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Multi-omic analyses can provide information on the potential for activity within a microbial community but often lack specificity to link functions to cell, primarily offer potential for function or rely on annotated databases. Functional assays are necessary for understanding in situ microbial activity to better describe and improve microbiome biology. Targeting enzyme activity through activity-based protein profiling enhances the accuracy of functional studies. Here, we introduce a pipeline of coupling activity-based probing with fluorescence-activated cell sorting, culturing, and downstream activity assays to isolate and examine viable populations of cells expressing a function of interest. We applied our approach to a soil microbiome using two activity-based probes to enrich for communities with elevated activity for lignocellulose-degradation phenotypes as determined by four fluorogenic kinetic assays. Our approach efficiently separated and identified microbial members with heightened activity for glycosyl hydrolases, and by expanding this workflow to various probes for other function, this process can be applied to unique phenotype targets of interest., (© 2023. ISME Publications B.V.)

Seafloor methane emissions can affect Earth's climate and ocean chemistry. Vast quantities of methane formed by microbial decomposition of organic matter are locked within gas hydrate and free gas on continental slopes, particularly in large areas with high sediment accumulations such as deep-sea fans. The release of methane in slope environments has frequently been associated with dissociation of gas hydrates near the edge of the gas hydrate stability zone on the upper slope, with discharges in greater water depths less understood. Here we show, using data from the Rio Grande Cone (western South Atlantic), that the intrinsic, gravity-induced downslope collapse of thick slope sediment accumulations creates structures that serve as pathways for gas migration, unlocking methane and causing seafloor emissions via giant gas flares in the water column. The observed emissions in the study region (up to 310 Mg year -1 ) are three times greater than estimates for the entire US North Atlantic margin and reveal the importance of collapsing sediment accumulations for ocean carbon cycling. Similar outgassing systems on the Amazon and Niger fans suggest that gravity tectonics on passive margins is a common yet overlooked mechanism driving massive seafloor methane emissions in sediment-laden continental slopes., (© 2023. The Author(s).)

Development of profiling strategies to provide high resolution understanding of enzymes involved in bacterial infections remains an important need. These strategies help resolve enzyme mechanisms of actions and can guide therapeutic development. We have developed a selective new activity-based probe (ABP) targeting a highly conserved surface bound enzyme, C5a peptidase, present in several pathogenic Streptococci . We demonstrate our probe inhibits C5a peptidase activity and enables detection of C5a peptidase expressing pathogens in microbial mixtures. Our profiling strategy selectively labels the pathogen by phenotype and enables specific isolation of the live bacteria providing a route for further in-depth investigation. This study paves the way towards a rapid detection, isolation, and characterization pipeline for existing and emerging strains of most common pathogenic Streptococci .

Aim: Numerous studies have shown that H 2 S serves as an acute oxygen sensor in a variety of cells. We hypothesize that H 2 S also serves in extended oxygen sensing., Methods: Here, we compare the effects of extended exposure (24-48 hours) to varying O 2 tensions on H 2 S and polysulphide metabolism in human embryonic kidney (HEK 293), human adenocarcinomic alveolar basal epithelial (A549), human colon cancer (HTC116), bovine pulmonary artery smooth muscle, human umbilical-derived mesenchymal stromal (stem) cells and porcine tracheal epithelium (PTE) using sulphur-specific fluorophores and fluorometry or confocal microscopy., Results: All cells continuously produced H 2 S in 21% O 2 and H 2 S production was increased at lower O 2 tensions. Decreasing O 2 from 21% to 10%, 5% and 1% O 2 progressively increased H 2 S production in HEK293 cells and this was partially inhibited by a combination of inhibitors of H 2 S biosynthesis, aminooxyacetate, propargyl glycine and compound 3. Mitochondria appeared to be the source of much of this increase in HEK 293 cells. H 2 S production in all other cells and PTE increased when O 2 was lowered from 21% to 5% except for HTC116 cells where 1% O 2 was necessary to increase H 2 S, presumably reflecting the hypoxic environment in vivo. Polysulphides (H 2 S n , where n = 2-7), the key signalling metabolite of H 2 S also appeared to increase in many cells although this was often masked by high endogenous polysulphide concentrations., Conclusion: These results show that cellular H 2 S is increased during extended hypoxia and they suggest this is a continuously active O 2 -sensing mechanism in a variety of cells., (© 2019 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Manganese porphyrins (MnPs), MnTE-2-PyP 5+ , MnTnHex-2-PyP 5+ and MnTnBuOE-2-PyP 5+ , are superoxide dismutase (SOD) mimetics and form a redox cycle between O 2 and reductants, including ascorbic acid, ultimately producing hydrogen peroxide (H 2 O 2 ). We previously found that MnPs oxidize hydrogen sulfide (H 2 S) to polysulfides (PS; H 2 S n , n = 2-6) in buffer. Here, we examine the effects of MnPs for 24 h on H 2 S metabolism and PS production in HEK293, A549, HT29 and bone marrow derived stem cells (BMDSC) using H 2 S (AzMC, MeRho-AZ) and PS (SSP4) fluorophores. All MnPs decreased intracellular H 2 S production and increased intracellular PS. H 2 S metabolism and PS production were unaffected by cellular O 2 (5% versus 21% O 2 ), H 2 O 2 or ascorbic acid. We observed with confocal microscopy that mitochondria are a major site of H 2 S production in HEK293 cells and that MnPs decrease mitochondrial H 2 S production and increase PS in what appeared to be nucleoli and cytosolic fibrillary elements. This supports a role for MnPs in the metabolism of H 2 S to PS, the latter serving as both short- and long-term antioxidants, and suggests that some of the biological effects of MnPs may be attributable to sulfur metabolism.

Understanding the roles that individual species or communities play within a microbiome is a significant challenge. The complexity and heterogeneity of microbiomes presents a challenge to researchers looking to unravel the function that microbiomes serve within larger environments. While identification of the species and proteins present in a microbiome can be accomplished through genomics approaches, strategies that report on enzyme activity are limited. In this chapter, we describe the application of small molecule chemical probes in the isolation and subsequent characterization of microbiome subpopulations based on enzymatic function. We will cover protocols for labeling microbes with appropriate probes, microbiome sample preparation, and using fluorescence-activated cell sorting to isolate subpopulations based on function. We hope that the strategies outlined here will serve as a resource for researchers studying the functional role that microbiomes play in the gut and soil., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hydrogen sulfide (H 2 S) is an important signaling molecule that provides protective activities in a variety of physiological and pathological processes. Among the different types of H 2 S donor compounds, thioamides have attracted attention due to prior conjugation to nonsteroidal anti-inflammatory drugs (NSAIDs) to access H 2 S-NSAID hybrids with significantly reduced toxicity, but the mechanism of H 2 S release from thioamides remains unclear. Herein, we reported the synthesis and evaluation of a class of thioamide-derived sulfenyl thiocarbamates ( SulfenylTCMs ) that function as a new class of H 2 S donors. These compounds are efficiently activated by cellular thiols to release carbonyl sulfide (COS), which is quickly converted to H 2 S by carbonic anhydrase (CA). In addition, through mechanistic investigations, we establish that COS-independent H 2 S release pathways are also operative. In contrast to the parent thioamide-based donors, the SulfenylTCMs exhibit excellent H 2 S releasing efficiencies of up to 90% and operate through mechanistically well-defined pathways. In addition, we demonstrate that the sulfenyl thiocarbamate group is readily attached to common NSAIDs, such as naproxen, to generate YZ-597 as an efficient H 2 S-NSAID hybrid, which we demonstrate releases H 2 S in cellular environments. Taken together, this new class of H 2 S donor motifs provides an important platform for new donor development.

Hydrogen sulfide (H 2 S) is a biologically active molecule that exhibits protective effects in a variety of physiological and pathological processes. Although several H 2 S-related biological effects have been discovered by using H 2 S donors, knowing how much H 2 S has been released from donors under different conditions remains challenging. Now, a series of γ-ketothiocarbamate (γ-KetoTCM) compounds that provide the first examples of colorimetric H 2 S donors and enable direct quantification of H 2 S release, were reported. These compounds are activated through a pH-dependent deprotonation/β-elimination sequence to release carbonyl sulfide (COS), which is quickly converted into H 2 S by carbonic anhydrase. The p-nitroaniline released upon donor activation provides an optical readout that correlates directly to COS/H 2 S release, thus enabling colorimetric measurement of H 2 S donation., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hydrogen sulfide (H 2 S) is an endogenously produced gas that is toxic at high concentrations. It is eliminated by a dedicated mitochondrial sulfide oxidation pathway, which connects to the electron transfer chain at the level of complex III. Direct reduction of cytochrome c (Cyt C) by H 2 S has been reported previously but not characterized. In this study, we demonstrate that reduction of ferric Cyt C by H 2 S exhibits hysteretic behavior, which suggests the involvement of reactive sulfur species in the reduction process and is consistent with a reaction stoichiometry of 1.5 mol of Cyt C reduced/mol of H 2 S oxidized. H 2 S increases O 2 consumption by human cells (HT29 and HepG2) treated with the complex III inhibitor antimycin A, which is consistent with the entry of sulfide-derived electrons at the level of complex IV. Cyt C-dependent H 2 S oxidation stimulated protein persulfidation in vitro, while silencing of Cyt C expression decreased mitochondrial protein persulfidation in a cell culture. Cyt C released during apoptosis was correlated with persulfidation of procaspase 9 and with loss of its activity. These results reveal a potential role for the electron transfer chain in general, and Cyt C in particular, for potentiating sulfide-based signaling.

Significance: Carbonyl sulfide (COS) is the most prevalent sulfur-containing gas in the Earth's atmosphere, and it plays important roles in the global sulfur cycle. COS has been implicated in origin of life peptide ligation, is the primary energy source for certain bacteria, and has been detected in mammalian systems. Despite this long and intertwined history with terrestrial biology, limited attention has focused on potential roles of COS as a biological mediator. Recent Advances: Although bacterial COS production is well documented, definitive sources of mammalian COS production have not been confirmed. Enzymatic COS consumption in mammals, however, is well documented and occurs primarily by carbonic anhydrase (CA)-mediated conversion to hydrogen sulfide (H 2 S). COS has been detected in ex vivo mammalian tissue culture, as well as in exhaled breath as a potential biomarker for different disease pathologies, including cystic fibrosis and organ rejection. Recently, chemical tools for COS delivery have emerged and are poised to advance future investigations into the role of COS in different biological contexts., Critical Issues: Possible roles of COS as an important biomolecule, gasotransmitter, or sulfide transport intermediate remain to be determined. Key advances in both biological and chemical tools for COS research are needed to further investigate these questions., Future Directions: Further evaluation of the biological roles of COS and disentangling the chemical biology of COS from that of H 2 S are needed to further elucidate these interactions. Chemical tools for COS delivery and modulation may provide a first avenue of investigative tools to answer many of these questions. Antioxid. Redox Signal. 28, 1516-1532.

Hydrogen sulfide (H2S) is an important biomolecule, and controllable H2S donors are needed to investigate H2S biological functions. Here we utilize cysteine-mediated addition/cyclization chemistry to unmask an acrylate-functionalized thiocarbamate and release carbonyl sulfide (COS), which is quickly converted to H2S by carbonic anhydrase (CA).

Carbonyl sulfide (COS) releasing scaffolds are gaining popularity as hydrogen sulfide (H 2 S) donors through exploitation of the carbonic anhydrase (CA)-mediated hydrolysis of COS to H 2 S. The majority of compounds in this emerging class of donors undergo triggerable decomposition (often referred to as self-immolation) to release COS, and a handful of different COS-releasing structures have been reported. One benefit of this donation strategy is that numerous caged COS-containing core motifs are possible and are poised for development into self-immolative COS/H 2 S donors. Because the intermediate release of COS en route to H 2 S donation requires CA, it is important that the COS donor motifs do not inhibit CA directly. In this work, we investigate the cytotoxicity and CA inhibition properties of different caged COS donor cores, as well as caged CO 2 and CS 2 motifs and non-self-immolative control compounds. None of the compounds investigated exhibited significant cytotoxicity or enhanced cell proliferation at concentrations up to 100 μM in A549 cells, but we identified four core structures that function as CA inhibitors, thus providing a roadmap for the future development of self-immolative COS/H 2 S donor motifs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hydrogen sulfide (H 2 S) is an important biological mediator, and synthetic H 2 S donating molecules provide an important class of investigative tools for H 2 S research. Here, we report esterase-activated H 2 S donors that function by first releasing carbonyl sulfide (COS), which is rapidly converted to H 2 S by the ubiquitous enzyme carbonic anhydrase (CA). We report the synthesis, self-immolative decomposition, and H 2 S release profiles of the developed scaffolds. In addition, the developed esterase-triggered COS/H 2 S donors exhibit higher levels of cytotoxicity than equivalent levels of Na 2 S or the common H 2 S donors GYY4137 and AP39. Using cellular bioenergetics measurements, we establish that the developed donors reduce cellular respiration and ATP synthesis in BEAS 2B human lung epithelial cells, which is consistent with COS/H 2 S inhibition of cytochrome c oxidase in the mitochondrial respiratory chain although not observed with common H 2 S donors at the same concentrations. Taken together, these results may suggest that COS functions differently than H 2 S in certain biological contexts or that the developed donors are more efficient at delivering H 2 S than other common H 2 S-releasing motifs.

Hydrogen sulfide (H 2 S) is an important biomolecule with high therapeutic potential. Here we leverage the inverse-electron demand Diels-Alder (IEDDA) click reaction between a thiocarbamate-functionalized trans-cyclooctene and a tetrazine to deliver carbonyl sulfide (COS), which is quickly converted to H 2 S by the uniquitous enzyme carbonic anhydrase (CA), thus providing a new strategy for bio-orthogonal COS/H 2 S donation.

Hydrogen sulfide (H2S) is an important biological signaling molecule, and chemical tools for H2S delivery and detection have emerged as important investigative methods. Key challenges in these fields include developing donors that are triggered to release H2S in response to stimuli and developing probes that do not irreversibly consume H2S. Here we report a new strategy for H2S donation based on self-immolation of benzyl thiocarbamates to release carbonyl sulfide, which is rapidly converted to H2S by carbonic anhydrase. We leverage this chemistry to develop easily modifiable donors that can be triggered to release H2S. We also demonstrate that this approach can be coupled with common H2S-sensing motifs to generate scaffolds which, upon reaction with H2S, generate a fluorescence response and also release caged H2S, thus addressing challenges of analyte homeostasis in reaction-based probes.

Objectives: The objective of the following study is to evaluate the interaction between the resin materials used in immediate dentin sealing (IDS) techniques and impression materials with two different techniques to eliminate the oxygen-inhibition layer., Materials and Methods: The occlusal dentin surface of 35 human molars was exposed. The teeth were used in two Groups: Group 1 - Impression with Express XT; Group 2 - Impression with Impregum. Groups 1 and 2 were divided into 14 subgroups: Groups 1a and 2a - Control groups; 1b and 2b - IDS with Clearfil SE Bond (CSE); 1c and 2c - IDS with CSE + additional polymerization with glycerin jelly; 1d and 2d - IDS with CSE + alcohol; 1e and 2e - IDS with CSE and Protect Liner F (PLF); 1f and 2f - IDS with CSE and PLF + additional polymerization with glycerin jelly; and 1g and 2g - IDS with CSE and PLF + alcohol. Five teeth were used in each experimental group, and the tooth surface was photographed using a digital camera., Results: Small quantity of unpolymerized impression material remained attached to the CSE or to the PLF in Groups 1b and 1e. Groups 1c and 1d prevented the interaction. Small quantity of polymerized impression material remained attached to the CSE or to the PLF for Groups 2b and 2e. The same interaction was observed for Groups 2c and 2d. For Groups 2c and 2f, no interactions were observed., Conclusion: Resin materials interacted with impression materials. The application of glycerin jelly and alcohol prevented the interaction of CSE with Express XT and PLF with Impregum; however, these treatments were not completely effective in preventing the interaction of CSE with Impregum and PLF with Express XT.