Fluorescent Indicator Research Articles

A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging

Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7× increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca2+ responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca2+ concentration using fluorescence lifetime imaging microscopy (FLIM).

Identifying sources and distribution of organic pollutants in a Moroccan river: Characterization of dissolved organic matter by absorption, excitation–emission fluorescence and chemometric analyses

This study investigates surface water contamination of Ben-Kazza River in Morocco, fed by effluents from an adjacent lagoon-based wastewater treatment plant (WWTP) and seasonally by industrial effluents, and which occasionally serves to irrigate agricultural fields. This study has two purpose: i) to track the main sources of contamination through the evolution of dissolved organic matter (DOM) characteristics along the watercourse, and ii) to characterize the WWTP influents and effluents with a focus on the efficiency of the lagoon treatment. We characterized a total of 495 water samples across the watercourse and from the inlet and outlet of the WWTP, using UV–visible absorption and excitation–emission fluorescence coupled with chemometric analyses. Absorption indicators and fluorescence indices were calculated and compared across sampling points. Results highlight spatial shifts together with temporal changes in DOM. PARAFAC identified components that varied between protein-like, humic-like and anthropogenic-like fluorophores along the river, permitted to trace the anthropogenic components and their sources. The lagoon treatment appeared to better remove fresh organic material than humic material: fluorescence intensity decreased by 68 % for peak T1 and by 22 % for peak C. Maximum fluorescence intensities (Fmax) decreased across all PARAFAC components, leading to more than 55 % reduction of ΣFmax.

Non-destructive estimation of flesh oil content in avocado (Persea americana Mill.) using fluorescence images from 365-nm UV light excitation.

The flesh oil content (OC) is a crucial commercial indicator of avocado maturity and directly correlates with its nutritional quality. To meet export standards and optimize edible characteristics, avocados must be harvested at the appropriate stage of physiological maturity. The significant variability in OC during maturation, without any external morphological indicators, poses a longstanding challenge. Currently, harvesting maturity is optimized through time-consuming, destructive laboratory methods like freeze-drying and chemical extraction, which use representative samples to estimate the maturity of entire orchards. In this study, for the first time, we employed fluorescence imaging of avocado skin using 365-nm UV polarized light excitation to estimate the OC in the 'Bacon' avocado cultivar. We developed a surface fluorescence index that strongly correlates with OC, achieving correlation coefficients up to -0.91. Our non-destructive and rapid approach achieved a cross-validation accuracy with an R2 value of 0.81, enabling the classification of avocados with low and high OC. This pioneering method shows considerable potential for further improvement and refinement. This study lays the groundwork for developing a portable, cost-effective, and real-time method for non-destructive in situ monitoring of avocado OC in the field and its integration into large-scale post-harvest grading systems.

Ligation-driven hyperbranched DNA assembly for label-free and sequence-specific measurement of RNA N6-methyladenosine in human breast tissues

N6-methyladenosine (m6A) is a critical post-transcriptional regulator, and it is linked to various biological functions (e.g., degradation, export, and translation) and multiple human diseases (e.g., cancers, immune disorders, and neurodegenerative diseases). The current methods for sequence-specific detection of m6A usually suffer from radioactive risk, large sample consumption, washing/separation steps, and false-positive signal. Herein, we develop a ligation-driven hyperbranched DNA assembly-based fluorescent biosensor for label-free and sequence-specific detection of m6A. In this assay, the unmethylated RNA can be identified and cleaved by m6A-sensitive endoribonuclease MazF. The retained intact m6A-RNA is complementary to the padlock probe to form a circular DNA, subsequently initiating ligation-driven hyperbranched rolling circle amplification (HRCA). The obtained HRCA products can be simply quantified with SYBR Gold as a fluorescent indicator. This method can be homogeneously carried out in an isothermal, label-free and antibody-free manner. The limit of detection can reach 0.079 pM. Moreover, this method can differentiate even 0.01 % m6A level in excess coexisting interferences, monitor cellular m6A RNA level with single-cell sensitivity, and differentiate m6A level in breast tumor tissues and normal adjacent tissues, with promising applications in m6A-releated epigenetic research and clinical diagnosis.

Studying Drosophila Larval Behavior in Agarose Channels.

Larvae of the fruit fly Drosophila melanogaster are a popular and tractable model system for studying the development and function of sensorimotor circuits, thanks to the relative numerical simplicity of their nervous system and the wealth of available genetic tools to manipulate the anatomy, activity, and function of specific cell types. Researchers studying the role of a particular gene or cell type in sensorimotor circuit activity or function may wish to observe the effects of an experimental manipulation during behavior in the intact animal. Observing these effects, which may include changes in the intracellular calcium concentration or movement of small numbers of neurons, muscles, etc., typically requires high-spatial-resolution imaging, which poses several difficulties in the freely crawling larva. Freely crawling larvae can move quickly and with changeable heading, making manual or automatic tracking challenging; additionally, they may make three-dimensional movements, such as rearing, that can degrade imaging focus. These challenges are potentially solvable using advanced imaging and algorithmic tracking setups, but cost, space, or development time may be prohibitive. This protocol describes a simple and cost-effective method for placing larvae inside agarose channels, thereby restricting larval crawling to a single dimension and enabling higher-magnification time-series imaging of fluorescently labeled structures during many cycles of locomotion. By using larvae that express fluorescent calcium indicators in cells of interest, researchers can apply this method to study the effects of experimental manipulations on neural or muscular activity during behavior in the intact animal.

A Diboronic Acid-Based Fluorescent Sensor Array for Rapid Identification of Lonicerae Japonicae Flos and Lonicerae Flos.

Lonicerae japonicae flos (LJF) and Lonicerae flos (LF) are traditional Chinese herbs that are commonly used and widely known for their medicinal properties and edibility. Although they may have a similar appearance and vary slightly in chemical composition, their effectiveness as medicine and their use in clinical settings vary significantly, making them unsuitable for substitution. In this study, a novel 2 × 3 six-channel fluorescent sensor array is proposed that uses machine learning algorithms in combination with the indicator displacement assay (IDA) method to quickly identify LJF and LF. This array comprises two coumarin-based fluorescent indicators (ES and MS) and three diboronic acid-substituted 4,4'-bipyridinium cation quenchers (Q1-Q3), forming six dynamic complexes (C1-C6). When these complexes react with the ortho-dihydroxy groups of phenolic acid compounds in LJF and LF, they release different fluorescent indicators, which in turn causes distinct fluorescence recovery. By optimizing eight machine learning algorithms, the model achieved 100% and 98.21% accuracy rates in the testing set and the cross-validation predictions, respectively, in distinguishing between LJF and LF using Linear Discriminant Analysis (LDA). The integration of machine learning with this fluorescent sensor array shows great potential in analyzing and detecting foods and pharmaceuticals that contain polyphenols.

A comparative study of optical and size properties of dissolved organic matter in the lower Mississippi River and Pearl River

Monthly water samples were collected from the lower Mississippi and Pearl Rivers between January 2009 and August 2011 to investigate the heterogeneity in the dynamic variations of dissolved organic carbon (DOC), colloidal organic carbon, chromophoric and fluorescence dissolved organic matter (CDOM and FDOM), PARAFAC-derived fluorescent components, and other optical properties including spectral slope, specific UV absorbance (SUVA), and fluorescence indices between the two contrasting river systems. The lower Mississippi River exhibits relatively lower concentrations of DOC (306 ± 41 μM C) and CDOM (27.9 ± 5.7 m−1 at 254 nm), featuring lower aromaticity (indicated by SUVA254) and apparent molecular weight (or higher spectral slope) with weak seasonal variability. The Pearl River, in contrast, has elevated levels of DOC (537 ± 212 μM C) and CDOM (66.4 ± 31.4 m−1), characterized by higher aromaticity, higher molecular weight, and significant seasonality, primarily originating from soil-derived allochthonous sources. The abundance of the >1 kDa colloidal DOC was, on average, 58 ± 3 % of the bulk DOC in the lower Mississippi River and 68 ± 6 % in the lower Pearl River. The >1 kDa high-molecular weight DOM (HMW-DOM) consistently had lower spectral slope and biological index (BIX) values, but higher humification index (HIX) values compared to both bulk DOM and low-molecular-weight DOM (LMW-DOM) counterparts. These trends could be representative of other similar large and small rivers. Four PARAFAC-derived fluorescent components (three humic-like and one protein-like) were identified for both rivers. A positive correlation between discharge and terrestrial humic-like fluorescent components indicated their dominant allochthonous sources, while the protein-like component decreased with increasing discharge, consistent with its autochthonic source and a dilution effect during high flow seasons. The occurrence of large flood events during the sampling period contributed to large DOC pulses, with DOM of higher aromaticity and HMW-DOM. This has important implications for coastal ocean ecosystems, which are increasingly impacted by river flooding events under changing climate conditions. Our results also provide an improved understanding of DOM dynamics in two representative rivers and establish a baseline dataset for future studies to assess changes in sources and composition of DOM and their impacts on the coastal ocean in response to climate, hydrological, and anthropogenic influences.

Seasonal changes of dissolved CO2 and its linkage with optical characteristics of DOM in groundwater in agricultural areas

Groundwater contains significant quantities of dissolved CO2, which are crucial contributors to the global carbon cycle. Dissolved organic matter (DOM) functions as a principal carbon and energy source for microbial communities within groundwater ecosystems, yet the relationship between dissolved CO2 and optical characteristics of DOM remains ambiguous. Here, we delineate the variations in seasonal and vertical distribution of dissolved CO2 concentrations and DOM optical characteristics, and assess their interconnections in the groundwater of an agricultural region. The results showed that the average concentration of dissolved CO2 in groundwater was 11,300 ± 5,788 ppm, with the highest levels observed in summer and the lowest in winter. Additionally, shallow wells exhibited higher concentrations of dissolved CO2 compared to deep wells. The mass balance calculation suggested that 40,092 t of CO2 are released annually due to the degassing of supersaturated CO2 from groundwater extraction. Three-dimensional fluorescence with parallel factor analysis (EEM-PARAFAC) identified five components of DOM in groundwater: Components C1, C2, C3 and C4 are humic-like substances, and C5 is a tryptophan-like substance associated with protein structures. The analysis of compositional features and fluorescence indices revealed that DOM is predominantly derived from microbial sources, with mixed contributions from terrestrial sources in groundwater. Principal component analysis (PCA) showed that first two axes accounted for 74.7 % of the variance in the seasonal variation of DOM optical characteristics and dissolved CO2 concentration, which reveals that DOM decomposition contributes to CO2 saturation in groundwater. However, a larger proportion of CO2 may be derived from leaching in the vadose zone during the rainy season or irrigation periods in shallow aquifer. This study enhanced the understanding of carbon dynamics within agricultural groundwater.

Characteristics of Dissolved Organic Matter as Affected by Decomposition of Different Organic Materials in Alpine Wetland

Dissolved organic matter (DOM) plays a significant role in the nutrient supply, energy flow, and pollutant transportation in the wetland ecosystem. However, little is known about the effect of the decomposition of different organic materials in alpine wetland water on the DOM characteristics. By conducting a 90-day decomposition experiment with the addition of different organic materials (peat soil, yak manure, and plant litter) alone or their combinations into alpine wetland water, we characterized the water DOM using three-dimension excitation-emission matrix spectroscopy. The results showed that the decomposition of organic materials significantly affected the chemical properties, sources, humification degree, and composition of the water DOM. The decomposition increased dissolved organic carbon and dissolved total nitrogen in the water. For most of the water samples, a fluorescence index ranging from 1.4 to 1.7 and a biological index of less than 0.8 may indicate that both autochthonous and allochthonous sources contributed to the water DOM, which may primarily rely on allochthonous sources. UVA (37.55–46.81% of total fluorescent components) and UVC fulvic-like substances (29.91–35.53% of total fluorescent components) dominated the water DOM compositions. Among the treatments, additions of peat soil and yak manure led to the highest and the lowest humification degree of the water DOM, respectively. For the treatment of the combination decomposition of all three organic materials, the yak manure may stimulate microbial activity and facilitate the decomposition of plant litter and peat soil and, therefore, boost the humic-like substances in the water DOM. These findings may help the development of wetland biomass management with the objective of maintaining alpine wetland ecosystem services.

Hyperglycemia adversely affects critical physiological events related to rat sperm capacitation

Hyperglycemia, the hallmark of diabetes mellitus (DM), is the main cause of DM-related systemic complications, including reproductive issues. Furthermore, the incidence of DM in males of reproductive ages is becoming an increasing concern, as the complexity of sperm capacitation (an essential process for fertilizing the egg) extends beyond conventional sperm parameters such as count, viability, and motility. Capacitation defects cause male infertility, and DM-related hyperglycemia may affect this process. We explore the effects of uncontrolled hyperglycemia on sperm using alloxan-induced hyperglycemic Wistar rats. In addition to assessing conventional sperm parameters, we also evaluated functional indicators, including hyperactivation (HA) with a pharmacological approach and assessed its effects with a computer-assisted sperm analysis (CASA); fluorescence indicators to monitor membrane potential (EmR, DiSC3(5)) and mitochondrial membrane potential (Ψ, JC-1); CatSper activity, using its ability to permeate Na+ ions, and ATP levels with the luciferin-luciferase reaction. We confirmed previous findings with our hyperglycemic model, which replicated the typical reduction on conventional sperm parameters. In sperm from hyperglycemic rats, we observed increased motility and HA levels after pharmacological treatment. Additionally, CatSper activity was unaffected by hyperglycemia, while EmR was hyperpolarized under non-capacitating condition. Finally, we noted a low percentage of hyperpolarized Ψ and reduced ATP content. This study highlights the significance of impact of hyperglycemia on sperm physiology and capacitation. We proposed that low ATP levels perturb energy state, signaling pathways, ion channels activity, motility, and HA. Our findings offer insight into DM-associated infertility and potential treatment strategies.

Components of dissolved organic carbon in relation to environmental factors in lakes along an altitudinal gradient in central China.

Dissolved organic carbon (DOC) is an important organic substance that affects biogeochemical processes and underwater light conditions in aquatic ecosystems. To explore altitudinal variations in DOC components and water quality, 66 surface water samples were collected along an altitudinal gradient (10-1857m) in three regions of central China, including a montane region (Enshi Prefecture) and two floodplain regions (Dongting Lake Basin and Wuhan City). DOC components were measured using a three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy, in conjunction with fluorescence regional integration (FRI) and fluorescence indices. Generally, lakes at high elevations (Enshi Prefecture) had higher DOC concentrations (a mean value of 6.43mgL-1) than floodplain lakes in Dongting Basin (4.51mgL-1) and Wuhan City (3.89mgL-1). Fluorescence index (FI, a range of 1.37-1.92) and biological index (BIX, a range of 0.47-0.74) indicated that DOC mainly originated from allochthonous sources in the three regions. Enshi Prefecture had relatively lower FI and BIX values, suggestive of a larger contribution of allochthonous carbon in this montane region in comparison with the floodplain regions. Humic-like substances were more abundant than other four fluorescent components, including tyrosine-like, tryptophan-like, fulvic acid-like, microbial-like substances. Spearman rank correlation analyses showed that tryptophane-like substances were positively correlated with K+, Mg2+, and Na+, while humic-like substances were negatively correlated with these cations. Taken together, the results can improve our understanding on altitudinal variations in DOC components and potential driving forces.

Response of alpine vegetation function to climate change in the Tibetan Plateau: A perspective from solar-induced chlorophyll fluorescence

Vegetation change in the Tibetan Plateau (TP) is a crucial indicator of climate change in alpine regions. Previous studies have reported an overall greening trend in the vegetation structure across the TP, especially in its northeastern part, in response to a warming climate. However, variations in the vegetation function and the possible drivers remain poorly understood. Considering the optimal temperature for plants in TP is usually higher than the current temperature, our hypothesis is the function and structure of alpine vegetation have changed synchronously over past few decades. To test this hypothesis, we analyzed satellite-observed solar-induced chlorophyll fluorescence (SIF) and leaf area index (LAI) in the Yellow River source (YRS) region in the northeastern TP to quantify the long-term trends in vegetation functional and structural states, respectively. The results suggest that from 1982 to 2018, SIF increased significantly in 77.71 % of the YRS area, resulting in a significant upward trend of 0.52 × 10−3 mW m−2 nm−1 sr−1 yr−1 (p < 0.001) for the regional-mean SIF. This represents a 16.1 % increase in SIF, which is close in magnitude to the increase in LAI over the same period. The synchronous changes between vegetation function and structure suggest that improved greenness corresponds to a similar level of change in carbon uptake across YRS. Additionally, we used a multiple regression approach to quantify the contribution of climatic factors to SIF changes in YRS. Our analyses show that the increases in SIF were primarily driven by rising temperatures. Spatially, temperature dominated SIF changes in most parts of YRS, except for certain dry parts in the northern and western YRS, where precipitation had a greater impact. Our results are crucial for a comprehensive understanding of climate regulations on vegetation structure and function in high-elevation regions.

Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level.

Intracellular temperature is a fundamental parameter in biochemical reactions. Genetically encoded fluorescent temperature indicators (GETIs) have been developed to visualize intracellular thermogenesis; however, the temperature sensitivity or localization capability in specific organelles should have been further improved to clearly capture when and where intracellular temperature changes at the subcellular level occur. Here, we developed a new GETI, gMELT, composed of donor and acceptor subunits, in which cyan and yellow fluorescent proteins, respectively, as a Förster resonance energy transfer (FRET) pair were fused with temperature-sensitive domains. The donor and acceptor subunits associated and dissociated in response to temperature changes, altering the FRET efficiency. Consequently, gMELT functioned as a fluorescence ratiometric indicator. Untagged gMELT was expressed in the cytoplasm, whereas versions fused with specific localization signals were targeted to the endoplasmic reticulum (ER) or mitochondria. All gMELT variations enabled more sensitive temperature measurements in cellular compartments than those in previous GETIs. The gMELTs, tagged with ER or mitochondrial targeting sequences, were used to detect thermogenesis in organelles stimulated chemically, a method previously known to induce thermogenesis. The observed temperature changes were comparable to previous reports, assuming that the fluorescence readout changes were exclusively due to temperature variations. Furthermore, we demonstrated how macromolecular crowding influences gMELT fluorescence given that this factor can subtly affect the fluorescence readout. Investigating thermogenesis with gMELT, accounting for factors such as macromolecular crowding, will enhance our understanding of intracellular thermogenesis phenomena.

Relief of pain in mice by an antibody with high affinity for cell adhesion molecule 1 on nerves

AimsCell adhesion molecule 1 (CADM1) is a member of the immunoglobulin superfamily and is abundantly expressed on nerve fibers. Recently, the anti-CADM1 ectodomain antibody 3E1 has proven useful as a drug delivery vector for CADM1-expressing cells in vitro. When injected subcutaneously into mice, whether 3E1 accumulates on nerve fibers and serves as an analgesic was examined. Main methodsInjected 3E1 was detected by immunohistochemistry and double immunofluorescence. Analgesic effects were verified by a formalin-induced chemical-inflammatory pain test and video-recorded behavior analysis that were performed 6, 12, and 24 h after antibody injection. Primary cultures of mouse dorsal root ganglion (DRG) cells were incubated with 3E1 and expressions of CADM1 and its key downstream molecules were examined by Western blot analyses and live cell imaging. DRG cells were loaded with a Ca2+ fluorescent indicator Fluo-8 and a femtosecond laser pulse was irradiated near the cell body to mechanically stimulate the nerves. Key findingsSubcutaneously injected 3E1 was widely localized almost exclusively on peripheral nerve fibers in the dermis. In formalin tests, 3E1-injected mice exhibited less pain-related behavior than control mice. When 3E1 was added to DRG cell cultures, it localized to neurites and resulted in decreased expression of CADM1, increased phosphorylation of Src and Akt, and CADM1-3E1 complex formation. Femtosecond laser-induced stimulation transmission along neurites was clearly visualized by Fluo-8 fluorescence in control cells, whereas it was markedly suppressed in 3E1-treated cells. Significance3E1 was suggested to be a potential long-acting analgesic based on its high affinity for CADM1.

Cell Cycle Modulation through Physical Confinement in Micrometer-Thick Hydrogel Sheaths.

Recently, surface engineering of the cell membrane with biomaterials has attracted great attention for various biomedical applications. In this study, we investigated the possibility of modulating cell cycle progression using alginate and gelatin-based hydrogel sheaths with a thickness of ∼1 μm. The hydrogel sheath was formed on cell surfaces through cross-linking catalyzed by horseradish peroxidase immobilized on the cell surface. The hydrogel sheath did not decrease the viability (>95% during 2 days of culture) of the human cervical carcinoma cell line (HeLa) expressing the fluorescent ubiquitination-based cell cycle indicator 2 (HeLa/Fucci2). Coating the HeLa/Fucci2 cells with the hydrogel sheath resulted in a cell cycle arrest in the G2/M phase, which can be caused by the reduced F-actin formation. As a result of this cell cycle arrest, an inhibition of cell growth was observed in the HeLa/Fucci2 cells. Taken together, our results demonstrate that the hydrogel sheath coating on the cell surface is a feasible approach to modulating cell cycle progression.

Machine learning and fluorosensing for estimation of maize nitrogen status at early growth-stages

Potential of mobile fluorescence sensor measurements have been in focus for quantifying plant nitrogen (N) variability early in the crop growing season. Real time estimation of such N status indicators at field scale would enable precision management of N fertilizers. In standard practice, linear regression analysis involves the use of several fluorescence channels and indices as predictive variables for estimating plant nitrogen content. Considering the multi-collinearity between these predictor variables, the conventional regression analysis (multiple linear regression) often fails to deliver a good range of prediction accuracies. Hence, machine learning regression techniques are utilized in this study to estimate N status indicators i.e., %N, above ground biomass, and N uptake at V6 and V9 growth stages of maize across three site-years in 2012 and 2013 crop growing seasons. The Multiplex®3 (FORCE-A) portable active fluorescence system was used to capture fluorescence information. Derived indices including four N balance indices (NBI_R, NBI_B, NBI_B, and NBI1), two chlorophyll indices (CHL and CHL1), and one flavonoid index (FLAV) were used as predictors. The independent site data were first utilized in a Support Vector Regression (SVR) model to assess the training and test accuracies in estimation of N status indicators considering a comparative analysis between V6 and V9 growth stages. The current research also involved assessing how well the machine learning-trained model could be applied to a different dataset and validated its performance in a cross-site experimental setting. Subsequently, cross-site comparisons of nitrogen status estimates were conducted to recommend the selection of machine learning strategies. These strategies include (1) Partial Least Square Regression, (2) Support Vector Regression, (3) Gaussian Process Regression, (4) Random Forest Regression, and (5) Artificial Neural Network. The comparative investigation demonstrated promising accuracy in estimating plant nitrogen content, above-ground biomass, and nitrogen uptake at the V6 stages of maize, with correlation coefficients in the moderate range (r = 0.72 ± 0.03) and Root Mean Square Error. Superior prediction accuracies were obtained at V9 growth stages than at V6. Among the various machine learning models assessed, Support Vector Regression consistently outperformed the others across the three site-year datasets, delivering error estimates well within acceptable ranges and demonstrating the lowest RMSE values for maize nitrogen indicators.

A GCaMP reporter mouse with chondrocyte specific expression of a green fluorescent calcium indicator

One of the major processes occurring during the healing of a fractured long bone is chondrogenesis, leading to the formation of the soft callus, which subsequently undergoes endochondral ossification and ultimately bridges the fracture site. Thus, understanding the molecular mechanisms of chondrogenesis can enhance our knowledge of the fracture repair process. One such molecular process is calciun (Ca++) signaling, which is known to play a critical role in the development and regeneration of multiple tissues, including bone, in response to external stimuli. Despite the existence of various mouse models for studying Ca++ signaling, none of them were designed to specifically examine the skeletal system or the various musculoskeletal cell types. As such, we generated a genetically engineered mouse model that is specific to cartilage (crossed with Col2a1 Cre mice) to study chondrocytes. Herein, we report on the characterization of this transgenic mouse line using conditional expression of GCaMP6f, a Ca++-indicator protein. Specifically, this mouse line exhibits increased GCaMP6f fluorescence following Ca++ binding in chondrocytes. Using this model, we show real-time Ca++ signaling in embryos, newborn and adult mice, as well as in fracture calluses. Further, robust expression of GCaMP6f in chondrocytes can be easily detected in embryos, neonates, adults, and fracture callus tissue sections. Finally, we also report on Ca++ signaling pathway gene expression, as well as real-time Ca++ transient measurements in fracture callus chondrocytes. Taken together, these mice provide a new experimental tool to study chondrocyte-specific Ca++ signaling during skeletal development and regeneration, as well as various in vitro perturbations.

Spontaneous and evoked angiotensin II sniffer cell activity in the lamina terminalis in vitro.

Angiotensin II (ANG II) has been shown to have central nervous system effects. Although tissue renin-angiotensin systems (RAS) have been demonstrated in multiple tissues, the existence of a brain RAS is still a matter of debate. These studies test for angiotensin release from brain slices prepared from adult male Sprague-Dawley rats and male and female renin knock-out rats using Chinese hamster ovary cells modified to express both the angiotensin II type 1 receptor and a fluorescent calcium indicator. Sniffer cells were placed on the slices and calcium transients were measured from those located on or adjacent to the median preoptic nucleus with and without stimulation of the subfornical organ. Bath application of tetrodotoxin (1 µM) significantly attenuated spontaneous events while abolishing evoked sniffer cell activity. Bath application of dl-AP4 (10 µM, glutamatergic antagonist) did not affect either spontaneous or evoked release. Incubating the slices with fluorocitrate to inactive astrocytes did not influence sniffer cell activity in the MnPO. Pharmacological experiments indicate that ANG II release is largely both renin (aliskiren 10 µM) and ACE-1 (captopril 100 µM) dependent. However, experiments with brain slices prepared from male and female Renin knock-out rats suggest that alternative synthetic pathways may exist. Finally, these studies demonstrate that increases in ANG II release are observed following 7 days of chronic intermittent hypoxia. These studies suggest the existence of a tissue-specific RAS in the brain that involves canonical and alternative ANG II synthetic pathways and is upregulated in an animal model of sleep apnea.NEW & NOTEWORTHY These studies used Chinese hamster ovary cells that were cloned to express an angiotensin receptor (At1ra) and a calcium indicator (R-GECO) to detect the release of angiotensin from brain slices containing the lamina terminalis of rats. Some of the experiments use tissue from renin knockout rats. The results support the existence of an angiotensin system in the brain that may involve alternative synthetic pathways and is upregulated by intermittent hypoxia.

Design of deep-red emissive forced intercalation-induced light-up peptide as an indicator for the HIV-1 TAR RNA-ligand assay: integration of benzo[c,d]indole-quinoline (BIQ) cyanine dye into Tat peptide.

We report on a deep-red emissive fluorogenic peptide probe for human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA as an indicator for fluorescence indicator displacement (FID) assay. The probe design is based on the concept of the forced intercalation of thiazole orange (TO) dyes (FIT) on the peptide backbone, as recently proposed by our group, where the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. Here, instead of green emissive TO, we utilized a deep-red emissive benzo[c,d]indole-quinoline (BIQ) cyanine dye developed previously by our group for imaging of nucleolar RNA in living cells. The developed 9-mer FIT peptide (RKKRR-BIQ-RRR; named BIQ-FiLuP) exhibits a significant off-on signaling ability for TAR RNA (λem = 660nm, I/I0 = 130-fold, Φfree = 0.0009, Φbound = 0.052), and the dissociation constant Kd reaches ca. 1nM. When used in FID assay, BIQ-FiLuP, like TO-based FiLuP, is able to distinguish between competitive and noncompetitive inhibitors, which has never been demonstrated with all previous indicators for TAR RNA. Deep-red emissive BIQ-FiLuP facilitates the evaluation of green to yellow emissive ligands without suffering from optical interference. The combination use with green emissive TO-based FiLuP (λem = 541nm) would cover the examination of a wide range of fluorescent test compounds.

Abstract Mo097: Inhibiting the Sarcomere Improves Diastolic Dysfunction in Patient-Derived hiPSC-CM Models of Pediatric Restrictive Cardiomyopathy

Introduction: Pediatric patients with Restrictive Cardiomyopathy (RCM) develop heart failure due to stiffening of the wall of the cardiac ventricle leading to diastolic dysfunction but preserved cardiac contractility. Treatment options are extremely limited, and the evaluation of new therapeutic strategies is hindered due to a lack of models that recapitulate this ventricular stiffening. Here, we evaluate two novel patient-derived human induced pluripotent stem cell (hiPSC) models of restrictive cardiomyopathy due to mutations in the thin filament, and evaluate separate sarcomere-targeted therapeutic strategies to improve diastolic function. Hypothesis: Directly decreasing the thin filament’s response to calcium will improve diastolic function in RCM models with less effect on systolic function than myosin inhibition. Methods: hiPSC-cardiomyocytes (hiPSC-CMs) were differentiated from hiPSC lines generated from two patients with pediatric RCM, as well as their isogenic controls. After replating on polyacrylamide gels and staining with fluorescent calcium indicators, diastolic tension, generated tension, and calcium sensitivity were measured at baseline and upon treatment with either a troponin calcium desensitizer (W7) or a myosin inhibitor (mavacamten). Results: hiPSC-CMs from both patients showed increased diastolic tension, generated tension, and calcium sensitivity compared to their isogenic controls. Treatment with Mavacamten improved diastolic function but severely compromised systolic function, while treatment with W7 improved diastolic function with less effect on systolic function. Conclusions: Directly inhibiting the sarcomere in models of RCM improved measures of diastolic dysfunction, but directly targeting the thin filament in these lines showed less effect on systolic function than myosin inhibition, suggesting cardiac troponin calcium desensitizers may be more beneficial in this severe diastolic disease.