Expanded Coverage of Phytocompounds by Mass Spectrometry Imaging Using On-Tissue Chemical Derivatization by 4-APEBA.

Probing the entirety of any species metabolome is an analytical grand challenge, especially on a cellular scale. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a common spatial metabolomics assay, but this technique has limited molecular coverage for several reasons. To expand the application space of spatial metabolomics, we developed an on-tissue chemical derivatization (OTCD) workflow using 4-APEBA for the confident identification of several dozen elusive phytocompounds. Overall, this new OTCD method enabled the annotation of roughly 280 metabolites, with only a 10% overlap in metabolic coverage when compared to analog negative ion mode MALDI-MSI on serial sections. We demonstrate that 4-APEBA outperforms other derivatization agents by providing: (1) broad specificity toward carbonyls, (2) low background, and (3) introduction of bromine isotopes. Notably, the latter two attributes also facilitate more confidence in our bioinformatics for data processing. The workflow detailed here trailblazes a path toward spatial hormonomics within plant samples, enhancing the detection of carboxylates, aldehydes, and plausibly other carbonyls. As such, several phytohormones, which have various roles within stress responses and cellular communication, can now be spatially profiled, as demonstrated in poplar root and soybean root nodule.

Ligand- and Structure-Based Analysis of Deep Learning-Generated Potential α2a Adrenoceptor Agonists.

The α2a adrenoceptor is a medically relevant subtype of the G protein-coupled receptor family. Unfortunately, high-throughput techniques aimed at producing novel drug leads for this receptor have been largely unsuccessful because of the complex pharmacology of adrenergic receptors. As such, cutting-edge in silico ligand- and structure-based assessment and de novo deep learning methods are well positioned to provide new insights into protein-ligand interactions and potential active compounds. In this work, we (i) collect a dataset of α2a adrenoceptor agonists and provide it as a resource for the drug design community; (ii) use the dataset as a basis to generate candidate-active structures via deep learning; and (iii) apply computational ligand- and structure-based analysis techniques to gain new insights into α2a adrenoceptor agonists and assess the quality of the computer-generated compounds. We further describe how such assessment techniques can be applied to putative chemical probes with a case study involving proposed medetomidine-based probes.

Interrogating Plant-Microbe Interactions with Chemical Tools: Click Chemistry Reagents for Metabolic Labeling and Activity-Based Probes.

Continued expansion of the chemical biology toolbox presents many new and diverse opportunities to interrogate the fundamental molecular mechanisms driving complex plant-microbe interactions. This review will examine metabolic labeling with click chemistry reagents and activity-based probes for investigating the impacts of plant-associated microbes on plant growth, metabolism, and immune responses. While the majority of the studies reviewed here used chemical biology approaches to examine the effects of pathogens on plants, chemical biology will also be invaluable in future efforts to investigate mutualistic associations between beneficial microbes and their plant hosts.

Structure Dependent Determination of Organophosphate Targets in Mammalian Tissues Using Activity-Based Protein Profiling.

Acute and chronic exposures to organophosphates (OPs), including agricultural pesticides, industrial chemicals, and chemical warfare agents, remain a significant worldwide health risk. The mechanisms by which OPs alter development and cognition in exposed individuals remain poorly understood, in part due to the large number of structurally diverse OPs and the wide range of affected proteins and signaling pathways. To investigate the influence of structure on OP targets in mammalian systems, we have developed a series of probes for activity-based protein profiling (ABPP) featuring two distinct reactive groups that mimic OP chemical reactivity. FOP features a fluorophosphonate moiety, and PODA and CODA utilize a dialkynyl phosphate ester; both reactive group types target serine hydrolase activity. As the oxon represents the highly reactive and toxic functional group of many OPs, the new probes described herein enhance our understanding of tissue-specific reactivity of OPs. Chemoproteomic analysis of mouse tissues treated with the probes revealed divergent protein profiles, demonstrating the influence of probe structure on protein targeting. These targets also vary in sensitivity toward different OPs. The simultaneous use of multiple probes in ABPP experiments may therefore offer more comprehensive coverage of OP targets; FOP consistently labeled more targets in both brain and liver than PODA or CODA, suggesting the dialkyne warhead is more selective for enzymes in major signaling pathways than the more reactive fluorophosphonate warhead. Additionally, the probes can be used to assess reactivation of OP-inhibited enzymes by N-oximes and may serve as diagnostic tools for screening of therapeutic candidates in a panel of protein targets. These applications will help clarify the short- and long-term effects of OP toxicity beyond acetylcholinesterase inhibition, investigate potential points of convergence for broad spectrum therapeutic development, and support future efforts to screen candidate molecules for efficacy in various model systems.

Fluorescent Molecular Probes for Detection of One-Electron Oxidants Photochemically Generated by Dissolved Organic Matter.

We report a dual probe system based on 4'-substituted biphenyl-2-carboxylic acids (BPAs) for analysis of photooxidants generated by dissolved organic matter. The BPA probes are converted to the corresponding benzocoumarins (BZCs) at different rates depending on the mechanism of oxidation; thus, two probes used simultaneously can differentiate strong triplet excited state sensitizers from hydroxylating species such as hydroxyl radical (•OH) present in dissolved organic matter (DOM). Comparison of the ratios of BZC-CH3 and BZC-CF3 product formation using model photooxidants such as NaNO2, a •OH precursor, and model triplet sensitizer lumichrome gave a range of 2 to 250. Application of these probes to DOM isolates and whole natural waters afforded intermediate ratios. Although the oxidation potential of BPAs (>ca. 1.80 V SHE) is significantly higher than the estimated average reduction potential of typical 3CDOM* samples, these results have demonstrated the presence of a small pool of oxidants in the selected DOM isolates and whole water samples that is capable of oxidizing aromatic carboxylates. As an analytical tool, this probe pair can be used between pH 4-6 without affecting the product formation ratio and may find applications in various systems involving complex mixtures of photochemically produced oxidants of differing natures.

Cell-trappable fluorescent probes for endogenous hydrogen sulfide signaling and imaging H2O2-dependent H2S production.

Hydrogen sulfide (H2S) is a reactive small molecule generated in the body that can be beneficial or toxic owing to its potent redox activity. In living systems, disentangling the pathways responsible for H2S production and their physiological and pathological consequences remains a challenge in part due to a lack of methods for monitoring changes in endogenous H2S fluxes. The development of fluorescent probes with appropriate selectivity and sensitivity for monitoring production of H2S at biologically relevant signaling levels offers opportunities to explore its roles in a variety of systems. Here we report the design, synthesis, and application of a family of azide-based fluorescent H2S indicators, Sulfidefluor-4, Sulfidefluor-5 acetoxymethyl ester, and Sulfidefluor-7 acetoxymethyl ester, which offer the unique capability to image H2S generated at physiological signaling levels. These probes are optimized for cellular imaging and feature enhanced sensitivity and cellular retention compared with our previously reported molecules. In particular, Sulfidefluor-7 acetoxymethyl ester allows for direct, real-time visualization of endogenous H2S produced in live human umbilical vein endothelial cells upon stimulation with vascular endothelial growth factor (VEGF). Moreover, we show that H2S production is dependent on NADPH oxidase-derived hydrogen peroxide (H2O2), which attenuates VEGF receptor 2 phosphorylation and establishes a link for H2S/H2O2 crosstalk.

Chemical probes for molecular imaging and detection of hydrogen sulfide and reactive sulfur species in biological systems.

Hydrogen sulfide (H2S), a gaseous species produced by both bacteria and higher eukaryotic organisms, including mammalian vertebrates, has attracted attention in recent years for its contributions to human health and disease. H2S has been proposed as a cytoprotectant and gasotransmitter in many tissue types, including mediating vascular tone in blood vessels as well as neuromodulation in the brain. The molecular mechanisms dictating how H2S affects cellular signaling and other physiological events remain insufficiently understood. Furthermore, the involvement of H2S in metal-binding interactions and formation of related RSS such as sulfane sulfur may contribute to other distinct signaling pathways. Owing to its widespread biological roles and unique chemical properties, H2S is an appealing target for chemical biology approaches to elucidate its production, trafficking, and downstream function. In this context, reaction-based fluorescent probes offer a versatile set of screening tools to visualize H2S pools in living systems. Three main strategies used in molecular probe development for H2S detection include azide and nitro group reduction, nucleophilic attack, and CuS precipitation. Each of these approaches exploits the strong nucleophilicity and reducing potency of H2S to achieve selectivity over other biothiols. In addition, a variety of methods have been developed for the detection of other reactive sulfur species (RSS), including sulfite and bisulfite, as well as sulfane sulfur species and related modifications such as S-nitrosothiols. Access to this growing chemical toolbox of new molecular probes for H2S and related RSS sets the stage for applying these developing technologies to probe reactive sulfur biology in living systems.

Elucidating the Gas-Phase Behavior of Nitazene Analog Protomers Using Structures for Lossless Ion Manipulations Ion Mobility-Orbitrap Mass Spectrometry.

2-Benzylbenzimidazoles, or "nitazenes", are a class of novel synthetic opioids (NSOs) that are increasingly being detected alongside fentanyl analogs and other opioids in drug overdose cases. Nitazenes can be 20× more potent than fentanyl but are not routinely tested for during postmortem or clinical toxicology drug screens; thus, their prevalence in drug overdose cases may be under-reported. Traditional analytical workflows utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS) often require additional confirmation with authentic reference standards to identify a novel nitazene. However, additional analytical measurements with ion mobility spectrometry (IMS) may provide a path toward reference-free identification, which would greatly accelerate NSO identification rates in toxicology laboratories. Presented here are the first IMS and collision cross section (CCS) measurements on a set of fourteen nitazene analogs using a structures for lossless ion manipulations (SLIM)-orbitrap MS. All nitazenes exhibited two high intensity baseline-separated IMS distributions, which fentanyls and other drug and druglike compounds also exhibit. Incorporating water into the electrospray ionization (ESI) solution caused the intensities of the higher mobility IMS distributions to increase and the intensities of the lower mobility IMS distributions to decrease. Nitazenes lacking a nitro group at the R1 position exhibited the greatest shifts in signal intensities due to water. Furthermore, IMS-MS/MS experiments showed that the higher mobility IMS distributions of all nitazenes possessing a triethylamine group produced fragment ions with m/z 72, 100, and other low intensity fragments while the lower mobility IMS distributions only produced fragment ions with m/z 72 and 100. The IMS, solvent, and fragmentation studies provide experimental evidence that nitazenes potentially exhibit three gas-phase protomers. The cyclic IMS capability of SLIM was also employed to partially resolve four sets of structurally similar nitazene isomers (e.g., protonitazene/isotonitazene, butonitazene/isobutonitazene/secbutonitazene), showcasing the potential of using high-resolution IMS separations in MS-based workflows for reference-free identification of emerging nitazenes and other NSOs.

Identification of Unique Fragmentation Patterns of Fentanyl Analog Protomers Using Structures for Lossless Ion Manipulations Ion Mobility-Orbitrap Mass Spectrometry.

The opioid crisis in the United States is being fueled by the rapid emergence of new fentanyl analogs and precursors that can elude traditional library-based screening methods, which require data from known reference compounds. Since reference compounds are unavailable for new fentanyl analogs, we examined if fentanyls (fentanyl + fentanyl analogs) could be identified in a reference-free manner using a combination of electrospray ionization (ESI), high-resolution ion mobility (IM) spectrometry, high-resolution mass spectrometry (MS), and higher-energy collision-induced dissociation (MS/MS). We analyzed a mixture containing nine fentanyls and W-15 (a structurally similar molecule) and found that the protonated forms of all fentanyls exhibited two baseline-separated IM distributions that produced different MS/MS patterns. Upon fragmentation, both IM distributions of all fentanyls produced two high intensity fragments, resulting from amine site cleavages. The higher mobility distributions of all fentanyls also produced several low intensity fragments, but surprisingly, these same fragments exhibited much greater intensities in the lower mobility distributions. This observation demonstrates that many fragments of fentanyls predominantly originate from one of two different gas-phase structures (suggestive of protomers). Furthermore, increasing the water concentration in the ESI solution increased the intensity of the lower mobility distribution relative to the higher mobility distribution, which further supports that fentanyls exist as two gas-phase protomers. Our observations on the IM and MS/MS properties of fentanyls can be exploited to positively differentiate fentanyls from other compounds without requiring reference libraries and will hopefully assist first responders and law enforcement in combating new and emerging fentanyls.

Mitochondrial hydrogen peroxide and defective cholesterol efflux prevent in vitro fertilization by cryopreserved inbred mouse sperm.

Recent advances in the cryopreservation of mouse sperm have resulted in dramatically improved in vitro fertilization (IVF) rates, but the biological mechanisms underlying the techniques remain unclear. Two different classes of compounds have been widely utilized to improve the IVF rates of cryopreserved mouse sperm: antioxidants and cyclodextrins. To determine how cryopreservation reduces mouse sperm IVF and how antioxidants and cyclodextrins mitigate this effect, we examined sperm function and oxidative damage after cryopreservation, with and without treatments, in mouse strains important for biomedical research. Our investigation revealed mouse strain-specific effects on IVF by modulation of oxidative stress and cholesterol efflux of cryopreserved sperm. Antioxidants improved IVF rates of C57Bl6/J cryopreserved mouse sperm by reducing hydrogen peroxide produced by sperm mitochondria and ameliorating peroxidative damage to the sperm acrosome. Enhancing cholesterol efflux with cyclodextrin restored capacitation-dependent sperm function and IVF after cryopreservation of C57Bl/6J, C57Bl/6N, and 129X1 mouse sperm. Our results highlight two accessible pathways for continued development of IVF techniques for mouse sperm and provide novel endpoints prognostic of IVF success. These insights may improve sperm cryopreservation methods of other mouse strains and species.

Research highlights: impacts of microplastics on plankton.

Each year, millions of metric tons of the plastic produced for food packaging, personal care products, fishing gear, and other human activities end up in lakes, rivers, and the ocean. The breakdown of these primary plastics in the environment results in microplastics, small fragments of plastic typically less than 1-5 mm in size. These synthetic particles have been detected in all of the world's oceans and also in many freshwater systems, accumulating in sediment, on shorelines, suspended in surface waters, and being ingested by plankton, fish, birds, and marine mammals. While the occurrence of plastics in surface waters has been surveyed in a number of studies, the impacts of microplastics on marine organisms are still being elucidated. This highlight features three recent publications that explore the interactions of microplastics with planktonic organisms to clarify the effects of these pollutants on some of the ocean's smallest and most important inhabitants.

Research highlights: laboratory studies of the formation and transformation of atmospheric organic aerosols.

Atmospheric particles are emitted from a variety of anthropogenic and natural precursors and have direct impacts on climate, by scattering solar irradiation and nucleating clouds, and on health, by causing oxidative stress in the lungs when inhaled. They may also form from gaseous precursors, creating complex mixtures of organic and inorganic material. The chemical composition and the physical properties of aerosols will evolve during their one-week lifetime which will consequently change their impact on climate and health. The heterogeneity of aerosols is difficult to model and thus atmospheric aerosol research strives to characterize the mechanisms involved in nucleating and transforming particles in the atmosphere. Recent advances in four laboratory studies of aerosol formation and aging are highlighted here.

Research highlights: challenges in the characterization, storage, and isolation of natural organic matter.

Dissolved organic matter (DOM) is a critical component of natural waters, providing an important nutrient source for the microbial community, binding and interacting with metals and pollutants, and serving as a major reservoir of carbon in the global carbon cycle. Differences in organic material origin as well as biotic and abiotic transformation processes produce DOM of varying composition. These highly complex mixtures are difficult to characterize due to the diversity of compounds found in DOM. This Highlight examines three recent publications that study the methods used to characterize DOM and how storage and handling of DOM samples may affect its composition. Collectively, these publications underscore the challenges that researchers face when working with natural organic matter and offer important considerations for natural organic matter handling and analysis.

Research highlights: natural passive samplers--plants as biomonitors.

In the past decade, interest in boosting the collection of data on environmental pollutants while reducing costs has spurred intensive research into passive samplers, instruments that monitor the environment through the free flow of chemical species. These devices, although relatively inexpensive compared to active sampling technologies, are often tailored for collection of specific contaminants or monitoring of a single phase, typically water or air. Plants as versatile, natural passive samplers have gained increased attention in recent years due to their ability to absorb a diverse range of chemicals from the air, water, and soil. Trees, lichens, and other flora have evolved exquisite biological features to facilitate uptake of nutrients and water from the ground and conduct gas exchange on an extraordinary scale, making them excellent monitors of their surroundings. Sampling established plant specimens in a region also provides both historical and spatial data on environmental contaminants at relatively low cost in a non-invasive manner. This Highlight presents several recent publications that demonstrate how plant biomonitoring can be used to map the distribution of a variety of pollutants and identify their sources.

Research highlights: elucidation of biogeochemical factors influencing methylmercury production.

Coal combustion and other human activities release inorganic mercury into the atmosphere at levels far greater than emissions from natural sources, significantly perturbing the global mercury cycle. Subsequent biogeochemical transformation of inorganic mercury to highly toxic methylmercury allows this heavy metal pollutant to enter the food web, where it bioaccumulates and can have severe impacts on animal and human populations. This Highlight features recent articles that examine in detail the effects of nutrient availability on the methylation-demethylation activity of microorganisms living in sediment with mercury contamination. By investigating differences in levels of sulfate, iron, organic matter, and other environmental factors, this research provides insight into the conditions that may favor methylmercury formation and thereby better inform remediation efforts in the future.

Research highlights: modelling to assess climate change impacts and promote development.

We highlight four recent articles on biophysical modelling for the Ecosystem Services and Poverty Alleviation (ESPA) Deltas project in the Ganges-Brahmaputra-Meghna (GBM) delta system. These publications are part of a themed collection in Environmental Science: Processes & Impacts and contribute to a larger body of collaborative work that aims to assess the impacts of changing climate, policy, and development efforts on vulnerable populations in the GBM delta.

Azide-based fluorescent probes: imaging hydrogen sulfide in living systems.

Hydrogen sulfide is a redox active sulfur species that is endogenously generated in mammalian systems as an antioxidant and signaling molecule to support cellular function. The fundamental and ubiquitous actions of hydrogen sulfide demand sensitive and specific methods to track this biomolecule as it is produced within living organisms with temporal and spatial regulation. In this context, the hydrogen sulfide-mediated reduction of an azide to an amine is a useful method for organic synthesis, and this reaction has successfully been exploited to yield biocompatible fluorescent probes for hydrogen sulfide detection in vitro and in cells. This chapter provides protocols and guidelines for applying azide-based fluorescence probes to detecting hydrogen sulfide in living systems, including a protocol that was used to detect endogenous hydrogen sulfide in living single cells using a confocal microscope.

Preparation and use of MitoPY1 for imaging hydrogen peroxide in mitochondria of live cells.

Mitochondria peroxy yellow 1 (MitoPY1) is a small-molecule fluorescent probe that selectively tracks to the mitochondria of live biological specimens and responds to local fluxes of hydrogen peroxide (H(2)O(2)) by a turn-on fluorescence enhancement. This bifunctional dye uses a triphenylphosphonium targeting group and a boronate-based molecular switch to selectively respond to H(2)O(2) over competing reactive oxygen species (ROS) within the mitochondria. MitoPY1 can be used to measure mitochondrial H(2)O(2) levels in both cell culture and tissue models. In this protocol, we describe the synthesis of MitoPY1 and how to use this chemical tool to visualize mitochondrial H(2)O(2) in live cells. The preparation of MitoPY1 is anticipated to take 7-10 d, and assays involving microscopy of cultured mammalian cells can be performed in 1-2 d.

Boronate-based fluorescent probes: imaging hydrogen peroxide in living systems.

Hydrogen peroxide, a reactive oxygen species with unique chemical properties, is produced endogenously in living systems as a destructive oxidant to ward off pathogens or as a finely tuned second messenger in dynamic cellular signaling pathways. In order to understand the complex roles that hydrogen peroxide can play in biological systems, new tools to monitor hydrogen peroxide in its native settings, with high selectivity and sensitivity, are needed. Knowledge of organic synthetic reactivity provides the foundation for the molecular design of selective, functional hydrogen peroxide probes. A palette of fluorescent and luminescent probes that react chemoselectively with hydrogen peroxide has been developed, utilizing a boronate oxidation trigger. These indicators offer a variety of colors and in cellulo characteristics and have been used to examine hydrogen peroxide in a number of experimental setups, including in vitro fluorometry, confocal fluorescence microscopy, and flow cytometry. In this chapter, we provide an overview of the chemical features of these probes and information on their behavior to help researchers select the optimal probe and application.

Two-photon fluorescence imaging of intracellular hydrogen peroxide with chemoselective fluorescent probes.

We present the application of two-photon fluorescence (TPF) imaging to monitor intracellular hydrogen peroxide (H2O2) production in brain cells. For selective imaging of H2O2 over other reactive oxygen species, we employed small-molecule fluorescent probes that utilize a chemoselective boronate deprotection mechanism. Peroxyfluor-6 acetoxymethyl ester detects global cellular H2O2 and mitochondria peroxy yellow 1 detects mitochondrial H2O2. Two-photon absorption cross sections for these H2O2 probes are measured with a mode-locked Ti:sapphire laser in the wavelength range of 720 to 1040 nm. TPF imaging is demonstrated in the HT22 cell line to monitor both cytoplasmic H2O2 and localized H2O2 production in mitochondria. Endogenous cytoplasmic H2O2 production is detected with TPF imaging in rat astrocytes modified with d-amino acid oxidase. The TPF H2O2 imaging demonstrated that these chemoselective probes are powerful tools for the detection of intracellular H2O2.