Novel Seed Size: A Novel Seed-Developing Gene in Glycine max.

Soybean-seed development is controlled in multiple ways, as in many known regulating genes. Here, we identify a novel gene, Novel Seed Size (NSS), involved in seed development, by analyzing a T-DNA mutant (S006). The S006 mutant is a random mutant of the GmFTL4pro:GUS transgenic line, with phenotypes with small and brown seed coats. An analysis of the metabolomics and transcriptome combined with RT-qPCR in the S006 seeds revealed that the brown coat may result from the increased expression of chalcone synthase 7/8 genes, while the down-regulated expression of NSS leads to small seed size. The seed phenotypes and a microscopic observation of the seed-coat integument cells in a CRISPR/Cas9-edited mutant nss1 confirmed that the NSS gene conferred small phenotypes of the S006 seeds. As mentioned in an annotation on the Phytozome website, NSS encodes a potential DNA helicase RuvA subunit, and no such genes were previously reported to be involved in seed development. Therefore, we identify a novel gene in a new pathway controlling seed development in soybeans.

Bronchial epithelium epithelial-mesenchymal plasticity forms aberrant basaloid-like cells in vitro.

Although epithelial-mesenchymal transition (EMT) is a common feature of fibrotic lung disease, its role in fibrogenesis is controversial. Recently, aberrant basaloid cells were identified in fibrotic lung tissue as a novel epithelial cell type displaying a partial EMT phenotype. The developmental origin of these cells remains unknown. To elucidate the role of EMT in the development of aberrant basaloid cells from the bronchial epithelium, we mapped EMT-induced transcriptional changes at the population and single-cell levels. Human bronchial epithelial cells grown as submerged or air-liquid interface (ALI) cultures with or without EMT induction were analyzed by bulk and single-cell RNA-Sequencing. Comparison of submerged and ALI cultures revealed differential expression of 8,247 protein coding (PC) and 1,621 long noncoding RNA (lncRNA) genes and revealed epithelial cell-type-specific lncRNAs. Similarly, EMT induction in ALI cultures resulted in robust transcriptional reprogramming of 6,020 PC and 907 lncRNA genes. Although there was no evidence for fibroblast/myofibroblast conversion following EMT induction, cells displayed a partial EMT gene signature and an aberrant basaloid-like cell phenotype. The substantial transcriptional differences between submerged and ALI cultures highlight that care must be taken when interpreting data from submerged cultures. This work supports that lung epithelial EMT does not generate fibroblasts/myofibroblasts and confirms ALI cultures provide a physiologically relevant system to study aberrant basaloid-like cells and mechanisms of EMT. We provide a catalog of PC and lncRNA genes and an interactive browser (https://bronc-epi-in-vitro.cells.ucsc.edu/) of single-cell RNA-Seq data for further exploration of potential roles in the lung epithelium in health and lung disease.

Gene Circuit Explorer (GeneEx): an interactive web-app for visualizing, simulating and analyzing gene regulatory circuits.

SUMMARY: GeneEx is an interactive web-app that uses an ODE-based mathematical modeling approach to simulate, visualize and analyze gene regulatory circuits (GRCs) for an explicit kinetic parameter set or for a large ensemble of random parameter sets. GeneEx offers users the freedom to modify many aspects of the simulation such as the parameter ranges, the levels of gene expression noise and the GRC network topology itself. This degree of flexibility allows users to explore a variety of hypotheses by providing insight into the number and stability of attractors for a given GRC. Moreover, users have the option to upload, and subsequently compare, experimental gene expression data to simulated data generated from the analysis of a built or uploaded custom circuit. Finally, GeneEx offers a curated database that contains circuit motifs and known biological GRCs to facilitate further inquiry into these. Overall, GeneEx enables users to investigate the effects of parameter variation, stochasticity and/or topological changes on gene expression for GRCs using a systems-biology approach. AVAILABILITY AND IMPLEMENTATION: GeneEx is available at https://geneex.jax.org. This web-app is released under the MIT license and is free and open to all users and there is no mandatory login requirement. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Elucidating cancer metabolic plasticity by coupling gene regulation with metabolic pathways.

Metabolic plasticity enables cancer cells to switch their metabolism phenotypes between glycolysis and oxidative phosphorylation (OXPHOS) during tumorigenesis and metastasis. However, it is still largely unknown how cancer cells orchestrate gene regulation to balance their glycolysis and OXPHOS activities. Previously, by modeling the gene regulation of cancer metabolism we have reported that cancer cells can acquire a stable hybrid metabolic state in which both glycolysis and OXPHOS can be used. Here, to comprehensively characterize cancer metabolic activity, we establish a theoretical framework by coupling gene regulation with metabolic pathways. Our modeling results demonstrate a direct association between the activities of AMPK and HIF-1, master regulators of OXPHOS and glycolysis, respectively, with the activities of three major metabolic pathways: glucose oxidation, glycolysis, and fatty acid oxidation. Our model further characterizes the hybrid metabolic state and a metabolically inactive state where cells have low activity of both glycolysis and OXPHOS. We verify the model prediction using metabolomics and transcriptomics data from paired tumor and adjacent benign tissue samples from a cohort of breast cancer patients and RNA-sequencing data from The Cancer Genome Atlas. We further validate the model prediction by in vitro studies of aggressive triple-negative breast cancer (TNBC) cells. The experimental results confirm that TNBC cells can maintain a hybrid metabolic phenotype and targeting both glycolysis and OXPHOS is necessary to eliminate their metabolic plasticity. In summary, our work serves as a platform to symmetrically study how tuning gene activity modulates metabolic pathway activity, and vice versa.

Automatic Rate and Rate-Integrating Mode-Switchable Axisymmetric Gyroscope.

This paper describes a novel rate and rate-integrating mode-switchable axisymmetric gyroscope. A precession angle tracking algorithm is developed to enable the gyro to switch automatically between rate and rate-integrating modes at preset rate points through a digital control system within the gyro. We also propose a vibrating amplitude control method for the rate-integrating mode that directly extracts the angular rate output to ensure switching stability. In rate mode, the bias instability and angle random walk of the gyro reach 0.106°/h and 0.011°/√h, respectively. Additionally, an input range of over ±5000°/s is measured in rate-integrating mode. The scale factor nonlinearity reaches approximately 116 ppm over the full-scale range. The control system implements effective steering control of the gyroscope, with a switching delay of 10 ms from rate mode to rate-integrating mode and 100 ms from rate-integrating to rate mode. The proposed system actualizes a new type of gyroscope with high accuracy and a wide input range, which combines the benefits of rate and rate-integrating modes.

An Efficient Agrobacterium rhizogenes-Mediated Hairy Root Transformation Method in a Soybean Root Biology Study.

The stable genetic transformation of soybean is time-consuming and inefficient. As a simple and practical alternative method, hairy root transformation mediated by Agrobacterium rhizogenes is widely applied in studying root-specific processes, nodulation, biochemical and molecular functions of genes of interest, gene editing efficiency of CRISPR/Cas9, and biological reactors and producers. Therefore, many laboratories have developed unique protocols to obtain hairy roots in composite plants composed of transgenic roots and wild-type shoots. However, these protocols still suffer from the shortcomings of low efficiency and time, space, and cost consumption. To address this issue, we developed a new protocol efficient regeneration and transformation of hairy roots (eR&T) in soybean, by integrating and optimizing the main current methods to achieve high efficiency in both hairy root regeneration and transformation within a shorter period and using less space. By this eR&T method, we obtained 100% regeneration of hairy roots for all explants, with an average 63.7% of transformation frequency, which promoted the simultaneous and comparative analysis of the function of several genes. The eR&T was experimentally verified Promoter:GUS reporters, protein subcellular localization, and CRISPR/Cas9 gene editing experiments. Employing this approach, we identified several novel potential regulators of nodulation, and nucleoporins of the Nup107-160 sub-complex, which showed development-dependent and tissue-dependent expression patterns, indicating their important roles in nodulation in soybean. Thus, the new eR&T method is an efficient and economical approach for investigating not only root and nodule biology, but also gene function.

Spatial Divergence of PHR-PHT1 Modules Maintains Phosphorus Homeostasis in Soybean Nodules.

Maintaining phosphorus (Pi) homeostasis in nodules is the key to nodule development and nitrogen fixation, an important source of nitrogen for agriculture and ecosystems. PHOSPHATE-TRANSPORTER1 (PHT1) and its regulator PHOSPHATE-STARVATION-RESPONSE1 (PHR1), which constitute the PHR1-PHT1 module, play important roles in maintaining Pi homeostasis in different organs. However, the PHR1-PHT1 module and its functions in nodules remain unknown. We identified one PHT1 (GmPHT1;11) and four PHR1 (GmPHR1) homologs in soybean (Glycine max) plants, which displayed specific expression patterns in different tissues in nodules, similar to previously reported GmPHT1;1 and GmPHT1;4 Through the integration of different approaches, GmPHR-GmPHT1 modules were confirmed. Combining our results and previous reports, we established multiple GmPHR-GmPHT1 modules acting in the infected or noninfected tissues in nodules. A single GmPHR had more than one GmPHT1 target, and vice versa. Therefore, overlapping and cross-talking modules monitored the wave of available Pi to maintain Pi homeostasis in nodules, which sequentially regulated nodule initiation and development. High levels of GmPHT1;11 enhanced Pi accumulation in nodules, increased nodule size, but decreased nodule number. Nitrogenase activity was also enhanced by GmPHT1;11 Our findings uncover GmPHR-GmPHT1 modules in nodules, which expands our understanding of the mechanism of maintaining Pi homeostasis in soybean plants.

Role of metabolic spatiotemporal dynamics in regulating biofilm colony expansion.

Cell fate determination is typically regulated by biological networks, yet increasing evidences suggest that cell-cell communication and environmental stresses play crucial roles in the behavior of a cell population. A recent microfluidic experiment showed that the metabolic codependence of two cell populations generates a collective oscillatory dynamic during the expansion of a Bacillus subtilis biofilm. We develop a modeling framework for the spatiotemporal dynamics of the associated metabolic circuit for cells in a colony. We elucidate the role of metabolite diffusion and the need of two distinct cell populations to observe oscillations. Uniquely, this description captures the onset and thereafter stable oscillatory dynamics during expansion and predicts the existence of damping oscillations under various environmental conditions. This modeling scheme provides insights to understand how cells integrate the information from external signaling and cell-cell communication to determine the optimal survival strategy and/or maximize cell fitness in a multicellular system.

Generalized Radon transform approach to target motion parameter estimation using a stationary underwater vector hydrophone.

Underwater platforms provide long-term detection of undersea targets. In this paper, we propose a method for the estimation of target motion parameters by submerged static acoustic detection equipment. The proposed method is based on the Radon transform of modeling the target moving in a uniform straight line. The heading angle, the time to the closest point of approach (CPA), and the ratio of velocity to the horizontal range of the target at the CPA to the sensor are obtained by applying the generalized Radon transform (GRT) to bearing-time records. The velocity of the target is determined by applying the GRT to the line-spectrum-time records. Furthermore, the motion trajectory of the target with respect to the detection equipment can be calculated from the above parameters. To validate the feasibility and performance of the proposed method, computer simulations and sea trials based on a fixed single vector measurement system were analyzed in this paper. The results suggest that the proposed method can accurately estimate the motion parameters and can calculate the trajectory of the moving vessel along a straight line at constant velocity.

Reduction of Coil-Crack Angle Sensitivity Effect Using a Novel Flux Feature of ACFM Technique.

Alternating current field measurement (ACFM) testing is one of the promising techniques in the field of non-destructive testing with advantages of the non-contact capability and the reduction of lift-off effects. In this paper, a novel crack detection approach was proposed to reduce the effect of the angled crack (cack orientation) by using rotated ACFM techniques. The sensor probe is composed of an excitation coil and two receiving coils. Two receiving coils are orthogonally placed in the center of the excitation coil where the magnetic field is measured. It was found that the change of the x component and the peak value of the z component of the magnetic field when the sensor probe rotates around a crack followed a sine wave shape. A customized accelerated finite element method solver programmed in MATLAB was adopted to simulate the performance of the designed sensor probe which could significantly improve the computation efficiency due to the small crack perturbation. The experiments were also carried out to validate the simulations. It was found that the ratio between the z and x components of the magnetic field remained stable under various rotation angles. It showed the potential to estimate the depth of the crack from the ratio detected by combining the magnetic fields from both receiving coils (i.e., the x and z components of the magnetic field) using the rotated ACFM technique.

Evaluation of Coating Thickness Using Lift-Off Insensitivity of Eddy Current Sensor.

Defect detection in ferromagnetic substrates is often hampered by nonmagnetic coating thickness variation when using conventional eddy current testing technique. The lift-off distance between the sample and the sensor is one of the main obstacles for the thickness measurement of nonmagnetic coatings on ferromagnetic substrates when using the eddy current testing technique. Based on the eddy current thin-skin effect and the lift-off insensitive inductance (LII), a simplified iterative algorithm is proposed for reducing the lift-off variation effect using a multifrequency sensor. Compared to the previous techniques on compensating the lift-off error (e.g., the lift-off point of intersection) while retrieving the thickness, the simplified inductance algorithms avoid the computation burden of integration, which are used as embedded algorithms for the online retrieval of lift-offs via each frequency channel. The LII is determined by the dimension and geometry of the sensor, thus eliminating the need for empirical calibration. The method is validated by means of experimental measurements of the inductance of coatings with different materials and thicknesses on ferrous substrates (dual-phase alloy). The error of the calculated coating thickness has been controlled to within 3% for an extended lift-off range of up to 10 mm.

Analysis of Tilt Effect on Notch Depth Profiling Using Thin-Skin Regime of Driver-Pickup Eddy-Current Sensor.

Electromagnetic eddy current sensors are commonly used to identify and quantify the surface notches of metals. However, the unintentional tilt of eddy current sensors affects results of size profiling, particularly for the depth profiling. In this paper, based on the eddy current thin-skin regime, a revised algorithm has been proposed for the analytical voltage or impedance of a tilted driver-pickup eddy current sensor scanning across a long ideal notch. Considering the resolution of the measurement, the bespoke driver-pickup, also termed as transmitter-receiver (T-R) sensor is designed with a small mean radius of 1 mm. In addition, the T-R sensor is connected to the electromagnetic instrument and controlled by a scanning stage with high spatial travel resolution, with a limit of 0.2 µm and selected as 0.25 mm. Experiments were conducted for imaging of an aluminium sheet with seven machined long notches of different depths using T-R sensor under different tilt angles. By fitting the measured voltage (both real and imaginary part) with proposed analytical algorithms, the depth profiling of notches is less affected by the tilt angle of sensors. From the results, the depth of notches can be retrieved within a deviation of 10% for tilt angles up to 60 degrees.

Modeling the response of a tumor-suppressive network to mitogenic and oncogenic signals.

Intrinsic tumor-suppressive mechanisms protect normal cells against aberrant proliferation. Although cellular signaling pathways engaged in tumor repression have been largely identified, how they are orchestrated to fulfill their function still remains elusive. Here, we built a tumor-suppressive network model composed of three modules responsible for the regulation of cell proliferation, activation of p53, and induction of apoptosis. Numerical simulations show a rich repertoire of network dynamics when normal cells are subject to serum stimulation and adenovirus E1A overexpression. We showed that oncogenic signaling induces ARF and that ARF further promotes p53 activation to inhibit proliferation. Mitogenic signaling activates E2F activators and promotes Akt activation. p53 and E2F1 cooperate to induce apoptosis, whereas Akt phosphorylates p21 to repress caspase activation. These prosurvival and proapoptotic signals compete to dictate the cell fate of proliferation, cell-cycle arrest, or apoptosis. The cellular outcome is also impacted by the kinetic mode (ultrasensitivity or bistability) of p53. When cells are exposed to serum deprivation and recovery under fixed E1A, the shortest starvation time required for apoptosis induction depends on the terminal serum concentration, which was interpreted in terms of the dynamics of caspase-3 activation and cytochrome c release. We discovered that caspase-3 can be maintained active at high serum concentrations and that E1A overexpression sensitizes serum-starved cells to apoptosis. This work elucidates the roles of tumor repressors and prosurvival factors in tumor repression based on a dynamic network analysis and provides a framework for quantitatively exploring tumor-suppressive mechanisms.

Testing the gene expression classification of the EMT spectrum.

The epithelial-mesenchymal transition (EMT) plays a central role in cancer metastasis and drug resistance-two persistent clinical challenges. Epithelial cells can undergo a partial or full EMT, attaining either a hybrid epithelial/mesenchymal (E/M) or mesenchymal phenotype, respectively. Recent studies have emphasized that hybrid E/M cells may be more aggressive than their mesenchymal counterparts. However, mechanisms driving hybrid E/M phenotypes remain largely elusive. Here, to better characterize the hybrid E/M phenotype (s) and tumor aggressiveness, we integrate two computational methods-(a) RACIPE-to identify the robust gene expression patterns emerging from the dynamics of a given gene regulatory network, and (b) EMT scoring metric-to calculate the probability that a given gene expression profile displays a hybrid E/M phenotype. We apply the EMT scoring metric to RACIPE-generated gene expression data generated from a core EMT regulatory network and classify the gene expression profiles into relevant categories (epithelial, hybrid E/M, mesenchymal). This categorization is broadly consistent with hierarchical clustering readouts of RACIPE-generated gene expression data. We also show how the EMT scoring metric can be used to distinguish between samples composed of exclusively hybrid E/M cells and those containing mixtures of epithelial and mesenchymal subpopulations using the RACIPE-generated gene expression data.

Breast cancer tumorigenicity is dependent on high expression levels of NAF-1 and the lability of its Fe-S clusters.

Iron-sulfur (Fe-S) proteins are thought to play an important role in cancer cells mediating redox reactions, DNA replication, and telomere maintenance. Nutrient-deprivation autophagy factor-1 (NAF-1) is a 2Fe-2S protein associated with the progression of multiple cancer types. It is unique among Fe-S proteins because of its 3Cys-1His cluster coordination structure that allows it to be relatively stable, as well as to transfer its clusters to apo-acceptor proteins. Here, we report that overexpression of NAF-1 in xenograft breast cancer tumors results in a dramatic augmentation in tumor size and aggressiveness and that NAF-1 overexpression enhances the tolerance of cancer cells to oxidative stress. Remarkably, overexpression of a NAF-1 mutant with a single point mutation that stabilizes the NAF-1 cluster, NAF-1(H114C), in xenograft breast cancer tumors results in a dramatic decrease in tumor size that is accompanied by enhanced mitochondrial iron and reactive oxygen accumulation and reduced cellular tolerance to oxidative stress. Furthermore, treating breast cancer cells with pioglitazone that stabilizes the 3Cys-1His cluster of NAF-1 results in a similar effect on mitochondrial iron and reactive oxygen species accumulation. Taken together, our findings point to a key role for the unique 3Cys-1His cluster of NAF-1 in promoting rapid tumor growth through cellular resistance to oxidative stress. Cluster transfer reactions mediated by the overexpressed NAF-1 protein are therefore critical for inducing oxidative stress tolerance in cancer cells, leading to rapid tumor growth, and drugs that stabilize the NAF-1 cluster could be used as part of a treatment strategy for cancers that display high NAF-1 expression.

Interrogating the topological robustness of gene regulatory circuits by randomization.

One of the most important roles of cells is performing their cellular tasks properly for survival. Cells usually achieve robust functionality, for example, cell-fate decision-making and signal transduction, through multiple layers of regulation involving many genes. Despite the combinatorial complexity of gene regulation, its quantitative behavior has been typically studied on the basis of experimentally verified core gene regulatory circuitry, composed of a small set of important elements. It is still unclear how such a core circuit operates in the presence of many other regulatory molecules and in a crowded and noisy cellular environment. Here we report a new computational method, named random circuit perturbation (RACIPE), for interrogating the robust dynamical behavior of a gene regulatory circuit even without accurate measurements of circuit kinetic parameters. RACIPE generates an ensemble of random kinetic models corresponding to a fixed circuit topology, and utilizes statistical tools to identify generic properties of the circuit. By applying RACIPE to simple toggle-switch-like motifs, we observed that the stable states of all models converge to experimentally observed gene state clusters even when the parameters are strongly perturbed. RACIPE was further applied to a proposed 22-gene network of the Epithelial-to-Mesenchymal Transition (EMT), from which we identified four experimentally observed gene states, including the states that are associated with two different types of hybrid Epithelial/Mesenchymal phenotypes. Our results suggest that dynamics of a gene circuit is mainly determined by its topology, not by detailed circuit parameters. Our work provides a theoretical foundation for circuit-based systems biology modeling. We anticipate RACIPE to be a powerful tool to predict and decode circuit design principles in an unbiased manner, and to quantitatively evaluate the robustness and heterogeneity of gene expression.

Custom edge-element FEM solver and its application to eddy-current simulation of realistic 2M-element human brain phantom.

Extensive research papers of three-dimensional computational techniques are widely used for the investigation of human brain pathophysiology. Eddy current analyzing could provide an indication of conductivity change within a biological body. A significant obstacle to current trend analyses is the development of a numerically stable and efficiency-finite element scheme that performs well at low frequency and does not require a large number of degrees of freedom. Here, a custom finite element method (FEM) solver based on edge elements is proposed using the weakly coupled theory, which separates the solution into two steps. First, the background field (the magnetic vector potential on each edge) is calculated and stored. Then, the electric scalar potential on each node is obtained by FEM based on Galerkin formulations. Consequently, the electric field and eddy current distribution in the object can be obtained. This solver is more efficient than typical commercial solvers since it reduces the vector eddy current equation to a scalar one, and reduces the meshing domain to just the eddy current region. It can therefore tackle complex eddy current calculations for models with much larger numbers of elements, such as those encountered in eddy current computation in biological tissues. An example is presented with a realistic human brain mesh of 2 million elements. In addition, with this solver, the equivalent magnetic field induced from the excitation coil is applied, and therefore there is no need to mesh the excitation coil. In combination, these significantly increase the efficiency of the solver. Bioelectromagnetics. 39:604-616, 2018. © 2018 Wiley Periodicals, Inc.

Jagged-Delta asymmetry in Notch signaling can give rise to a Sender/Receiver hybrid phenotype.

Notch signaling pathway mediates cell-fate determination during embryonic development, wound healing, and tumorigenesis. This pathway is activated when the ligand Delta or the ligand Jagged of one cell interacts with the Notch receptor of its neighboring cell, releasing the Notch Intracellular Domain (NICD) that activates many downstream target genes. NICD affects ligand production asymmetrically--it represses Delta, but activates Jagged. Although the dynamical role of Notch-Jagged signaling remains elusive, it is widely recognized that Notch-Delta signaling behaves as an intercellular toggle switch, giving rise to two distinct fates that neighboring cells adopt--Sender (high ligand, low receptor) and Receiver (low ligand, high receptor). Here, we devise a specific theoretical framework that incorporates both Delta and Jagged in Notch signaling circuit to explore the functional role of Jagged in cell-fate determination. We find that the asymmetric effect of NICD renders the circuit to behave as a three-way switch, giving rise to an additional state--a hybrid Sender/Receiver (medium ligand, medium receptor). This phenotype allows neighboring cells to both send and receive signals, thereby attaining similar fates. We also show that due to the asymmetric effect of the glycosyltransferase Fringe, different outcomes are generated depending on which ligand is dominant: Delta-mediated signaling drives neighboring cells to have an opposite fate; Jagged-mediated signaling drives the cell to maintain a similar fate to that of its neighbor. We elucidate the role of Jagged in cell-fate determination and discuss its possible implications in understanding tumor-stroma cross-talk, which frequently entails Notch-Jagged communication.

Modeling putative therapeutic implications of exosome exchange between tumor and immune cells.

Development of effective strategies to mobilize the immune system as a therapeutic modality in cancer necessitates a better understanding of the contribution of the tumor microenvironment to the complex interplay between cancer cells and the immune response. Recently, effort has been directed at unraveling the functional role of exosomes and their cargo of messengers in this interplay. Exosomes are small vesicles (30-200 nm) that mediate local and long-range communication through the horizontal transfer of information, such as combinations of proteins, mRNAs and microRNAs. Here, we develop a tractable theoretical framework to study the putative role of exosome-mediated cell-cell communication in the cancer-immunity interplay. We reduce the complex interplay into a generic model whose three components are cancer cells, dendritic cells (consisting of precursor, immature, and mature types), and killer cells (consisting of cytotoxic T cells, helper T cells, effector B cells, and natural killer cells). The framework also incorporates the effects of exosome exchange on enhancement/reduction of cell maturation, proliferation, apoptosis, immune recognition, and activation/inhibition. We reveal tristability-possible existence of three cancer states: a low cancer load with intermediate immune level state, an intermediate cancer load with high immune level state, and a high cancer load with low immune-level state, and establish the corresponding effective landscape for the cancer-immunity network. We illustrate how the framework can contribute to the design and assessments of combination therapies.

HKOH-1: A Highly Sensitive and Selective Fluorescent Probe for Detecting Endogenous Hydroxyl Radicals in Living Cells.

The hydroxyl radical (. OH), one of the most reactive and deleterious reactive oxygen species (ROS), has been suggested to play an essential role in many physiological and pathological scenarios. However, a reliable and robust method to detect endogenous . OH is currently lacking owing to its extremely high reactivity and short lifetime. Herein we report a fluorescent probe HKOH-1 with superior in vitro selectivity and sensitivity towards . OH. With this probe, we have calibrated and quantified the scavenging capacities of a wide range of reported . OH scavengers. Furthermore, HKOH-1r, which was designed for better cellular uptake and retention, has performed robustly in detection of endogenous . OH generation by both confocal imaging and flow cytometry. Furthermore, this probe has been applied to monitor . OH generation in HeLa cells in response to UV light irradiation. Therefore, HKOH-1 could be used for elucidating . OH related biological functions.