Nanomolar Sensitivity Research Articles

Multispectral open-air intraoperative fluorescence imaging.

Intraoperative fluorescence imaging informs decisions regarding surgical margins by detecting and localizing signals from fluorescent reporters, labeling targets such as malignant tissues. This guidance reduces the likelihood of undetected malignant tissue remaining after resection, eliminating the need for additional treatment or surgery. The primary challenges in performing open-air intraoperative fluorescence imaging come from the weak intensity of the fluorescence signal in the presence of strong surgical and ambient illumination, and the auto-fluorescence of non-target components, such as tissue, especially in the visible spectral window (400-650 nm). In this work, a multispectral open-air fluorescence imaging system is presented for translational image-guided intraoperative applications, which overcomes these challenges. The system is capable of imaging weak fluorescence signals with nanomolar sensitivity in the presence of surgical illumination. This is done using synchronized fluorescence excitation and image acquisition with real-time background subtraction. Additionally, the system uses a liquid crystal tunable filter for acquisition of multispectral images that are used to spectrally unmix target fluorescence from non-target auto-fluorescence. Results are validated by preclinical studies on murine models and translational canine oncology models.

Investigation of Drug-Induced Hepatotoxicity and Its Remediation Pathway with Reaction-Based Fluorescent Probes.

Drug-induced liver injury (DILI) is considered a serious problem related to public health, due to its unpredictability and acute response. The level of peroxynitrite (ONOO-) generated in liver has long been regarded as a biomarker for the prediction and measurement of DILI. Herein we present two reaction-based fluorescent probes (Naph-ONOO- and Rhod-ONOO-) for ONOO- through a novel and universally applicable mechanism: ONOO--mediated deprotection of α-keto caged fluorophores. Among them, Rhod-ONOO- can selectively accumulate and react in mitochondria, one of the main sources of ONOO-, with a substantial lower nanomolar sensitivity of 43 nM. The superior selectivity and sensitivity of two probes enable real-time imaging of peroxynitrite generation in lipopolysaccharide-stimulated live cells, with a remarkable difference from cells doped with other interfering reactive oxygen species, in either one- or two-photon imaging modes. More importantly, we elucidated the drug-induced hepatotoxicity pathway with Rhod-ONOO- and revealed that CYP450/CYP2E1-mediated enzymatic metabolism of acetaminophen leads to ONOO- generation in liver cells. This is the first time to showcase the drug-induced hepatotoxicity pathways by use of a small-molecule fluorescent probe. We hence conclude that fluorescent probes can engender a deeper understanding of reactive species and their pathological revelations. The reaction-based fluorescent probes will be a potentially useful chemical tool to assay drug-induced hepatotoxicity.

Abstract 4147A: Potent anti-cancer activity of the imipridone ONC206: A selective dopamine D2-like receptor antagonist

Abstract DRD2 is a G protein-coupled receptor (GPCR) that is overexpressed in many cancers, controls an array of pro-survival signaling pathways, and its antagonism causes anti-cancer effects. ONC201, the founding member of the imipridone class of anti-cancer compounds, is a small molecule DRD2 antagonist that is in Phase I/II advanced cancer clinical trials. In this study, we evaluated the binding target and antitumor activity of ONC206, a chemical analogue of ONC201. An orphan small molecule target prediction algorithm revealed that ONC206, like ONC201, antagonizes DRD2. Experimental GPCR profiling using the PathHunter® β-Arrestin assay, determined that ONC206 selectively antagonizes the D2-like (DRD2/3/4), but not the D1-like (DRD 1/5), subfamily of dopamine receptors. ONC206 possesses a ~10-fold increased affinity for DRD2 compared to ONC201 with a Ki of ~320nM with selectivity that was superior to approved antipsychotics. The increased association rate for the ONC206-DRD2 interaction was responsible for the increased affinity, whereas the dissociation rate was similar to ONC201 and atypical antipsychotics that are well tolerated. TCGA analysis and immunohistochemistry of patient-derived tissue microarrays revealed DRD2 was overexpressed in neuroblastoma, sarcoma and pheochromocytoma specimens relative to normal tissues. In vitro efficacy profiling of ONC206 in the Genomic of Drug Sensitivity in Cancer collection of cell lines revealed broad efficacy across most tumor types (GI50 <78-889nM). Bone cancer and neuroblastoma were identified as the most ONC206-responsive solid tumor types that were comparatively less responsive to ONC201. Within bone cancer cell lines, Ewing’s sarcoma (n=16) was the most sensitive to ONC206 with nanomolar sensitivity (GI50 168-303nM) that was superior to ONC201. ONC206 was highly efficacious in neuroblastoma (n=35, GI50 87-589nM) including cell lines derived from metastatic sites and with MYCN amplification associated with poor prognosis. In the PC12 rat pheochromocytoma cell line ONC206 (GI50 200nM) was superior to ONC201. ONC206 time-course experiments revealed anti-cancer effects occurring at 48-72 post-treatment, similar to ONC201. In support of a wide therapeutic window, ONC206 reduced the viability of normal fibroblasts (HFF-1) at relatively high doses (GI50 > 5µM). Efficacy evaluations in MHH-ES-1 athymic nude mice xenografts demonstrated that ONC206 (100 mg/kg PO every 10 days) causes significant tumor growth inhibition that was comparable to methotrexate (400 mg/kg, IP) while being better tolerated. In summary, ONC206 is an imipridone that acts as a selective antagonist of DRD2 at nanomolar concentrations and has broad-spectrum anti-tumor activity. ONC206 may address tumor types where the properties of ONC201 do not permit for complete therapeutic engagement in vivo. Citation Format: Varun Vijay Prabhu, Neel Madhukar, Jessica Wagner, Rohinton Tarapore, Mathew Garnett, Ultan McDermott, Cyril Benes, Neil Charter, Sean Deacon, Alexander VanEngelenburg, Olivier Elemento, Wafik El-Deiry, Martin Stogniew, Wolfgang Oster, Joshua Allen. Potent anti-cancer activity of the imipridone ONC206: A selective dopamine D2-like receptor antagonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4147A. doi:10.1158/1538-7445.AM2017-4147A

Monitoring single-synapse glutamate release and presynaptic calcium concentration in organised brain tissue

Brain function relies in large part on Ca2+-dependent release of the excitatory neurotransmitter glutamate from neuronal axons. Establishing the causal relationship between presynaptic Ca2+ dynamics and probabilistic glutamate release is therefore a fundamental quest across neurosciences. Its progress, however, has hitherto depended primarily on the exploration of either cultured nerve cells or giant central synapses accessible to direct experimental probing in situ. Here we show that combining patch-clamp with time-resolved imaging of Ca2+ −sensitive fluorescence lifetime of Oregon Green BAPTA-1 (Tornado-FLIM) enables readout of single spike-evoked presynaptic Ca2+ concentration dynamics, with nanomolar sensitivity, in individual neuronal axons in acute brain slices. In parallel, intensity Tornado imaging of a locally expressed extracellular optical glutamate sensor iGluSnFr provides direct monitoring of single-quantum, single-synapse glutamate releases in situ. These two methods pave the way for simultaneous registration of presynaptic Ca2+ dynamics and transmitter release in an intact brain at the level of individual synapses.

Transition-Metal-Free Oxidative C(sp2 )-H Hydroxylation of Terpyridines: A HOMO-Raising Strategy for the Construction of a New Super-Stable Terpyridine Chromophores.

Direct functionalization of terpyridines is an increasingly important topic in the field of dyes and catalysis as well as supramolecular chemistry, but its synthesis and transformation is usually challenging. Herein, a HOMO-raising strategy is reported for the construction of a super-stable novel terpyridine chromophores, in which the selective oxidation of terpyridines at its 3-position was determined successfully to the synthesis of phenol-functionalization of terpyridines (TPyOHs) bearing a hydrogen bonding group. The corresponding TPyOHs displayed strong aggregation-induced emission and exhibited highly selective and visual detection of ZnII cation with a record green terpyridine-based luminophore with nanomolar sensitivity (125 nm).

Intracavity folding of a perylene dye affords a high-affinity complex with cucurbit[8]uril.

A fluorescent perylene dye with two aromatic units was designed for binding to cucurbit[8]uril. The binding affinity of the complexes increased about 3 orders of magnitude compared to the dye without a secondary aromatic unit. The high affinity allows the complexes to act as fluorescent probes for detection of strong binding guests with nanomolar sensitivity.

DNA aptamer probes for detection of estrogen receptor α positive carcinomas

Estrogen receptor alpha (ERα) also known as NR3A1 (nuclear receptor subfamily 3, group A, member 1) is a ligand-activated transcription factor. It is an important biomarker for breast cancer metastasis. In the present study, we report a novel DNA aptamer candidate against estrogen receptor (ER) alpha structure. Theenriched aptamercandidate was obtained after 14 iterative cycles of invitroprotein-SELEX process. Isothermal calorimetry study suggests the nanomolar sensitivity of the candidate ER_Apt1 to its target protein. Fluorescence- and chemiluminescence-binding assays confirm the specificity of the candidate aptamer to ER alpha positive breast cancer cell line. Comparative analysis of ER_Apt1 to ER alpha monoclonal antibody wasalso performed to analyze the expression of ER alpha in various malignant cancer cell line. Cytochemical and immunohistochemistry assay indicates its potential use as a diagnostic agent against ERα positive carcinomas. The nucleotide aptamer sequences described in the present study can be used for the detection, treatment, prophylaxis and diagnosis of ERα-related disorder.

Abstract 2458: Targeting checkpoint kinase 1 (CHK1) with the small molecule inhibitor LY2606368 mesylate monohydrate in models of high-risk pediatric cancer yields significant antitumor effects

Abstract CHK1 is a serine/threonine protein kinase essential for S-phase and G2/M cell cycle checkpoint regulation following DNA damage. Targeted inhibition of CHK1 in several tumor types increases DNA damage and replication stress, culminating in cell death through mitotic catastrophe. Recent studies have identified CHK1 as a therapeutic target in several pediatric tumor types. We evaluated the antitumor efficacy of LY2606368 mesylate monohydrate (“LY”), a checkpoint kinase 1 (CHK1)/CHK2 inhibitor currently in early phase clinical trials for adult solid cancers, in a panel of pediatric tumor cell lines and mouse models of embryonal tumors and pediatric sarcoma. In vitro effects of LY were assessed via Cell Titer Glo, immunoblotting, and cell cycle analysis by flow cytometry. For in vivo studies, mice bearing cell-derived (CDX) or patient-derived xenografts (PDX) of several pediatric tumor types were treated with four weekly cycles of 10 mg/kg LY BID for 3 consecutive days, followed by a 4 day dosing holiday. Tumor volume and body weight were measured 2x weekly. Xenograft tumor health following LY, chemotherapy, or combination treatment was evaluated by fluorescent immunohistochemistry (IHC) for a panel of markers for cell proliferation (Ki67), apoptosis (TUNEL), and angiogenesis (CD31, smooth muscle actin [SMA], MECA32). Single digit nanomolar sensitivity to LY was observed in the majority of pediatric cancer cell lines evaluated in vitro. A more detailed analysis of LY-treated neuroblastoma and pediatric sarcoma cell lines showed increased DNA damage, CHK1 phosphorylation, and MAPK pathway activation. Significant single agent LY activity was observed in mouse models of neuroblastoma and pediatric sarcoma, but not in models of hepatoblastoma or retinoblastoma. Acquired resistance to LY was observed in the ST162 and SJCRH30 models of alveolar rhabdomyosarcoma. Interestingly, more stroma was observed following LY single agent treatment as measured by CD31, SMA, and MECA32 IHC staining; co-treatment with chemotherapy reduced the amount of SMA expressing-cells. Overall, our data demonstrate that LY is highly effective as a single agent in murine in vivo models of human neuroblastoma and several pediatric sarcoma subtypes. Current studies include further evaluation of the LY mechanism of action; investigation into the mechanism of intrinsic and acquired resistance; and identification of possible biomarkers for LY sensitivity. Citation Format: Caitlin D. May, Richard Beckmann, Wayne Blosser, Michele Dowless, Alle VanWye, Teresa Burke, Gerard Oakley, Jennifer Stephens, Julie Stewart, Beverly Falcon, Louis Stancato. Targeting checkpoint kinase 1 (CHK1) with the small molecule inhibitor LY2606368 mesylate monohydrate in models of high-risk pediatric cancer yields significant antitumor effects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2458.

Comparison of Cherenkov excited fluorescence and phosphorescence molecular sensing from tissue with external beam irradiation

Ionizing radiation delivered by a medical linear accelerator (LINAC) generates Cherenkov emission within the treated tissue. A fraction of this light, in the 600–900 nm wavelength region, propagates through centimeters of tissue and can be used to excite optical probes in vivo, enabling molecular sensing of tissue analytes. The success of isolating the emission signal from this Cherenkov excitation background is dependent on key factors such as: (i) the Stokes shift of the probe spectra; (ii) the excited state lifetime; (iii) the probe concentration; (iv) the depth below the tissue surface; and (v) the radiation dose used. Previous studies have exclusively focused on imaging phosphorescent dyes, rather than fluorescent dyes. However there are only a few biologically important phosphorescent dyes and yet in comparison there are thousands of biologically relevant fluorescent dyes. So in this study the focus was a study of efficacy of Cherenkov-excited luminescence using fluorescent commercial near-infrared probes, IRDye 680RD, IRDye 700DX, and IRDye 800CW, and comparing them to the well characterized phosphorescent probe Oxyphor PtG4, an oxygen sensitive dye. Each probe was excited by Cherenkov light from a 6 MV external radiation beam, and measured in continuous wave or time-gated modes. The detection was performed by spectrally resolving the luminescence signals, and measuring them with spectrometer-based separation on an ICCD detector. The results demonstrate that IRDye 700DX and PtG4 allowed for the maximal signal to noise ratio. In the case of the phosphorescent probe, PtG4, with emission decays on the microsecond (μs) time scale, time-gated acquisition was possible, and it allowed for higher efficacy in terms of the probe concentration and detection depth. Phantoms containing the probe at 5 mm depth could be detected at concentrations down to the nanoMolar range, and at depths into the tissue simulating phantom near 3 cm. In vivo studies showed that 5 nmol of dye was readily detected with radiation doses less than 5 cGy. Since concentration, radiation dose and depth each contribute to the level of the detected signal, it may be possible to improve any of these parameters at expense of the others. This paradigm of nanoMolar sensitivity for optical reporters in vivo introduces the concept of molecular sensing of tumors during therapy or diagnostically with biologically relevant concentrations of fluorescent reporters.

A novel biosensor to study cAMP dynamics in cilia and flagella.

The cellular messenger cAMP regulates multiple cellular functions, including signaling in cilia and flagella. The cAMP dynamics in these subcellular compartments are ill-defined. We introduce a novel FRET-based cAMP biosensor with nanomolar sensitivity that is out of reach for other sensors. To measure cAMP dynamics in the sperm flagellum, we generated transgenic mice and reveal that the hitherto methods determining total cAMP levels do not reflect changes in free cAMP levels. Moreover, cAMP dynamics in the midpiece and principal piece of the flagellum are distinctively different. The sole cAMP source in the flagellum is the soluble adenylate cyclase (SACY). Although bicarbonate-dependent SACY activity requires Ca(2+), basal SACY activity is suppressed by Ca(2+). Finally, we also applied the sensor to primary cilia. Our new cAMP biosensor features unique characteristics that allow gaining new insights into cAMP signaling and unravel the molecular mechanisms underlying ciliary function in vitro and in vivo.

Sustained-release synthetic biomarkers for monitoring thrombosis and inflammation using point-of-care compatible readouts.

Postoperative infection and thromboembolism represent significant sources of morbidity and mortality but cannot be easily tracked after hospital discharge. Therefore, a molecular test that could be performed at home would significantly impact disease management. Our lab has previously developed intravenously delivered 'synthetic biomarkers' that respond to dysregulated proteases to produce a urinary signal. These assays, however, have been limited to chronic diseases or acute diseases initiated at the time of diagnostic administration. Here, we formulate a subcutaneously administered sustained release system by using small PEG scaffolds (<10 nm) to promote diffusion into the bloodstream over a day. We demonstrate the utility of a thrombin sensor to identify thrombosis and an MMP sensor to measure inflammation. Finally, we developed a companion paper ELISA using printed wax barriers with nanomolar sensitivity for urinary reporters for point-of-care detection. Our approach for subcutaneous delivery of nanosensors combined with urinary paper analysis may enable facile monitoring of at-risk patients.

EPR studies on the kinetics of the α-hydroxyethyl radical generated by Fenton-like chemistry

Flow systems, either stopped or continuous, have long been at the core of kinetic studies of chemical reactions. Such flow systems need to be coupled with appropriate spectroscopic or otherwise techniques for the detection of the chemical species studied. If paramagnetic species are formed or consumed during the investigated reaction, electron paramagnetic resonance (EPR) with its nanomolar sensitivity can be the spectroscopic method of choice. However, not much literature is available on the application of EPR to quantitatively study kinetics of chemical reactions in the liquid state. Herein, we report the characterisation of the commercially available mixing resonator ER 4117 MX from Bruker using the TEMPO-dithionite reaction as a standard. Furthermore, this setup was used to study the kinetics of the Fenton-like system of TiCl3/H2O2 and ethanol forming theα-hydroxyethyl radical. Potential contributions of reactions with O2, H2O2, Ti3+/4+, and self-recombination in the decay of theα-hydroxyethyl radical were investigated and the bimolecular decay was shown to be the dominant decay pathway, with a decay rate constant of 6.6×108M−1s−1. This study shows the effectiveness and capabilities of EPR as a direct, sensitive and in-situ method in kinetic studies.

A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters

DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag(+) ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection.

A ratiometric fluorescent probe for detection of biogenic primary amines with nanomolar sensitivity.

An ultrasensitive ratiometric fluorescent sensor made of an N,N-dimethylaminonaphthalene anhydride moiety for detection of aliphatic primary amines is reported. Biogenic amines at nanomolar concentration is detected with the additional ability to discriminate between primary, secondary and tertiary amines by using both UV-Visible and fluorescence spectroscopy.

Monitoring interfacial lectin binding with nanomolar sensitivity using a plasmon field effect transistor

By immobilizing glycopolymers onto the surface of the recently developed plasmonic field effect transistor (FET), the recognition between lectins and surface-immobilized glycopolymers can be detected over a wide dynamic range (10-10 to 10-4 M) in an environment that resembles the glycocalyx. The binding to the sensor surface by various lectins was tested, and the selectivities and relative binding affinity trends observed in solution were maintained on the sensor surface, and the significantly higher avidities are attributed to cluster-glycoside effects that occur on the surface. The combination of polymer surface chemistry and optoelectronic output in this device architecture produces amongst the highest reported detection sensitivity for ConA. This work demonstrates the benefits that arise from combining emerging device architectures and soft-matter systems to create cutting edge nanotechnologies that lend themselves to fundamental biological studies and integration into point-of-use diagnostics and sensors.

Purcell Factor: A Tunable Metric for Plasmon-Coupled Fluorescence Emission Enhancements in Cermet Nanocavities

We demonstrate an important approach to correlate Purcell factor (PF) and surface plasmon-coupled emission (SPCE) enhancements with the use of finite-difference time-domain (FDTD) simulations and time-correlated single-photon counting (TCSPC) studies of a radiating dipole in cermet nanocavities. We observed >50-fold fluorescence enhancement with high directionality and polarization of Rhodamine 6G (Rh6G) emission trapped in the nanocavity created between the titanium-based ceramic nanoparticle and metallic silver thin film. Compositional variation with hybrid nanoparticles, TiC0N1 (TiN), TiC0.5N0.5 (TiCN), and TiC1N0 (TiC), brought about enhanced PFs and tunable fluorescence enhancements that were used for mobile-phone-based detection of tryptophan with nanomolar sensitivity. We hope that this study opens the door to next-gen plasmonics with the ability to tune and enhance the hot-spot electromagnetic field intensity of alternative plasmonic materials, as hybrid synergy spacers in the SPCE platform.

Lithographically Patterned PEDOT Nanowires for the Detection of Iron(III) with Nanomolar Sensitivity.

Arrays of nanowires of an electronically conductive polymeric affinity medium tailored to the detection of Fe(III) are prepared, and their properties for detecting Fe(III) are evaluated. This polymeric affinity medium consists of poly(3,4-ethylenedioxythiophene) (PEDOT) into which an iron chelator, deferoxamine (DFA), has been doped during the polymerization process. PEDOT-DFA nanowires are potentiostatically deposited from a solution containing both EDOT and DFA using lithographically patterned nanowire electrodeposition (LPNE). The through-nanowire electrical resistance of PEDOT-DFA nanowires is measured as a function of the Fe(III) concentration. In parallel with measurements on PEDOT-DFA nanowire arrays, the electrochemical impedance of PEDOT-DFA films is characterized as a function of the Fe(III) concentration and the frequency of the impedance measurement in order to better understand the mechanism of transduction. PEDOT-DFA nanowires detect Fe(III) from 10(-4) to 10(-8) M with a limit of detection of 300 pM (calculated) and 10 nM (measured).

Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction.

BackgroundLow molecular weight haptens (<1000 Da) cannot be recognized by the immune system unless conjugated to larger carrier molecules. Antibodies to these exceptionally small antigens can still be generated with exquisite sensitivity. A detailed understanding at the molecular level of this incredible ability of antibodies to recognize haptens, is still limited compared to other antigen classes.MethodsDifferent hapten targets with a broad range of structural flexibility and polarity were conjugated to carrier proteins, and utilized in sheep immunization. Three antibody libraries were constructed and used as potential pools to isolate specific antibodies to each target. The isolated antibodies were analysed in term of CDR length, canonical structure, and binding site shape and electrostatic potential.ResultsThe simple, chemically naïve structure of squalane (SQA) was recognized with micromolar sensitivity. An increase in structural rigidity of the hydrophobic and cyclic coprostane (COP) did not improve this binding sensitivity beyond the micromolar range, whilst the polar etioporphyrin (POR) was detected with nanomolar sensitivity. Homoserine lactone (HSL) molecules, which combine molecular flexibility and polarity, generated super-sensitive (picomolar) interactions. To better understand this range of antibody-hapten interactions, analyses were extended to examine the binding loop canonical structures and CDR lengths of a series of anti-hapten clones. Analyses of the pre and post- selection (panning of the phage displayed libraries) sequences revealed more conserved sites (123) within the post-selection sequences, when compared to their pre-selection counterparts (28). The strong selection pressure, generated by panning against these haptens resulted in the isolation of antibodies with significant sequence conservation in the FW regions, and suitable binding site cavities, representing only a relatively small subset of the available full repertoire sequence and structural diversity. As part of this process, the important influence of CDR H2 on antigen binding was observed through its direct interaction with individual antigens and indirect impact on the orientation and the pocket shape, when combined with CDRs H3 and L3. The binding pockets also displayed electrostatic surfaces that were complementary to the hydrophobic nature of COP, SQA, and POR, and the negatively charged HSL.ConclusionsThe best binding antibodies have shown improved capacity to recognize these haptens by establishing complementary binding pockets in terms of size, shape, and electrostatic potential.

Profiling of Free Fatty Acids Using Stable Isotope Tagging Uncovers a Role for Saturated Fatty Acids in Neuroexocytosis

The phospholipase-catalyzed release of free fatty acids (FFAs) from phospholipids is implicated in many critical biological processes such as neurotransmission, inflammation, and cancer. However, determining the individual change in FFAs generated during these processes has remained challenging due to the limitations of current methods, and has hampered our understanding of these key mediators. Here, we developed an "iTRAQ"-like method for profiling FFAs by stable isotope tagging (FFAST), based on the differential labeling of the carboxyl group and designed to resolve analytical variance, through a multiplexed assay in cells and subcellular fractions. With nanomolar sensitivity, this method revealed a spectrum of saturated FFAs elicited during stimulation of exocytosis that was identical in neurons and neurosecretory cells. Purified secretory vesicles also generated these FFAs when challenged with cytosol. Our multiplex method will be invaluable to assess the range of FFAs generated in other physiological and pathological settings.

Agrochemical control of plant water use using engineered abscisic acid receptors.

Rising temperatures and lessening fresh water supplies are threatening agricultural productivity and have motivated efforts to improve plant water use and drought tolerance. During water deficit, plants produce elevated levels of abscisic acid (ABA), which improves water consumption and stress tolerance by controlling guard cell aperture and other protective responses. One attractive strategy for controlling water use is to develop compounds that activate ABA receptors, but agonists approved for use have yet to be developed. In principle, an engineered ABA receptor that can be activated by an existing agrochemical could achieve this goal. Here we describe a variant of the ABA receptor PYRABACTIN RESISTANCE 1 (PYR1) that possesses nanomolar sensitivity to the agrochemical mandipropamid and demonstrate its efficacy for controlling ABA responses and drought tolerance in transgenic plants. Furthermore, crystallographic studies provide a mechanistic basis for its activity and demonstrate the relative ease with which the PYR1 ligand-binding pocket can be altered to accommodate new ligands. Thus, we have successfully repurposed an agrochemical for a new application using receptor engineering. We anticipate that this strategy will be applied to other plant receptors and represents a new avenue for crop improvement.