Osteosarcoma mechanobiology and therapeutic targets.

Osteosarcoma is one of the most common primary tumours of the bone, with a 5-year survival rate of less than 20% after the development of metastases. Osteosarcoma is highly predisposed in Paget's disease of the bone, and both have common characteristic skeletal features due to rapid bone remodelling. Osteosarcoma prognosis is location dependent, which further emphasizes the likely contribution of the bone microenvironment in its pathogenesis. Mechanobiology describes the processes involved when mechanical cues from the changing physical microenvironment of the bone are transduced to biological pathways through mechanosensitive cellular components. Mechanobiology-driven therapies have been used to curb tumour progression by direct alteration of the physical microenvironment or inhibition of metastasis-associated mechanosensitive proteins. This review emphasizes the contribution of mechanobiology to the progression of osteosarcoma and sheds light on current mechanobiology-based therapies and potential new targets for improving disease management. Additionally, the many different 3D models currently used to study osteosarcoma mechanobiology are summarized.

Chromatin hierarchical branching visualized at the nanoscale by electron microscopy.

Chromatin is spatially organized in a hierarchical manner by virtue of single nucleosomes condensing into higher order chromatin structures, conferring various mechanical properties and biochemical signals. These higher order chromatin structures regulate genomic function by organization of the heterochromatin and euchromatin landscape. Less is known about its transition state from higher order heterochromatin to the lower order nucleosome form, and there is no information on its physical properties. We have developed a facile method of electron microscopy visualization to reveal the interphase chromatin in eukaryotic cells and its organization into hierarchical branching structures. We note that chromatin hierarchical branching can be distinguished at four levels, clearly indicating the stepwise transition from heterochromatin to euchromatin. The protein-DNA density across the chromatin fibers decreases during the transition from compacted heterochromatin to dispersed euchromatin. Moreover, the thickness of the chromatin ranges between 10 to 270 nm, and the controversial 30 nm chromatin fiber exists as a prominent intermediate structure. This study provides important insights into higher order chromatin organization which plays a key role in diseases such as cancer.

Multiplexable fluorescence lifetime imaging (FLIM) probes for Abl and Src-family kinases.

Many commonly employed strategies to map kinase activities in live cells require expression of genetically encoded proteins (e.g. FRET sensors). In this work, we describe the development and preliminary application of a set of cell-penetrating, fluorophore labelled peptide substrates for fluorescence lifetime imaging (FLIM) of Abl and Src-family kinase activities. These probes do not rely on FRET pairs or genetically-encoded protein expression. We further demonstrate probe multiplexing and pixel-by-pixel quantification to estimate the relative proportion of modified probe, suggesting that this strategy will be useful for detailed mapping of single cell and subcellular dynamics of multiple kinases concurrently in live cells.

The transition structure of chromatin fibers at the nanoscale probed by cryogenic electron tomography.

The naked DNA inside the nucleus interacts with proteins and RNAs forming a higher order chromatin structure to spatially and temporally control transcription in eukaryotic cells. The 30 nm chromatin fiber is one of the most important determinants of the regulation of eukaryotic transcription. However, the transition of chromatin from the 30 nm inactive higher order structure to the actively transcribed lower order nucleosomal arrays is unclear, which limits our understanding of eukaryotic transcription. Using a method to extract near-native eukaryotic chromatin, we revealed the chromatin structure at the transitional state from the 30 nm chromatin to multiple nucleosomal arrays by cryogenic electron tomography (cryo-ET). Reproducible electron microscopy images revealed that the transitional structure is a branching structure that the 30 nm chromatin hierarchically branches into lower order nucleosomal arrays, indicating chromatin compaction at different levels to control its accessibility during the interphase. We further observed that some of the chromatin fibers on the branching structure have a helix ribbon structure, while the others randomly twist together. Our finding of the chromatin helix ribbon structure on the extracted native chromatin revealed by cryo-ET indicates a complex higher order chromatin organization beyond the beads-on-a-string structure. The hierarchical branching and helix ribbon structure may provide mechanistic insights into how chromatin organization plays a central role in transcriptional regulation and other DNA-related biological processes during diseases such as cancer.

Prenatal and ancestral exposure to di(2-ethylhexyl) phthalate alters gene expression and DNA methylation in mouse ovaries.

Di(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer and known endocrine disrupting chemical, which causes transgenerational reproductive toxicity in female rodents. However, the mechanisms of action underlying the transgenerational toxicity of DEHP are not understood. Therefore, this study determined the effects of prenatal and ancestral DEHP exposure on various ovarian pathways in the F1, F2, and F3 generations of mice. Pregnant CD-1 dams were orally exposed to corn oil (vehicle control) or DEHP (20 µg/kg/day-750 mg/kg/day) from gestation day 10.5 until birth. At postnatal day 21 for all generations, ovaries were removed for gene expression analysis of various ovarian pathways and for 5-methyl cytosine (5-mC) quantification. In the F1 generation, prenatal DEHP exposure disrupted the expression of cell cycle regulators, the expression of peroxisome-proliferator activating receptors, and the percentage of 5-mC compared to control. In the F2 generation, exposure to DEHP decreased the expression of steroidogenic enzymes, apoptosis factors, and ten-eleven translocation compared to controls. It also dysregulated the expression of phosphoinositide 3-kinase (PI3K) factors. In the F3 generation, ancestral DEHP exposure decreased the expression of steroidogenic enzymes, PI3K factors, cell cycle regulators, apoptosis factors, Esr2, DNA methylation mediators, and the percentage of 5-mC compared to controls. Overall, the data show that prenatal and ancestral DEHP exposure greatly suppress gene expression of pathways required for folliculogenesis and steroidogenesis in the ovary in a transgenerational manner and that gene expression may be influenced by DNA methylation. These results provide insight into some of the mechanisms of DEHP-mediated toxicity in the ovary across generations.

Epigenetic Process Monitoring in Live Cultures with Peptide Biosensors.

Acetyltransferase is a member of the transferase group responsible for transferring an acetyl group from acetyl-CoA to amino group of a histone lysine residue. Past efforts on histone acetylation monitoring involved biochemical analysis that do not provide spatiotemporal information in a dynamic format. We propose a novel approach to monitor acetyltransferase acetylation in live single cells using time correlated single photon counting fluorescence lifetime imaging (TCSPC-FLIM) with peptide biosensors. Utilizing 2D and 3D cultures we show that the peptide sensor has a specific response to acetyltransferase enzyme activity in a fluorescence lifetime dependent manner ( P < 0.001). Our FLIM biosensor concept enables real-time longitudinal measurement of acetylation activity with high spatial and temporal resolution in live single cells to monitor cell function or evaluate drug effects to treat cancer or neurological diseases.

Real-Time Multiplex Kinase Phosphorylation Sensors in Living Cells.

Phosphorylation is an important post-translational modification implicated in cellular signaling and regulation. However, current methods to study protein phosphorylation by various kinases lack spatiotemporal resolution or the ability to simultaneously observe in real time the activity of multiple kinases in live cells. We present a peptide biosensor strategy with time correlated single photon counting-fluorescence lifetime imaging (TCSPC-FLIM) to interrogate the spatial and temporal dynamics of VEGFR-2 and AKT phosphorylation activity in real time in live 2D and 3D cell culture models at single cell resolution. By recording the increase in fluorescence lifetime due to a change in the solvatochromic environment of the sensor upon phosphorylation, we demonstrate that spatiotemporal maps of protein kinase activity can be obtained. Our results suggest that fluorescence lifetime imaging of peptide biosensors can be effectively and specifically used to monitor and quantify phosphorylation of multiple kinases in live cells.

Toxicological effects of trichloroethylene exposure on immune disorders.

Trichloroethylene (TCE) is one of the most common ground water contaminants in USA. Even though recent regulation mandates restricted utilization of TCE, its use is not completely prohibited, especially in industrial and manufacturing processes. The risk of TCE on human health is an ongoing field of study and its implications on certain diseases such as cancer has been recognized and well-documented. However, the link between TCE and immune disorders is still an under-studied area. Studies on the risk of TCE on the immune system is usually focused on certain immune class disorders, but consensus on the impact of TCE on the immune system has not been established. This review presents representative work that investigates the effect of TCE on immune disorders and highlights future opportunities. We attempt to provide a broader perspective of the risks of TCE on the immune system and human health.

Monitoring focal adhesion kinase phosphorylation dynamics in live cells.

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase essential for a diverse set of cellular functions. Current methods for monitoring FAK activity in response to an extracellular stimulus lack spatiotemporal resolution and/or the ability to perform multiplex detection. Here we report on a novel approach to monitor the real-time kinase phosphorylation activity of FAK in live single cells by fluorescence lifetime imaging.

Multiplex single-cell quantification of rare RNA transcripts from protoplasts in a model plant system.

Here we demonstrate multiplex and simultaneous detection of four different rare RNA species from plant, Arabidopsis thaliana, using surface-enhanced Raman spectroscopy (SERS) and gold nanoprobes at single-cell resolution. We show the applicability of nanoparticle-based Raman spectroscopic sensor to study intracellular RNA copies. First, we demonstrate that gold-nanoparticles decorated with Raman probes and carrying specific nucleic acid probe sequences can be uptaken by the protoplasts. We confirm the internalization of gold nanoprobes by transmission electron microscopy, inductively-coupled plasma-mass spectrometry and fluorescence imaging. Second, we show the utility of a SERS platform to monitor individual alternatively spliced (AS) variants and miRNA copies within single cells. Finally, the distinctive spectral features of Raman-active dyes were exploited for multiplex analysis of AtPTB2, AtDCL2, miR156a and miR172a. Furthermore, single-cell studies were validated by in vitro quantification and evaluation of nanotoxicity of gold probes. Raman tag functionalized gold nanosensors yielded an approach for the tracking of rare RNAs within the protoplasts. The SERS-based approach for quantification of RNAs has the capability to be a highly sensitive, accurate and discerning method for single-cell studies including AS variants quantification and rare miRNA detection in specific plant species.

Non-fluorescent quantification of single mRNA with transient absorption microscopy.

Single molecule detection is confounded by the background signals from the biological environment, such as autofluorescence, Rayleigh scattering, or turbidity in cells and tissues. In this article, we report on the utilization of gold nanoparticles (AuNPs) as an orthogonal probe for non-fluorescence detection of single molecules with a transient absorption microscopy (TAM). The developed system and concepts were validated by quantitative evaluation of human epidermal receptor 2 (Her2) mRNA in cancer cells and tissues at single copy sensitivity. Results from TAM suggest that the average number of Her2 copies in SK-BR-3 and MCF-7 breast cancer cells is 203.19 ± 80.48, and 11.29 ± 4.47, respectively. Furthermore, TAM offers excellent signal-to-noise ratio in detecting mRNA in clinical tissues, indicating a significantly higher expression of Her2 genes in breast cancer tissues than that of normal tissues. Our single cell quantification TAM strategy was validated with a fluorescence in situ hybridization approach. Our demonstration shows that TAM has the potential to provide a new dimension in biomarker quantification at single molecule sensitivity in turbid biological environments providing a strong basis for clinical monitoring.

Point-of-care test for cervical cancer in LMICs.

Cervical cancer screening using Papanicolaou's smear test has been highly effective in reducing death from this disease. However, this test is unaffordable in low- and middle-income countries, and its complexity has limited wide-scale uptake. Alternative tests, such as visual inspection with acetic acid or Lugol's iodine and human papillomavirus DNA, are sub-optimal in terms of specificity and sensitivity, thus sensitive and affordable tests with high specificity for on-site reporting are needed. Using proteomics and bioinformatics, we have identified valosin-containing protein (VCP) as differentially expressed between normal specimens and those with cervical intra-epithelial neoplasia grade 2/3 (CIN2/CIN3+) or worse. VCP-specific immunohistochemical staining (validated by a point-of-care technology) provided sensitive (93%) and specific (88%) identification of CIN2/CIN3+ and may serve as a critical biomarker for cervical-cancer screening. Future efforts will focus on further refinements to enhance analytic sensitivity and specificity of our proposed test, as well as on prototype development.

Kinase phosphorylation monitoring with i-motif DNA cross-linked SERS probes.

We propose an ultrasensitive SERS-based peptide biosensor platform to monitor phosphorylation catalyzed by kinase in a dynamic format. The developed SERS strategy has a short response time with potential to monitor phosphorylation in live cells.

Rapid and unbiased extraction of chromatin associated RNAs from purified native chromatin.

An ultra fast and unbiased method that uses salicylic acid coated magnetic nanoparticles (SAMNPs) and magnetophoretic chromatography is developed to extract chromatin associated RNAs (CARs). The SAMNPs were first used for enriching cells from the cell culture media and further used for capturing chromatin after cells were lysed. The formed SAMNPs-chromatin complexes were transferred to a viscous polyethylene glycol (PEG) solution stored in a 200-µl pipette tip. Due to the difference in viscosities, a bi-layer liquid was formed inside the pipette tip. The SAMNPs-chromatin complexes were separated from the free SAMNPs and free RNA-SAMNPs complexes by applying an external magnetic field. The CARs were further extracted from the SAMNP-chromatin complexes directly. The extracted CARs were reverse transcribed as cDNA and further characterized by real-time qPCR. The total assay time taken for cell separation, chromatin purification and chromatin associated RNAs extraction can be accomplished in less than 2h.

Single-cell screening and quantification of transcripts in cancer tissues by second-harmonic generation microscopy.

Fluorescence-based single molecule techniques to interrogate gene expression in tissues present a very low signal-to-noise ratio due to the strong autofluorescence and other background signals from tissue sections. This report presents a background-free method using second-harmonic generation (SHG) nanocrystals as probes to quantify the messenger RNA (mRNA) of human epidermal growth receptor 2 (Her2) at single molecule resolution in specific phenotypes at single-cell resolution directly in tissues. Coherent SHG emission from individual barium titanium oxide (BTO) nanoprobes was demonstrated, allowing for a stable signal beyond the autofluorescence window. Her2 surface marker and Her2 mRNA were specifically labeled with BTO probes, and Her2 mRNA was quantified at single copy sensitivity in Her2 expressing phenotypes directly in cancer tissues. Our approach provides the first proof of concept of a cross-platform strategy to probe tissues at single-cell resolution in situ.

Trichloroethylene sensing in water based on SERS with multifunctional Au/TiO2 core-shell nanocomposites.

Herein we report on a rapid and highly sensitive scheme to detect trichloroethylene (TCE), an environmental contaminant, by surface enhanced Raman scattering (SERS) with multifunctional Au/TiO2 core-shell nanocomposites as SERS substrates. A facile approach to fabricate TiO2 shell around gold core nanocomposites is proposed as sensors for TCE detection by SERS. During detection, TCE was first oxidized due to the photocatalytic activity of the TiO2 shell and the increase in SERS intensity due to the product of TCE photooxidation can be used to determine the concentration of TCE. It should be noted that the SERS of the Raman label, 4-mercaptopyridine (4-MPy) modified onto the gold nanoparticle (GNP) core is in proportion to the product of TCE photooxidation. After optimizing the sample pH, enrichment of the analyte, and the UV exposure time, the methodology developed accomplishes an excellent limit of detection (LOD) (0.038 µM, i.e.∼5 ppb) for TCE in water. Our unique approach based on the synthesized SERS composite to detect TCE, a chlorinated environmental contaminant directly in water could pave the way for the development of a multifunctional nanosensor platform to monitor TCE and the catalytic reactions in a multiplex format.

Modified graphene oxide sensors for ultra-sensitive detection of nitrate ions in water.

Nitrate ions is a very common contaminant in drinking water and has a significant impact on the environment, necessitating routine monitoring. Due to its chemical and physical properties, it is hard to directly detect nitrate ions with high sensitivity in a simple and inexpensive manner. Herein with amino group modified graphene oxide (GO) as a sensing element, we show a direct and ultra-sensitive method to detect nitrate ions, at a lowest detected concentration of 5 nM in river water samples, much lower than the reported methods based on absorption spectroscopy. Furthermore, unlike the reported strategies based on absorption spectroscopy wherein the nitrate concentration is determined by monitoring an increase in aggregation of gold nanoparticles (GNPs), our method evaluates the concentration of nitrate ions based on reduction in aggregation of GNPs for monitoring in real samples. To improve sensitivity, several optimizations were performed, including the assessment of the amount of modified GO required, concentration of GNPs and incubation time. The detection methodology was characterized by zeta potential, TEM and SEM. Our results indicate that an enrichment of modified GO with nitrate ions contributed to excellent sensitivity and the entire detection procedure could be completed within 75 min with only 20 µl of sample. This simple and rapid methodology was applied to monitor nitrate ions in real samples with excellent sensitivity and minimum pretreatment. The proposed approach paves the way for a novel means to detect anions in real samples and highlights the potential of GO based detection strategy for water quality monitoring.

Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage.

Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed.

Epitaxial superlattices with titanium nitride as a plasmonic component for optical hyperbolic metamaterials.

Titanium nitride (TiN) is a plasmonic material having optical properties resembling gold. Unlike gold, however, TiN is complementary metal oxide semiconductor-compatible, mechanically strong, and thermally stable at higher temperatures. Additionally, TiN exhibits low-index surfaces with surface energies that are lower than those of the noble metals which facilitates the growth of smooth, ultrathin crystalline films. Such films are crucial in constructing low-loss, high-performance plasmonic and metamaterial devices including hyperbolic metamaterials (HMMs). HMMs have been shown to exhibit exotic optical properties, including extremely high broadband photonic densities of states (PDOS), which are useful in quantum plasmonic applications. However, the extent to which the exotic properties of HMMs can be realized has been seriously limited by fabrication constraints and material properties. Here, we address these issues by realizing an epitaxial superlattice as an HMM. The superlattice consists of ultrasmooth layers as thin as 5 nm and exhibits sharp interfaces which are essential for high-quality HMM devices. Our study reveals that such a TiN-based superlattice HMM provides a higher PDOS enhancement than gold- or silver-based HMMs.