Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

Plant cell-surface GIPC sphingolipids sense salt to trigger Ca2+ influx.

Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca2+ concentration, which activate Ca2+-binding proteins and upregulate the Na+/H+ antiporter in order to remove Na+. Salt-induced increases in Ca2+ have long been thought to be involved in the detection of salt stress, but the molecular components of the sensing machinery remain unknown. Here, using Ca2+-imaging-based forward genetic screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca2+]i increases 1 (moca1), and identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca2+ spikes and waves, Na+/H+ antiporter activation, and regulation of growth. Na+ binds to GIPCs to gate Ca2+ influx channels. This salt-sensing mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and could be used to improve salt resistance in crops.

OSCA1 is an osmotic specific sensor: a method to distinguish Ca2+ -mediated osmotic and ionic perception.

Genetic mutants defective in stimulus-induced Ca2+ increases have been gradually isolated, allowing the identification of cell-surface sensors/receptors, such as the osmosensor OSCA1. However, determining the Ca2+ -signaling specificity to various stimuli in these mutants remains a challenge. For instance, less is known about the exact selectivity between osmotic and ionic stresses in the osca1 mutant. Here, we have developed a method to distinguish the osmotic and ionic effects by analyzing Ca2+ increases, and demonstrated that osca1 is impaired primarily in Ca2+ increases induced by the osmotic but not ionic stress. We recorded Ca2+ increases induced by sorbitol (osmotic effect, OE) and NaCl/CaCl2 (OE + ionic effect, IE) in Arabidopsis wild-type and osca1 seedlings. We assumed the NaCl/CaCl2 total effect (TE) = OE + IE, then developed procedures for Ca2+ imaging, image analysis and mathematic fitting/modeling, and found osca1 defects mainly in OE. The osmotic specificity of osca1 suggests that osmotic and ionic perceptions are independent. The precise estimation of these two stress effects is applicable not only to new Ca2+ -signaling mutants with distinct stimulus specificity but also the complex Ca2+ signaling crosstalk among multiple concurrent stresses that occur naturally, and will enable us to specifically fine tune multiple signal pathways to improve crop yields.

Flg22-induced Ca2+ increases undergo desensitization and resensitization.

The flagellin epitope flg22, a pathogen-associated molecular pattern (PAMP), binds to the receptor-like kinase FLAGELLIN SENSING2 (FLS2), and triggers Ca2+ influx across the plasma membrane (PM). The flg22-induced increases in cytosolic Ca2+ concentration ([Ca2+ ]i ) (FICA) play a crucial role in plant innate immunity. It's well established that the receptor FLS2 and reactive oxygen species (ROS) burst undergo sensitivity adaptation after flg22 stimulation, referred to as desensitization and resensitization, to prevent over responses to pathogens. However, whether FICA also mount adaptation mechanisms to ensure appropriate and efficient responses against pathogens remains poorly understood. Here, we analysed systematically [Ca2+ ]i increases upon two successive flg22 treatments, recorded and characterized rapid desensitization but slow resensitization of FICA in Arabidopsis thaliana. Pharmacological analyses showed that the rapid desensitization might be synergistically regulated by ligand-induced FLS2 endocytosis as well as the PM depolarization. The resensitization of FICA might require de novo FLS2 protein synthesis. FICA resensitization appeared significantly slower than FLS2 protein recovery, suggesting additional regulatory mechanisms of other components, such as flg22-related Ca2+ permeable channels. Taken together, we have carefully defined the FICA sensitivity adaptation, which will facilitate further molecular and genetic dissection of the Ca2+ -mediated adaptive mechanisms in PAMP-triggered immunity.

Plasmonic nanobiosensors for detection of microRNA cancer biomarkers in clinical samples.

MicroRNAs (miRNAs) play an important role in the regulation of biological processes and have demonstrated great potential as biomarkers for the early detection of various diseases, including esophageal adenocarcinoma (EAC) and Barrett's esophagus (BE), the premalignant metaplasia associated with EAC. Herein, we demonstrate the direct detection of the esophageal cancer biomarker, miR-21, in RNA extracted from 17 endoscopic tissue biopsies using the nanophotonics technology our group has developed, termed the inverse molecular sentinel (iMS) nanobiosensor, with surface-enhanced Raman scattering (SERS) detection. The potential of this label-free, homogeneous biosensor for cancer diagnosis without the need for target amplification was demonstrated by discriminating esophageal cancer and Barrett's esophagus from normal tissue with notable diagnostic accuracy. This work establishes the potential of the iMS nanobiosensor for cancer diagnostics via miRNA detection in clinical samples without the need for target amplification, validating the potential of this assay as part of a new diagnostic strategy. Combining miRNA diagnostics with the nanophotonics technology will result in a paradigm shift in achieving a general molecular analysis tool that has widespread applicability for cancer research as well as detection of cancer. We anticipate further development of this technique for future use in point-of-care testing as an alternative to histopathological diagnosis as our method provides a quick result following RNA isolation, allowing for timely treatment.

Inverse Molecular Sentinel-Integrated Fiberoptic Sensor for Direct and in Situ Detection of miRNA Targets.

Molecular advances have been made in analysis systems for a wide variety of applications ranging from biodiagnostics, biosafety, bioengineering, and biofuel research applications. There are, however, limited practical tools necessary for in situ and accurate detection of nucleic acid targets during field work. New technology is needed to translate these molecular advances from laboratory settings into the real-life practical monitoring realm. The exquisite characteristics (e.g., sensitivity and adaptability) of plasmonic nanosensors have made them attractive candidates for field-ready sensing applications. Herein, we have developed a fiber-based plasmonic sensor capable of direct detection (i.e., no washing steps required) of nucleic acid targets, which can be detected simply by immerging the sensor in the sample solution. This sensor is composed of an optical fiber that is decorated with plasmonic nanoprobes based on silver-coated gold nanostars (AuNS@Ag) to detect target nucleic acids using the surface-enhanced Raman scattering (SERS) sensing mechanism of nanoprobes referred to as inverse molecular sentinels (iMS). These fiber-optrodes can be reused for several detection-regeneration cycles (>6). The usefulness and applicability of the iMS fiber-sensors was tested by detecting target miRNA in extracts from leaves of plants that were induced to have different expression levels of miRNA targets. These fiber-optrodes enable direct detection of miRNA in plant tissue extract without the need for complex assays by simply immersing the fiber in the sample solution. The results indicate the fiber-based sensors developed herein have the potential to be a powerful tool for field and in situ analysis of nucleic acid samples.

Tailoring the Core-Satellite Nanoassembly Architectures by Tuning Internanoparticle Electrostatic Interactions.

The use of plasmonic nanoplatforms has received increasing interest in a wide variety of fields ranging from theranostics to environmental sensing to plant biology. In particular, the development of plasmonic nanoparticles into ordered nanoclusters has been of special interest due to the new chemical functionalities and optical responses that they can introduce. However, achieving predetermined nanocluster architectures from bottom-up approaches in the colloidal solution state still remains a great challenge. Herein, we report a one-pot assembly approach that provides flexibility in precise control of core-satellite nanocluster architectures in the colloidal solution state. We found that the pH of the assembly medium plays a vital role in the hierarchy of the nanoclusters. The architecture along with the size of the satellite gold nanoparticles determines the optical responses of nanoclusters. Using electron microscopy and optical spectroscopy, we introduce a set of design rules for the synthesis of distinct architectures of silica-core gold satellites nanoclusters in the colloidal solution state. Our findings provide insight into advancing the colloidal solution state nanoclusters formation with predictable architectures and optical properties.

Fano resonance in a gold nanosphere with a J-aggregate coating.

We present a facile method to induce J-aggregate formation on gold nanospheres in colloidal solution using polyvinylsulfate. The nanoparticle J-aggregate complex results in an absorption spectrum with a split lineshape due to plasmon-exciton coupling, i.e. via the formation of upper and lower plexcitonic branches. The use of nanoparticles with different plasmon resonances alters the position of the upper plexcitonic band while the lower band remains at the same wavelength. This splitting is investigated theoretically, and shown analytically to arise from Fano resonance between the plasmon of the gold nanoparticles and exciton of the J-aggregates. A theoretical simulation of a J-aggregate coated and uncoated gold nanosphere produces an absorption spectrum that shows good agreement with the experimentally measured spectra.

Surface-Enhanced Raman Spectroscopy-Based Detection of Micro-RNA Biomarkers for Biomedical Diagnosis Using a Comparative Study of Interpretable Machine Learning Algorithms.

Surface-enhanced Raman spectroscopy (SERS) has wide diagnostic applications due to narrow spectral features that allow multiplex analysis. We have previously developed a multiplexed, SERS-based nanosensor for micro-RNA (miRNA) detection called the inverse molecular sentinel (iMS). Machine learning (ML) algorithms have been increasingly adopted for spectral analysis due to their ability to discover underlying patterns and relationships within large and complex data sets. However, the high dimensionality of SERS data poses a challenge for traditional ML techniques, which can be prone to overfitting and poor generalization. Non-negative matrix factorization (NMF) reduces the dimensionality of SERS data while preserving information content. In this paper, we compared the performance of ML methods including convolutional neural network (CNN), support vector regression, and extreme gradient boosting combined with and without NMF for spectral unmixing of four-way multiplexed SERS spectra from iMS assays used for miRNA detection. CNN achieved high accuracy in spectral unmixing. Incorporating NMF before CNN drastically decreased memory and training demands without sacrificing model performance on SERS spectral unmixing. Additionally, models were interpreted using gradient class activation maps and partial dependency plots to understand predictions. These models were used to analyze clinical SERS data from single-plexed iMS in RNA extracted from 17 endoscopic tissue biopsies. CNN and CNN-NMF, trained on multiplexed data, performed most accurately with RMSElabel = 0.101 and 9.68 × 10-2, respectively. We demonstrated that CNN-based ML shows great promise in spectral unmixing of multiplexed SERS spectra, and the effect of dimensionality reduction on performance and training speed.

Accurate in vivo tumor detection using plasmonic-enhanced shifted-excitation Raman difference spectroscopy (SERDS).

For the majority of cancer patients, surgery is the primary method of treatment. In these cases, accurately removing the entire tumor without harming surrounding tissue is critical; however, due to the lack of intraoperative imaging techniques, surgeons rely on visual and physical inspection to identify tumors. Surface-enhanced Raman scattering (SERS) is emerging as a non-invasive optical alternative for intraoperative tumor identification, with high accuracy and stability. However, Raman detection requires dark rooms to work, which is not consistent with surgical settings. Methods: Herein, we used SERS nanoprobes combined with shifted-excitation Raman difference spectroscopy (SERDS) detection, to accurately detect tumors in xenograft murine model. Results: We demonstrate for the first time the use of SERDS for in vivo tumor detection in a murine model under ambient light conditions. We compare traditional Raman detection with SERDS, showing that our method can improve sensitivity and accuracy for this task. Conclusion: Our results show that this method can be used to improve the accuracy and robustness of in vivo Raman/SERS biomedical application, aiding the process of clinical translation of these technologies.

Direct and Label-Free Detection of MicroRNA Cancer Biomarkers using SERS-Based Plasmonic Coupling Interference (PCI) Nanoprobes.

MicroRNAs (miRNAs), small noncoding endogenous RNA molecules, are emerging as promising biomarkers for early detection of various diseases and cancers. Practical screening tools and strategies to detect these small molecules are urgently needed to facilitate the translation of miRNA biomarkers into clinical practice. In this study, a label-free biosensing technique based on surface-enhanced Raman scattering (SERS), referred to as plasmonic coupling interference (PCI), was applied for the multiplex detection of miRNA biomarkers. The sensing mechanism of the PCI technique relies on the formation of a nanonetwork consisting of nanoparticles with Raman labels located between adjacent nanoparticles that are interconnected by DNA duplexes. Because of the plasmonic coupling effect of adjacent nanoparticles in the nanonetwork, the Raman labels exhibit intense SERS signals. Such effect can be modulated by the addition of miRNA targets of interest that act as inhibitors to interfere with the formation of this nanonetwork, resulting in a diminished SERS signal. In this study, the PCI technique is theoretically analyzed, and the multiplex capability for detection of multiple miRNA cancer biomarkers is demonstrated, establishing the great potential of PCI nanoprobes as a useful diagnostic tool for medical applications.

Plasmonic gold nanostar-mediated photothermal immunotherapy for brain tumor ablation and immunologic memory.

Brain tumors present unique therapeutic challenges and they include glioblastoma (GBM) and metastases from cancers of other organs. Current treatment options are limited and include surgical resection, radiation therapy, laser interstitial thermal therapy and chemotherapy. Although much research has been done on the development of immune-based treatment platforms, only limited success has been demonstrated. Herein, we demonstrate a novel treatment of GBM through the use of plasmonic gold nanostars (GNS) as photothermal inducers for synergistic immuno photothermal nanotherapy (SYMPHONY), which combines treatments using gold nanostar and laser-induced photothermal therapy with checkpoint blockade immunotherapy. In the treatment of a murine flank tumor model with the CT-2A glioma cell line, SYMPHONY demonstrated the capability of producing long-term survivors that rejects rechallenge with cancer cells, heralding the successful emergence of immunologic memory. This study is the first to investigate the use of this novel therapy for the treatment of GBM in a murine model.

Plasmonic Nanoprobes for in Vivo Multimodal Sensing and Bioimaging of MicroRNA within Plants.

Monitoring gene expression within whole plants is critical for many applications ranging from plant biology to agricultural biotechnology and biofuel development; however, no method currently exists for in vivo monitoring of genomic targets in plant systems without requiring sample extraction. Herein, we report a unique multimodal method based on plasmonic nanoprobes capable of in vivo imaging and biosensing of microRNA biotargets within whole plant leaves by integrating three different and complementary techniques: surface-enhanced Raman scattering (SERS), X-ray fluorescence (XRF), and plasmonics-enhanced two-photon luminescence (TPL). The method developed uses plasmonic nanostars, which not only provide large Raman signal enhancement but also allow for localization and quantification by XRF and plasmonics-enhanced TPL, owing to gold content and high two-photon luminescence cross sections. Our method uses inverse molecular sentinel nanoprobes for SERS bioimaging of microRNA within Arabidopsis thaliana leaves to provide a dynamic SERS map of detected microRNA targets while also quantifying nanoprobe concentrations using XRF and TPL. The nanoprobes were observed to occupy the intercellular spaces upon infiltration into the leaf tissues. This report lays the foundation for the use of plasmonic nanoprobes for in vivo functional imaging of nucleic acid biotargets in whole plants, a tool that will revolutionize bioengineering research by allowing the study of these biotargets with previously unmet spatial and temporal resolution, 200 µm and 30 min, respectively.

Gold nanoparticles-mediated photothermal therapy and immunotherapy.

Cancer has been a significant threat to human health with more than eight million deaths each year in the world. Therefore, there is a significant need for novel technologies to effectively treat cancer and ultimately reduce cancer recurrences, treatment costs, number of radical cystectomies and mortality. A promising therapeutic platform for cancer is offered by nanoparticle-mediated therapy. This review highlights the development and applications of various nanoparticle platforms for photo-induced hyperthermia and immunotherapy. Taking advantage of gold's high biocompatibility, gold nanoparticles (GNPs) can be injected intravenously and accumulate preferentially in cancer cells due to the enhanced permeability and retention effect. Various gold nanoplatforms including nanospheres, nanoshells, nanorods, nanocages and nanostars have been used for effective photothermal treatment of various cancers. GNPs have also been used in immunotherapies, involving cancer antigen and immune adjuvant delivery as well as combination therapies with photothermal therapy. Among GNPs platforms, gold nanostars (GNS) have great therapeutic potential due to their unique star-shaped geometry that dramatically enhances light absorption and provides high photon-to-heat conversion efficiency due to the plasmonic effect. This photothermal process can be exploited to specifically ablate tumors and, more importantly, to amplify the antitumor immune response following the highly immunogenic thermal death of cancer cells. GNS-mediated photothermal therapy combined with checkpoint immunotherapy has been found to reverse tumor-mediated immunosuppression, thereby leading to the treatment of not only primary tumors but also cancer metastasis, as well as to induce effective long-lasting immunity, in other words, an anticancer 'vaccine' effect.

Shining Gold Nanostars: From Cancer Diagnostics to Photothermal Treatment and Immunotherapy.

Cancer has been a significant threat to human health with more than eight million deaths each year in the world. There is an urgent need to develop novel methods to improve cancer management. Biocompatible gold nanostars (GNS) with tip-enhanced electromagnetic and optical properties have been developed and applied for multifunctional cancer diagnostics and therapy (theranostics). The GNS platform can be used for multiple sensing, imaging and treatment modalities, such as surface-enhanced Raman scattering, two-photon photoluminescence, magnetic resonance imaging and computed tomography as well as photothermal therapy and immunotherapy. GNS-mediated photothermal therapy combined with checkpoint immunotherapy has been found to reverse tumor-mediated immunosuppression, leading to the treatment of not only primary tumors but also cancer metastasis as well as inducing effective long-lasting immunity, i.e. an anticancer 'vaccine' effect.

Photothermal ablation of inflammatory breast cancer tumor emboli using plasmonic gold nanostars.

Inflammatory breast cancer (IBC) is rare, but it is the most aggressive subtype of breast cancer. IBC has a unique presentation of diffuse tumor cell clusters called tumor emboli in the dermis of the chest wall that block lymph vessels causing a painful, erythematous, and edematous breast. Lack of effective therapeutic treatments has caused mortality rates of this cancer to reach 20%-30% in case of women with stage III-IV disease. Plasmonic nanoparticles, via photothermal ablation, are emerging as lead candidates in next-generation cancer treatment for site-specific cell death. Plasmonic gold nanostars (GNS) have an extremely large two-photon luminescence cross-section that allows real-time imaging through multiphoton microscopy, as well as superior photothermal conversion efficiency with highly concentrated heating due to its tip-enhanced plasmonic effect. To effectively study the use of GNS as a clinically plausible treatment of IBC, accurate three-dimensional (3D) preclinical models are needed. Here, we demonstrate a unique in vitro preclinical model that mimics the tumor emboli structures assumed by IBC in vivo using IBC cell lines SUM149 and SUM190. Furthermore, we demonstrate that GNS are endocytosed into multiple cancer cell lines irrespective of receptor status or drug resistance and that these nanoparticles penetrate the tumor embolic core in 3D culture, allowing effective photothermal ablation of the IBC tumor emboli. These results not only provide an avenue for optimizing the diagnostic and therapeutic application of GNS in the treatment of IBC but also support the continuous development of 3D in vitro models for investigating the efficacy of photothermal therapy as well as to further evaluate photothermal therapy in an IBC in vivo model.

Human Adipose-Derived Stem Cells Labeled with Plasmonic Gold Nanostars for Cellular Tracking and Photothermal Cancer Cell Ablation.

BACKGROUND: Gold nanostars are unique nanoplatforms that can be imaged in real time and transform light energy into heat to ablate cells. Adipose-derived stem cells migrate toward tumor niches in response to chemokines. The ability of adipose-derived stem cells to migrate and integrate into tumors makes them ideal vehicles for the targeted delivery of cancer nanotherapeutics. METHODS: To test the labeling efficiency of gold nanostars, undifferentiated adipose-derived stem cells were incubated with gold nanostars and a commercially available nanoparticle (Qtracker), then imaged using two-photon photoluminescence microscopy. The effects of gold nanostars on cell phenotype, proliferation, and viability were assessed with flow cytometry, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide metabolic assay, and trypan blue, respectively. Trilineage differentiation of gold nanostar-labeled adipose-derived stem cells was induced with the appropriate media. Photothermolysis was performed on adipose-derived stem cells cultured alone or in co-culture with SKBR3 cancer cells. RESULTS: Efficient uptake of gold nanostars occurred in adipose-derived stem cells, with persistence of the luminescent signal over 4 days. Labeling efficiency and signal quality were greater than with Qtracker. Gold nanostars did not affect cell phenotype, viability, or proliferation, and exhibited stronger luminescence than Qtracker throughout differentiation. Zones of complete ablation surrounding the gold nanostar-labeled adipose-derived stem cells were observed following photothermolysis in both monoculture and co-culture models. CONCLUSIONS: Gold nanostars effectively label adipose-derived stem cells without altering cell phenotype. Once labeled, photoactivation of gold nanostar-labeled adipose-derived stem cells ablates neighboring cancer cells, demonstrating the potential of adipose-derived stem cells as a vehicle for the delivery of site-specific cancer therapy.

Folate Receptor-Targeted Theranostic Nanoconstruct for Surface-Enhanced Raman Scattering Imaging and Photodynamic Therapy.

We report the synthesis of a folate receptor (FR)-targeted theranostic nanocomposite for surface-enhanced Raman scattering (SERS) imaging and photodynamic therapy (PDT). FR-specific SERS detection and PDT are demonstrated in vitro using two FR-positive cancer cell lines and one FR-negative cancer cell lines.

Multiplexed Detection of MicroRNA Biomarkers Using SERS-Based Inverse Molecular Sentinel (iMS) Nanoprobes.

MicroRNAs (miRNAs) have demonstrated great promise as a novel class of biomarkers for early detection of various cancers, including breast cancer. However, due to technical difficulties in detecting these small molecules, miRNAs have not been adopted into routine clinical practice for early diagnostics. Thus, it is important to develop alternative detection strategies that could offer more advantages over conventional methods. Here, we demonstrate the application of a "turn-on" SERS sensing technology, referred to as "inverse Molecular Sentinel (iMS)" nanoprobes, as a homogeneous assay for multiplexed detection of miRNAs. This SERS nanoprobe involves the use of plasmonic-active nanostars as the sensing platform. The "OFF-to-ON" signal switch is based on a nonenzymatic strand-displacement process and the conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. This technique was previously used to detect a synthetic DNA sequence of interest. In this study, we modified the design of the nanoprobe to be used for the detection of short (22-nt) miRNA sequences. The demonstration of using iMS nanoprobes to detect miRNAs in real biological samples was performed with total small RNA extracted from breast cancer cell lines. The multiplex capability of the iMS technique was demonstrated using a mixture of the two differently labeled nanoprobes to detect miR-21 and miR-34a miRNA biomarkers for breast cancer. The results of this study demonstrate the feasibility of applying the iMS technique for multiplexed detection of short miRNAs molecules.