Reactive Probe Research Articles

Front Cover: Site‐Specific Small Molecule Labeling of an Internal Loop in JC Polyomavirus Pentamers Using the π‐Clamp‐Mediated Cysteine Conjugation (ChemBioChem 21/2021)

Few methods exist to site-specifically label loops of globular proteins without interfering with the function of the protein. A modular π-clamp reactive perfluoro-aromatic probe was synthesized in only two-steps and used to selectively modify an internal, surface exposed loop of the major capsid protein of JC polyomavirus (VP1). The perfluoro-biphenyl probe reacts selectively with the cysteine in the π-clamp (Phe-Cys-Pro-Phe) over 6 native cysteines present in VP1. Using microscopy, fluorescently labeled π-clamp VP1 can be observed binding and entering human glial cells. More information can be found in the Communication by F. M. Rossi, C. D. S. Nelson et al.

Proteomic Analysis Reveals IRAK4 As a Therapeutic Target in Chronic Lymphocytic Leukemia

Dimethylfumarate (DMF) and other electrophilic compounds are highly toxic to CLL cells in vitro and in xenograft animal models [Wu et al., 2010; Wu et al., 2016]. To study the mechanism of action, competitive isotopic tandem Orthogonal Proteolysis Activity Based Protein Profiling (isoTOP ABPP) - a chemical proteomic platform, which utilizes broad-spectrum cysteine reactive probes, - was applied to quantitatively map DMF protein targets in whole proteomes. Cells from 10 CLL patients were treated with 30 μM DMF for 4h followed by an isoTOP ABPP sample preparation and analysis workflow. Based on reproducible high isoTOP ABPP ratios across multiple biological replicates, cysteine 13 of interleukin-1 receptor-associated kinase 4 (IRAK4) was identified as a prominent target of DMF in CLL patient cells.IRAK4 plays a critical role in Toll-like receptor (TLR) signal transduction. Engagement of IRAK4 with the signaling adaptor protein MyD88 in TLRs activates downstream NF-κB and type-I IFN pathways. Approximately 15-20% of CLL patients have dominant activating mutations of MyD88, and nearly all patients with the CLL-related disease Waldenstrom's Macroglobulinemia have such mutations. Hence, we sought to evaluate known inhibitors of IRAK4 kinase activity for their effect on CLL cells. Multiple IRAK4 inhibitors are currently in clinical development, primarily for the treatment of autoimmune conditions. We used PF06650833, a specific inhibitor of IRAK4, previously tested in phase 1 trials for patients with systemic lupus erythematosus, to evaluate its effect on CLL cells viability. In vitro , PF06650833 reduced CLL cell viability in a dose-dependent manner. Reduction in IRAK4 and IκBα protein levels were also observed in samples treated with at least 15 µM PF06650833 by Western blot analysis. In vivo studies provide a better opportunity to evaluate the effect of this drug in blocking microenvironment signaling. We utilized a murine xenograft model in which primary CLL cells are transplanted into the peritoneum of Rag2/gamma chain immunodeficient mice. Each mouse received a single intraperitoneal injection of 1-10 mg/kg PF06650833 one day after transplantation. Cells were recovered 6 days after the treatment and analyzed by flow cytometry, showing remarkable reduction in CLL survival in a dose-dependent manner.In conclusion, unbiased proteomics analysis of a non-specific drug, DMF, identified IRAK4 as a target in cells from CLL patients. While the exact mode of action for DMF and PF06650833 might differ (disruption of higher IRAK4 complexes, inhibition of kinase dependent signaling or other), our results suggest that IRAK4 represents a potential druggable target in the management of patients with CLL. [Display omitted] DisclosuresChoi:Gilead, Genentech, Abbvie, and PCYC: Speakers Bureau; AbbVie: Other: Institutional research funding; AbbVie, Genentech, and PCYC: Consultancy.

Hinokitiol chelates intracellular iron to retard fungal growth by disturbing mitochondrial respiration

IntroductionThe increasing morbidity of fungal infections and the prevalence of drug resistance highlighted the discovery of novel antifungal agents and investigation of their modes of action. Iron chelators have been used to treat superficial fungal infections or potentiate the efficacy of certain antifungal drugs. Hinokitiol exhibits potent antifungal activity and iron-chelating ability. However, their relationships have not been established. ObjectivesThis study aims to explore the selectivity of hinokitiol against fungal cells and mammalian cells and determine the role of iron-chelating for the antifungal activity of hinokitiol. MethodsIron probe FeRhonox-1 was used to determine intracellular Fe2+ content. 5-Cyano-2,3-ditolyl tetrazolium chloride probe and Cell Counting Kit-8 were used to detect the mitochondrial respiratory activities. Quantitative real-time PCR and rescue experiments were performed to determine the effect of iron on the antifungal activity of hinokitiol. The effects of hinokitiol on fungal mitochondria were further evaluated using reactive oxygen species probes and several commercial Assay Kits. The ability of hinokitiol to induce resistance in Candida species was carried out using a serial passage method. The in vivo therapeutic effect of hinokitiol was evaluated using Galleria mellonella as an infectious model. ResultsHinokitiol was effective against a panel of Candida strains with multiple azole-resistant mechanisms and persistently inhibited Candida albicans growth. Mechanism investigations revealed that hinokitiol chelated fungal intracellular iron and inhibited the respiration of fungal cells but had minor effects on mammalian cells. Hinokitiol further inhibited the activities of mitochondrial respiratory chain complexes I and II and reduced mitochondrial membrane potential, thereby decreasing intracellular ATP synthesis and increasing detrimental intracellular reductive stress. Moreover, hinokitiol exhibited low potential for inducing resistance in several Candida species and greatly improved the survival of Candida-infected Galleria mellonella. ConclusionsThese findings suggested the potential application of hinokitiol as an iron chelator to treat fungal infections.

Effect of Food Colorants Supplementation on Reactive Oxygen Species, Antioxidant Vitamins Level and DNA Damage

There are various undesirable products generated from endogenous aerobic metabolism such as reactive oxygen species (ROS). Physiological and biochemical lesions are caused by ROS and which give rise to oxidative damages towards DNA, proteins and lipids which ultimately lead to cell death. This study was aimed to examine the effect of oral administration of food colorants (tartrazine and curcumin) on the oxidants and antioxidants level in blood and fecal of rats after 15, 30, and 45 days. Two doses were used based on the admissible daily intake (ADI) of 9.6 and 96 (high) mg/kg/body weight for tartrazine, 3.85 and 38.5 6 mg/kg/body weight for curcumin. The results showed that oral administration of tartrazine had significantly increased the total oxidant level, arylesterase, glutathione reductase, and MDA whereas there was significantly decreased the total antioxidants level, catalase, glutathione peroxidase in plasma and fecal after 30 and 45 days. Vitamin E and C were decreased in plasma. Fecal showed high level of vitamin A. High dose of tartrazine caused alteration in the aldehyde reactive probe (ARP) sites of DNA showing the DNA damage. After 45 day, significant increment was observed in the level of AST in low and high curcumin treated group. Whereas, elevations of arylestrase were seen in high curcumin group after 45 day. High dose of curcumin significantly (P≤ 0.05) decreased the concentration of vitamin C after 45 days of treatment and increased the level of vitamin E in plasma of treated groups after 30 and 45 days of treatment. The present study showed that the ADI and doses up to 10 times higher than ADI showed negative effects on antioxidant level and demonstrated the importance of using appropriate doses of food colorants such as tartrazine and curcumin in different processed food products.

(Invited) Interfacial Chemistry As an Enabling Tool in the Development of Transition Metal Phosphide Electrocatalysts

We are interested in achieving control of heterogeneous interfaces to promote inner-sphere electron-proton-transfer reactions using insights from molecular chemistry. Toward this end, we are developing solution-phase methods for the synthesis and post-synthetic modification of colloidal nanostructures with low overpotentials and high activity for multi-electron, multi-proton electrocatalytic reactions. Ligand exchange, surface etching, and covalent modification will be presented as complementary methods for altering the catalytic activity of colloidal transition metal phosphide electrocatalysts. A combination of electrochemical characterization, structural analysis, and electronic structure characterization will be presented. Together with stoichiometric probes of inner-sphere reactivity, these data will serve to reveal mechanistic details and design principles for catalysis in these systems.

Fluorescently Labeled Cellulose Nanofibers for Environmental Health and Safety Studies.

An optimal methodology for locating and tracking cellulose nanofibers (CNFs) in vitro and in vivo is crucial to evaluate the environmental health and safety properties of these nanomaterials. Here, we report the use of a new boron-dipyrromethene (BODIPY) reactive fluorescent probe, meso-DichlorotriazineEthyl BODIPY (mDTEB), tailor-made for labeling CNFs used in simulated or in vivo ingestion exposure studies. Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) was used to confirm covalent attachment and purity of mDTEB-labeled CNFs. The photoluminescence properties of mDTEB-labeled CNFs, characterized using fluorescence spectroscopy, include excellent stability over a wide pH range (pH2 to pH10) and high quantum yield, which provides detection at low (μM) concentrations. FLIM analysis also showed that lignin-like impurities present on the CNF reduce the fluorescence of the mDTEB-labeled CNF, via quenching. Therefore, the chemical composition and the methods of CNF production affect subsequent studies. An in vitro triculture, small intestinal, epithelial model was used to assess the toxicity of ingested mDTEB-labeled CNFs. Zebrafish (Danio rerio) were used to assess in vivo environmental toxicity studies. No cytotoxicity was observed for CNFs, or mDTEB-labeled CNFs, either in the triculture cells or in the zebrafish embryos.

A Workflow towards the Reproducible Identification and Quantitation of Protein Carbonylation Sites in Human Plasma.

Protein carbonylation, a marker of excessive oxidative stress, has been studied in the context of multiple human diseases related to oxidative stress. The variety of post-translational carbonyl modifications (carbonyl PTMs) and their low concentrations in plasma challenge their reproducible identification and quantitation. However, carbonyl-specific biotinylated derivatization tags (e.g., aldehyde reactive probe, ARP) allow for targeting carbonyl PTMs by enriching proteins and peptides carrying these modifications. In this study, an oxidized human serum albumin protein model (OxHSA) and plasma from a healthy donor were derivatized with ARP, digested with trypsin, and enriched using biotin-avidin affinity chromatography prior to nano reversed-phase chromatography coupled online to electrospray ionization tandem mass spectrometry with travelling wave ion mobility spectrometry (nRPC-ESI-MS/MS-TWIMS). The presented workflow addresses several analytical challenges by using ARP-specific fragment ions to reliably identify ARP peptides. Furthermore, the reproducible recovery and relative quantitation of ARP peptides were validated. Human serum albumin (HSA) in plasma was heavily modified by a variety of direct amino acid oxidation products and adducts from reactive carbonyl species (RCS), with most RCS modifications being detected in six hotspots, i.e., Lys10, Lys190, Lys199, Lys281, Lys432, and Lys525 of mature HSA.

Recognition of Surface Oxygen Intermediates on NiFe Oxyhydroxide Oxygen-Evolving Catalysts by Homogeneous Oxidation Reactivity.

NiFe oxyhydroxide is one of the most promising oxygen evolution reaction (OER) catalysts for renewable hydrogen production, and deciphering the identity and reactivity of the oxygen intermediates on its surface is a key challenge but is critical to the catalyst design for improving the energy efficiency. Here, we screened and utilized in situ reactive probes that can selectively target specific oxygen intermediates with high rates to investigate the OER intermediates and pathway on NiFe oxyhydroxide. Most importantly, the oxygen atom transfer (OAT) probes (e.g., 4-(diphenylphosphino) benzoic acid) could efficiently inhibit the OER kinetics by scavenging the OER intermediates, exhibiting lower OER currents, larger Tafel slopes, and larger kinetic isotope effect (KIE) values, while probes with other reactivities demonstrated much smaller effects. Combining the OAT reactivity with electrochemical kinetic and operando Raman spectroscopic techniques, we identified a resting Fe═O intermediate in the Ni-O scaffold and a rate-limiting O-O chemical coupling step between a Fe═O moiety and a vicinal bridging O. DFT calculation further revealed a longer Fe═O bond formed on the surface and a large kinetic energy barrier of the O-O chemical coupling step, corroborating the experimental results. These results point to a new direction of liberating lattice O and expediting O-O coupling for optimizing NiFe-based OER electrocatalyst.

Sulphide activity-dependent multicolor emission dye and its applications in in vivo imaging.

Reactive sulfur species (RSS) play pivotal roles in various pathological and physiological processes. There exists an intricate relevance in generation and metabolism among these substances. Although they are nucleophilic, there are still some differences in their reactivity. There are many methods to detect them by using reactive fluorescent probes, but the systematic study of their reactivity is still lacking. In our study, we designed a multiple reaction site fluorescent probe based on benzene conjugated benzopyrylium and NBD. The study revealed that besides both biothiols and hydrogen sulfide, sulfur dioxide (SO2) can cleave the ether bond. There are two reaction forms for GSH with low reactivity: cutting the ether bond and adding the conjugated double bond of benzopyrylium. Nevertheless, Cys/Hcy with higher activity can further rearrange with NBD after cutting the ether bond. In addition, SO2 can not only cleave the ether bond, but also continue to add the conjugated double bond of benzopyrylium. The above processes lead to multicolor emission of the probe, thus realizing the characteristic analysis of different sulfides. Thus the probe can be used for the detection of sulfide in mitochondria, and further for the imaging of sulfide in cells and zebrafish. This effective analysis method will provide a broad application prospect for practical applications.

A strongly Lewis-acidic and fluorescent borenium cation supported by a tridentate formazanate ligand.

Lewis acids are highly sought after for their applications in sensing, small-molecule activation, and catalysis. When combined with π-conjugated molecular frameworks, Lewis acids with unique optoelectronic properties can be realized. Here, we use a tridentate formazanate ligand to create a planar, redox-active, fluorescent, and strongly Lewis-acidic borenium cation. We also demonstrate that this compound can act as a colourimetric probe for reactivity.

Short-chain reactive probes as tools to unravel the Pseudomonas aeruginosa quorum sensing regulon.

In recent years, the world has seen a troubling increase in antibiotic resistance among bacterial pathogens. In order to provide alternative strategies to combat bacterial infections, it is crucial deepen our understanding into the mechanisms that pathogens use to thrive in complex environments. Most bacteria use sophisticated chemical communication systems to sense their population density and coordinate gene expression in a collective manner, a process that is termed “quorum sensing” (QS). The human pathogen Pseudomonas aeruginosa uses several small molecules to regulate QS, and one of them is N-butyryl-l-homoserine lactone (C4-HSL). Using an activity-based protein profiling (ABPP) strategy, we designed biomimetic probes with a photoreactive group and a ‘click’ tag as an analytical handle. Using these probes, we have identified previously uncharacterized proteins that are part of the P. aeruginosa QS network, and we uncovered an additional role for this natural autoinducer in the virulence regulon of P. aeruginosa, through its interaction with PhzB1/2 that results in inhibition of pyocyanin production.

α-Methylene-β-Lactone Scaffold for Developing Chemical Probes at the Two Ends of the Selectivity Spectrum.

The utilities of an α-methylene-β-lactone (MeLac) moiety as a warhead composed of multiple electrophilic sites are reported. We demonstrate that a MeLac-alkyne not only reacts with diverse proteins as a broadly reactive measurement probe, but also recruits reduced endogenous glutathione (GSH) to assemble a selective chemical probe of GSH-β-lactone (GSH-Lac)-alkyne in live cells. Tandem mass spectrometry reveals that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael or acyl addition. A peptide-centric proteomics platform demonstrates that the proteomic selectivity profiles of orlistat and parthenolide, which have distinct reactivities, are measurable by MeLac-alkyne as a high-coverage probe. The GSH-Lac-alkyne selectively probes the glutathione S-transferase P responsible for multidrug resistance. The assembly of the GSH-Lac probe exemplifies a modular and scalable route to develop selective probes with different recognizing moieties.

S2564 Cyanoacrylate Embolization Following EGD Resulting in Fulminant Hepatic Failure in a Patient With Cirrhosis

INTRODUCTION: Esophagogastric varices are a common complication of portal hypertension and can present with life-threatening bleeding. Definitive endoscopic therapy is via band ligation or sclerotherapy. The former is preferred for esophageal varices, but efficacy is lower in gastric varices (GV). Sclerotherapy with cyanoacrylate (CA) has shown better efficacy and is now recommended as first line therapy for bleeding GV. Studies on long-term efficacy and complications remain limited. CASE DESCRIPTION/METHODS: A 62-year-old woman with NASH cirrhosis (MELD 11) presented with hematemesis. She denied any history of SBP, varices, or encephalopathy. She endorsed a previous history of COVID-19 and had reactive IgG but PCR probe for SARS-CoV-2 was negative. She underwent EGD and was found to have oozing GV along the lesser curvature, which were treated with 4cc of CA achieving hemostasis. The following night she had altered mentation and the blood lactate was increased to 7.2 mmol/L. AST and ALT were also increased. She received broad spectrum antibiotics, and a CT angiogram showed evidence of embolization of CA into the left lobe of the liver. On day 3 her level of consciousness declined and she was intubated for airway compromise. An MRCP confirmed the presence of CA within the left hepatic lobe with associated ischemia. The lactate increased to 20 mmol/L and the blood ammonia level to 700 mcg/dL, with MELD 45. Continuous hemodialysis was started for anuric renal failure. She underwent evaluation for liver transplantation, but cerebral edema and multiorgan failure with refractory acidosis occurred and she died on day 7. DISCUSSION: We present a case of GV treated with CA and the subsequent embolization of CA into the left lobe of the liver. This precipitated acute on chronic liver failure with features of fulminant hepatic failure (FHF) complicated by severe hyperammonemia, cerebral edema, multiorgan failure, and death. Although she had a recent diagnosis of COVID-19, the time course, relatively normal initial inflammatory markers, and imaging suggest that CA embolization was likely the injury that led to fulminant hepatic failure. Given the lack of case reports of CA embolization to the liver causing infarction and few cases to the brain or distant vessels, further research on its long-term safety is warranted. Another novel aspect to this case is the development of FHF in a patient with known cirrhosis.

Comparison of UV/H2O2, UV/PMS, and UV/PDS in Destruction of Different Reactivity Compounds and Formation of Bromate and Chlorate.

In this study, we compared the decontamination kinetics of various target compounds and the oxidation by-products (bromate and chlorate) of PMS, PDS, and H2O2 under UV irradiation (UV/PMS, UV/PDS, UV/H2O2). Probes of different reactivity with hydroxyl and sulfate radicals, such as benzoic acid (BA), nitrobenzene (NB), and trichloromethane (TCM), were selected to compare the decontamination efficiency of the three oxidation systems. Experiments were performed under acidic, neutral, and alkaline pH conditions to obtain a full-scale comparison of UV/peroxides. Furthermore, the decontamination efficiency was also compared in the presence of common radical scavengers in water bodies [bicarbonate, carbonate, and natural organic matter (NOM)]. Finally, the formation of oxidation by-products, bromate, and chlorate, was also monitored in comparison in pure water and tap water. Results showed that UV/H2O2 showed higher decontamination efficiency than UV/PDS and UV/PMS for BA degradation while UV/H2O2 and UV/PMS showed better decontamination performance than UV/PDS for NB degradation under acidic and neutral conditions. UV/PMS was the most efficient among the three processes for BA and NB degradation under alkaline conditions, while UV/PDS was the most efficient for TCM degradation under all pH conditions. In pure water, both bromate and chlorate were formed in UV/PDS, small amounts of bromate and rare chlorate were observed in UV/PMS, and no detectable bromate and chlorate were formed in UV/H2O2. In tap water, no bromate and chlorate were detectable for all three systems.

GuttaFlow® Bioseal Cytotoxicity Assessment: In Vitro Study.

The sealers used for root canal treatment should be biocompatible for the peri-radicular tissues, to evaluate the cytotoxic effects of GuttaFlow® bioseal sealer and to compare them with AH26® epoxy resin. Culture media were conditioned with the GuttaFlow® bioseal and AH26® pellets. MDPC-23 odontoblast cell cultures were treated with conditioned medium and serial dilutions. To evaluate the metabolic activity and cellular viability, the MTT and SRB assays were performed. To determine the production of reactive oxygen species, the DHE and DCF-DA probes were used. Cell cycle and cell-death types were assessed by cytometry, and to evaluate the mineralization capacity, the Alizarin Red S coloration was used. Statistical analysis was performed using analysis of variance (ANOVA) when normality was found and Kruskal-Wallis on the opposite case. For the comparison with normality values, the Student t-test was used. Cells exposed to the GuttaFlow® bioseal conditioned medium maintained high metabolic activities, except at higher concentrations. Likewise, viability was maintained, but a significant decrease was observed after exposure to the highest concentration (p < 0.001), associated with cell death by late apoptosis and necrosis. When cell cultures were exposed to AH26®, metabolic activity was highly compromised, resulting in cell death. An imbalance in the production of peroxides and superoxide anion was observed. GuttaFlow® bioseal showed higher biocompatibility than AH26®.

Heterogeneous adaptation of cysteine reactivity to a covalent oncometabolite

An important context in which metabolism influences tumorigenesis is the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a disease in which mutation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) causes hyperaccumulation of fumarate. This electrophilic oncometabolite can alter gene activity at the level of transcription, via reversible inhibition of epigenetic dioxygenases, as well as posttranslationally, via covalent modification of cysteine residues. To better understand the potential for metabolites to influence posttranslational modifications important to tumorigenesis and cancer cell growth, here we report a chemoproteomic analysis of a kidney-derived HLRCC cell line. Using a general reactivity probe, we generated a data set of proteomic cysteine residues sensitive to the reduction in fumarate levels caused by genetic reintroduction of active FH into HLRCC cell lines. This revealed a broad up-regulation of cysteine reactivity upon FH rescue, which evidence suggests is caused by an approximately equal proportion of transcriptional and posttranslational modification-mediated regulation. Gene ontology analysis highlighted several new targets and pathways potentially modulated by FH mutation. Comparison of the new data set with prior studies highlights considerable heterogeneity in the adaptive response of cysteine-containing proteins in different models of HLRCC. This is consistent with emerging studies indicating the existence of cell- and tissue-specific cysteine-omes, further emphasizing the need for characterization of diverse models. Our analysis provides a resource for understanding the proteomic adaptation to fumarate accumulation and a foundation for future efforts to exploit this knowledge for cancer therapy.

Acrolein in cigarette smoke attenuates the innate immune responses mediated by surfactant protein D

BackgroundSurfactant proteins (SP) A and D belong to collectin family proteins, which play important roles in innate immune response in the lung. We previously demonstrated that cigarette smoke (CS) increases the acrolein modification of SP-A, thereby impairing the innate immune abilities of this protein. In this study, we focused on the effects of CS and its component, acrolein, on the innate immunity role of another collectin, SP-D. MethodsTo determine whether aldehyde directly affects SP-D, we examined the lungs of mice exposed to CS for 1 week and detected aldehyde-modified SP-D using an aldehyde reactive probe. The structural changes in CS extract (CSE) or acrolein-exposed recombinant human (h)SP-D were determined by western blot, liquid chromatography-electrospray ionization tandem mass spectrometry, and blue native-polyacrylamide gel electrophoresis analyses. Innate immune functions of SP-D were determined by bacteria growth and macrophage phagocytosis. ResultsAldehyde-modified SP-D as well as SP-A was detected in the lungs of mice exposed to CS for 1 week. Exposure of hSP-D to CSE or acrolein induced an increased higher-molecular -weight of hSP-D and acrolein induced modification of five lysine residues in hSP-D. These modifications led to disruption of the multimer structure of SP-D and attenuated its ability to inhibit bacterial growth and activate macrophage phagocytosis. ConclusionCS induced acrolein modification in SP-D, which in turn induced structural and functional defects in SP-D. General SignificanceThese results suggest that CS-induced structural and functional defects in SP-D contribute to the dysfunction of innate immune responses in the lung following CS exposure.

A reactive probe for Co2+ ion detection based on a catalytic decomposition process and its fluorescence imaging in living cells.

A novel reactive fluorescent probe for cobalt ions was prepared based on integration of thiourea functional groups, coumarin, and naphthalimide fluorophores. There was no fluorescence observed for the probe itself, however, in the presence of cobalt ions, catalytic decomposition occurred for the probe and coumarin molecular fragments were produced that emitted blue fluorescence. This enabled the probe to be used as a 'turn on' reagent for detection of cobalt ions. Under physiological pH conditions and in appropriate solvent systems, an obvious fluorescence enhancement for cobalt ions was observed in selective experiments. Competition experiments indicated that cobalt ions could still induce fluorescence enhancement in the presence of other metal ions. Sensitivity experiments showed that the detection limit for cobalt ions was 6.0 nM. Dynamics research demonstrated that the catalytic process was a pseudo-first-order reaction and the reaction constant (kobs ) was calculated to be 1.49 × 10-2 min-1 . In addition, the mechanism of catalytic decomposition could be demonstrated using electrospray ionization mass spectrometry and thin layer chromatography experiments. Cell fluorescence imaging experiments demonstrated that the probe could be used to detect cobalt ions in living HeLa cells.

Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture.

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.

Estimating surface electric fields using reactive formic acid probes and SEM image brightness analysis

By changing the electrical bias imposed on a Ni catalyst attached to an external circuit, the selectivity of catalytic formic acid decomposition is shown to change—favoring CO2/H2 production under negative bias and CO/H2O production under positive bias. A method for estimating the strength of externally generated surface electric fields by measuring this selectivity change is presented and used to approximate field strengths on the order of 0.20 V/nm. A COMSOL model of the catalyst was created which indicated that the presence of Ni particles increased field strength and uniformity across the catalyst. Comparing this model to SEM imaging of the catalyst verified that the field strengths are highest on the surface of the catalyst particles, pore edges, and microscopic defects. The methods developed herein may be useful to future reaction engineers seeking to incorporate applied electric fields into process designs.