanding, targeting, and preventing these critical biophysical traits and their underlying molecular mechanisms may provide a new approach to prevent metastatic behavior. Cancer Prev Res; 10(9); 514-24. ©2017 AACRSee related editorial by Hynds and Janes, p. 491.

Atomic force microscopy correlates antimetastatic potentials of HepG2 cell line with its redox/energy status: effects of curcumin and Khaya senegalensis.

OBJECTIVE: The fatality of cancer is mostly dependent on the possibility of occurrence of metastasis. Thus, if the development of metastasis can be prevented through novel therapeutic strategies targeted against this process, then the success of cancer treatment will drastically increase. In this study, therefore, we evaluated the antimetastatic potentials of an extract of Khaya senegalensis and curcumin on the metastatic liver cell line HepG2, and also assessed the anticancer property of the extract. METHODS: Cells were cultured and treated with graded concentrations of test substances for 24, 48, or 72 h with provisions made for negative controls. Treated cells were assessed as follows: nanotechnologically - atomic force microscopy (AFM) was used to determine cell stiffness; biochemically - cell cytotoxicity, glutathione level and adenosine triphosphate status, caspase activation and mitochondrial toxicity were considered; and microbiologically - a carrot disk assay was used to assess the anticancer property of the extract of K. senegalensis. RESULTS: Curcumin and K. senegalensis increased the cell stiffness by 2.6- and 4.0-fold respectively, indicating their antimetastatic effects. Corresponding changes in redox (glutathione level) and energy (adenosine triphosphate) status of the cells were also demonstrated. Further mechanistic studies indicated that curcumin was not mitotoxic in HepG2 cells unlike the K. senegalensis extract. In addition, the extract potently inhibited the Agrobacterium tumefaciens-induced genetic transformation based on carrot disk assay. CONCLUSION: Cell elasticity measurement data, using AFM, strongly suggested, for the first time, that both curcumin and the extract of K. senegalensis exhibited antimetastatic properties on HepG2 cells.

Two dimensional electrophysiological characterization of human pluripotent stem cell-derived cardiomyocyte system.

Stem cell-derived cardiomyocytes provide a promising tool for human developmental biology, regenerative therapies, disease modeling, and drug discovery. As human pluripotent stem cell-derived cardiomyocytes remain functionally fetal-type, close monitoring of electrophysiological maturation is critical for their further application to biology and translation. However, to date, electrophysiological analyses of stem cell-derived cardiomyocytes has largely been limited by biologically undefined factors including 3D nature of embryoid body, sera from animals, and the feeder cells isolated from mouse. Large variability in the aforementioned systems leads to uncontrollable and irreproducible results, making conclusive studies difficult. In this report, a chemically-defined differentiation regimen and a monolayer cell culture technique was combined with multielectrode arrays for accurate, real-time, and flexible measurement of electrophysiological parameters in translation-ready human cardiomyocytes. Consistent with their natural counterpart, amplitude and dV/dtmax of field potential progressively increased during the course of maturation. Monolayer culture allowed for the identification of pacemaking cells using the multielectrode array platform and thereby the estimation of conduction velocity, which gradually increased during the differentiation of cardiomyocytes. Thus, the electrophysiological maturation of the human pluripotent stem cell-derived cardiomyocytes in our system recapitulates in vivo development. This system provides a versatile biological tool to analyze human heart development, disease mechanisms, and the efficacy/toxicity of chemicals.

High-Speed Atomic Force Microscopy Revealing Contamination in DNA Purification Systems.

Motivated by reports of low-level DNA contamination in popular commercial DNA purification kits, we employed a novel high-speed atomic force microscopy (HS-AFM) method to detect and characterize particulate and polymeric contaminants in four such systems: Qiagen MinElute PCR Purification, Zymo Research DNA Clean and Concentrator-5, Invitrogen ChargeSwitch-Pro PCR Purification, and Beckman Coulter AMPure XP. HS-AFM avoids amplification artifacts present in PCR or in the sequencing of amplified products, and it requires no chemical labels and easily achieves near-single-molecule sensitivity. Using this technique, we found trace levels of filamentous contamination, similar in appearance to dsDNA, in eluates from the Zymo, Qiagen, and ChargeSwitch kits. Conversely, we detected no contaminants in magnetic bead-based AMPure XP solutions. Eluates from the Zymo kits also tested positive for DNA in fluorescent intercalator dye and whole genome amplification (WGA) assays. Qiagen kits tested positive in the fluorescence assay but negative in the WGA assay. Both ChargeSwitch and AMPure XP tested negative in the fluorescence assay while the WGA results for these two kits were ambiguous. Taken together, our findings suggest AMPure XP would be the best choice for analyses requiring very high analytical stringency. While HS-AFM alone does not provide chemical specificity, it is a potentially valuable tool for characterizing and quantifying trace contaminants in molecular biology reagents and instruments in cases where conventional techniques fail.

Mitochondrial Ca(2+) uptake by the voltage-dependent anion channel 2 regulates cardiac rhythmicity.

Tightly regulated Ca(2+) homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca(2+) handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca(2+) extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca(2+) uptake and accelerates the transfer of Ca(2+) from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca(2+) sparks and thereby inhibits Ca(2+) overload-induced erratic Ca(2+) waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca(2+) uptake in the regulation of cardiac rhythmicity.

Fine Needle Elastography (FNE) device for biomechanically determining local variations of tissue mechanical properties.

Diseased tissues exhibit changes in mechanical properties and thus possess clinical diagnostic significance. We report the design and development of a Fine Needle Elastography (FNE) prototype device integrated with Fine Needle Aspiration Cytology (FNAC) needle that allows for quantitative and sensitive assessment of tissues and materials based on local variations in elastic, friction, and cutting forces on needle insertion. A piezoelectric force-sensor at the base of FNA needle measures the forces opposing needle penetration with micrometer scale resolution. Measurement precision (±5 µm) and axial resolution (~20 µm) of FNE device was tested using control mm size gelatin matrices and unripe pear in assessing needle penetration resistance, force heterogeneity and optimization of needle penetration velocity. Further, we demonstrated the usefulness of FNE in quantitative, biomechanical differentiation of simulated thyroid tumor nodules in an ultrasound neck phantom. Fluid or solid nodules were probed in the phantom study coupled with ultrasound guidance. Our data shows significantly higher force variations (1-D force heterogeneity; HF,a=6.5 mN, HF,q=8.25 mN and stiffness heterogeneity; HS,a=0.0274 kN/m, HS,q=0.0395 kN/m) in solid nodules compared either to fluid nodules or to regions corresponding to healthy thyroid tissue within the ultrasound phantom. The results suggest future applications of in vivo FNE biopsies based on force heterogeneity to diagnose thyroid tumors in areas where ultrasound instrumentation or access to a qualified pathologist for FNAC are unavailable, as well as an ancillary diagnostic tool in thyroid cancer management.