Aerobic bacterial methane synthesis.

Reports of biogenic methane (CH4) synthesis associated with a range of organisms have steadily accumulated in the literature. This has not happened without controversy and in most cases the process is poorly understood at the gene and enzyme levels. In marine and freshwater environments, CH4 supersaturation of oxic surface waters has been termed the "methane paradox" because biological CH4 synthesis is viewed to be a strictly anaerobic process carried out by O2-sensitive methanogens. Interest in this phenomenon has surged within the past decade because of the importance of understanding sources and sinks of this potent greenhouse gas. In our work on Yellowstone Lake in Yellowstone National Park, we demonstrate microbiological conversion of methylamine to CH4 and isolate and characterize an Acidovorax sp. capable of this activity. Furthermore, we identify and clone a gene critical to this process (encodes pyridoxylamine phosphate-dependent aspartate aminotransferase) and demonstrate that this property can be transferred to Escherichia coli with this gene and will occur as a purified enzyme. This previously unrecognized process sheds light on environmental cycling of CH4, suggesting that O2-insensitive, ecologically relevant aerobic CH4 synthesis is likely of widespread distribution in the environment and should be considered in CH4 modeling efforts.

Arsenate-Induced Changes in Bacterial Metabolite and Lipid Pools during Phosphate Stress.

Agrobacterium tumefaciens GW4 is a heterotrophic arsenite-oxidizing bacterium with a high resistance to arsenic toxicity. It is now a model organism for studying the processes of arsenic detoxification and utilization. Previously, we demonstrated that under low-phosphate conditions, arsenate [As(V)] could enhance bacterial growth and be incorporated into biomolecules, including lipids. While the basic microbial As(V) resistance mechanisms have been characterized, global metabolic responses under low phosphate remain largely unknown. In the present work, the impacts of As(V) and low phosphate on intracellular metabolite and lipid profiles of GW4 were quantified using liquid chromatography-mass spectroscopy (LC-MS) in combination with transcriptional assays and the analysis of intracellular ATP and NADH levels. Metabolite profiling revealed that oxidative stress response pathways were altered and suggested an increase in DNA repair. Changes in metabolite levels in the tricarboxylic acid (TCA) cycle along with increased ATP are consistent with As(V)-enhanced growth of A. tumefaciens GW4. Lipidomics analysis revealed that most glycerophospholipids decreased in abundance when As(V) was available. However, several glycerolipid classes increased, an outcome that is consistent with maximizing growth via a phosphate-sparing phenotype. Differentially regulated lipids included phosphotidylcholine and lysophospholipids, which have not been previously reported in A. tumefaciens The metabolites and lipids identified in this study deepen our understanding of the interplay between phosphate and arsenate on chemical and metabolic levels.IMPORTANCE Arsenic is widespread in the environment and is one of the most ubiquitous environmental pollutants. Parodoxically, the growth of certain bacteria is enhanced by arsenic when phosphate is limited. Arsenate and phosphate are chemically similar, and this behavior is believed to represent a phosphate-sparing phenotype in which arsenate is used in place of phosphate in certain biomolecules. The research presented here uses a global approach to track metabolic changes in an environmentally relevant bacterium during exposure to arsenate when phosphate is low. Our findings are relevant for understanding the environmental fate of arsenic as well as how human-associated microbiomes respond to this common toxin.

Electronic Cigarette Refill Liquids: Nicotine Content, Presence of Child-Resistant Packaging, and in-Shop Compounding.

PURPOSE: To expand on our 2015 study of the nicotine content accuracy of e-liquids, including salts, and the presence of child-resistant packaging. We also describe compounding in shop (CIS). DESIGN AND METHODS: We analyzed samples from 35 shops. CIS processing was observed. Descriptive statistics summarized the data, and inference was performed. RESULTS: Actual nicotine content was significantly less than the identified content, on average, with a mean percent deviation 34.0% below the identified content. Only 3.8% of the samples' actual nicotine content was within 10% of the identified content; the maximum deviation was 213.2%. Of eight uniquely packaged samples, including designs resembling pop cans, ice cream cones, etc., the mean percent deviation was -39.6%; none were within 10% of the identified content. Eight shops compounded samples. After removing outlier values, significant differences were found in the percent deviations between the CIS and non-CIS free-base samples. A significantly higher percentage of CIS samples had nicotine content > 10% above the identified content, and none were within 10%. One shop visually estimated the nicotine quantities to add, e-liquids were not always relabeled to reflect new nicotine levels, and protective materials were not always worn during compounding. Child-resistant packaging was not present for one third of the samples. CONCLUSIONS: Labeling of nicotine content in e-liquids remains inaccurate, child-resistant packaging is inconsistent, and CIS is problematic. Effective e-liquid regulation is needed to protect public health. PRACTICE IMPLICATIONS: Nurses should educate families about the serious health risks of e-liquids and advocate for increased e-liquid regulations.

Quorum sensing inhibition as a promising method to control biofilm growth in metalworking fluids.

Microbial contamination in metalworking systems is a critical problem. This study determined the microbial communities in metalworking fluids (MWFs) from two machining shops and investigated the effect of quorum sensing inhibition (QSI) on biofilm growth. In both operations, biofilm-associated and planktonic microbial communities were dominated by Pseudomonadales (60.2-99.7%). Rapid recolonization was observed even after dumping spent MWFs and meticulous cleaning. Using Pseudomonas aeruginosa PAO1 as a model biofilm organism, patulin (40 µM) and furanone C-30 (75 µM) were identified as effective QSI agents. Both agents had a substantially higher efficacy compared to α-amylase (extracellular polymeric substance degrading enzyme) and reduced biofilm formation by 63% and 76%, respectively, in MWF when compared to untreated controls. Reduced production of putatively identified homoserine lactones and quinoline in MWF treated with QS inhibitors support the effect of QSI on biofilm formation. The results highlight the effectiveness of QSI as a potential strategy to eradicate biofilms in MWFs.

Neutrophil Immunomodulatory Activity of Natural Organosulfur Compounds.

Organosulfur compounds are bioactive components of garlic essential oil (EO), mustard oil, Ferula EOs, asafoetida, and other plant and food extracts. Traditionally, garlic (Allium sativum) is used to boost the immune system; however, the mechanisms involved in the putative immunomodulatory effects of garlic are unknown. We investigated the effects of garlic EO and 22 organosulfur compounds on human neutrophil responses. Garlic EO, allyl propyl disulfide, dipropyl disulfide, diallyl disulfide, and allyl isothiocyanate (AITC) directly activated Ca2+ flux in neutrophils, with the most potent being AITC. Although 1,3-dithiane did not activate neutrophil Ca2+ flux, this minor constituent of garlic EO stimulated neutrophil reactive oxygen species (ROS) production. In contrast, a close analog (1,4-dithiane) was unable to activate neutrophil ROS production. Although 1,3-dithiane-1-oxide also stimulated neutrophil ROS production, only traces of this oxidation product were generated after a 5 h treatment of HL60 cells with 1,3-dithiane. Evaluation of several phosphatidylinositol-3 kinase (PI3K) inhibitors with different subtype specificities (A-66, TGX 221, AS605240, and PI 3065) showed that the PI3K p110δ inhibitor PI 3065 was the most potent inhibitor of 1,3-dithiane-induced neutrophil ROS production. Furthermore, 1,3-dithiane enhanced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), glycogen synthase kinase 3 α/ß (GSK-3α/ß), and cAMP response element binding (CREB) protein in differentiated neutrophil-like HL60 cells. Density functional theory (DFT) calculations confirmed the reactivity of 1,3-dithiane vs. 1,4-dithiane, based on the frontier molecular orbital analysis. Our results demonstrate that certain organosulfur compounds can activate neutrophil functional activity and may serve as biological response modifiers by augmenting phagocyte functions.

Indium-Mediated Stereoselective Allylation.

Stereoselective indium-mediated organic reactions have enjoyed tremendous growth in the last 25 years. This is in part due to the insensitivity of allylindium to moisture, affording facile and practical reaction conditions coupled with outstanding functional group tolerance and minimal side reactions. Despite the plethora of articles about allylindium, there is much yet to be discovered and exploited for efficient and sustainable synthesis. In this Account, we describe indium-mediated synthetic methods for the preparation of chiral amines with the aim to present a balance of practical method development, novel asymmetric chemistry, and mechanistic understanding that impact multiple chemical and materials science disciplines. In 2005, we demonstrated the indium-mediated allylation of chiral hydrazones with complete diastereoselectivity (>99:1) and quantitative yields. Further, we revealed the first example of enantioselective indium-mediated allylation of hydrazones using catalytic (R)-3,3'-bis(trifluoromethyl)-BINOL ligands to afford homoallylic amines with high enantioselectivity. The use of enantiopure perfluoroalkylsulfonate BINOLs greatly improved the indium-mediated allylation of N-acylhydrazones with exquisite enantiocontrol (99% yield, 99% ee). This laboratory has also investigated indium-mediated asymmetric intramolecular cyclization in the presence of amino acid additives to deliver biologically relevant chromanes with excellent diastereoselectivity (dr >99:1). The effect of amino acid additives (N-Boc-glycine) was further investigated during the indium-mediated allylation of isatins with allyl bromide to yield homoallylic alcohols in excellent yields in a short time with a wide range of functional group tolerance. Critical mechanistic insight was gained, and evidence suggests that the additive plays two roles: (1) to increase the rate of formation of allylindium from allyl bromide and In(0) and (2) to increase the nucleophilicity of the allylindium reagent, probably through disruption of aggregates and coordination to the metal. We recently reported the palladium-catalyzed umpolung allylation of hydrazones with allyl acetates in the presence of indium(I) iodide (InI) with excellent diastereoselectivity (up to 99:1). The conversion was found to be inversely proportional to the phosphine concentration, providing insight into the mechanism of the critical redox transmetalation process that has implications for other Pd-catalyzed umpolung-type allylation processes. A detailed overview of the work in our lab is presented with the intention of stimulating further research interest in organoindium chemistry and its application in organic synthesis.

Electronic Cigarette Refill Liquids: Child-Resistant Packaging, Nicotine Content, and Sales to Minors.

UNLABELLED: To determine the accuracy of the labeled quantity of the nicotine content of the e-liquids sold in unlicensed vape stores, whether the packaging of e-liquids sold within the vape stores was child-resistant, whether minors were present within vape stores, and whether sales to minors occurred. This study was conducted across North Dakota prior to implementation of a new e-cigarette state law and provided a baseline assessment before enactment of the new legal requirements. DESIGN AND METHODS: We tested samples of e-liquids and performed observations in 16 stores that were selling e-cigarettes but were not legally required to be licensed for tobacco retail. The e-liquids were analyzed for nicotine content using a validated high-performance liquid chromatography method for nicotine analysis. RESULTS: Of the 70 collected e-liquid samples that claimed to contain nicotine, 17% contained more than the labeled quantity and 34% contained less than the labeled quantity by 10% or more, with one sample containing 172% more than the labeled quantity. Of the 94 e-liquid containers sampled, only 35% were determined to be child-resistant. Minors were present in stores, although no sales to minors occurred. CONCLUSIONS: Mislabeling of nicotine in e-liquids is common and exposes the user to the harmful effects of nicotine. The lack of child-resistant packaging for this potentially toxic substance is a serious public health problem. E-cigarettes should be included in the legal definition of tobacco products, child-resistant packaging and nicotine labeling laws should be enacted and strictly enforced, and vape stores should be licensed by states.

Anti-Coagulant and Antimicrobial Recombinant Heparin-Binding Major Ampullate Spidroin 2 (MaSp2) Silk Protein.

Governed by established structure-property relationships, peptide motifs comprising major ampullate spider silk confer a balance of strength and extensibility. Other biologically inspired small peptide motifs correlated to specific functionalities can be combined within these units to create designer silk materials with new hybrid properties. In this study, a small basic peptide, (ARKKAAKA) known to both bind heparin and mimic an antimicrobial peptide, was genetically linked to a protease-resistant, mechanically robust silk-like peptide, MaSp2. Purified fusion proteins (four silk domains and four heparin-binding peptide repeats) were expressed in E. coli. Successful fusion of a MaSp2 spider silk peptide with the heparin-binding motif was shown using a variety of analytical assays. The ability of the fusion peptide to bind heparin was assessed with ELISA and was further tested for its anticoagulant property using aPTT assay. Its intrinsic property to inhibit bacterial growth was evaluated using zone of inhibition and crystal violet (CV) assays. Using this strategy, we were able to link the two types of genetic motifs to create a designer silk-like protein with improved hemocompatibility and antimicrobial properties.

Coumarin-suberoylanilide hydroxamic acid as a fluorescent probe for determining binding affinities and off-rates of histone deacetylase inhibitors.

Histone deacetylases (HDACs) are intimately involved in epigenetic regulation and, thus, are one of the key therapeutic targets for cancer, and two HDAC inhibitors, namely suberoylanilide hydroxamic acid (SAHA) and romidepsin, have been recently approved for cancer treatment. Because the screening and detailed characterization of HDAC inhibitors has been time-consuming, we synthesized coumarin-SAHA (c-SAHA) as a fluorescent probe for determining the binding affinities (K(d)) and the dissociation off-rates (k(off)) of the enzyme-inhibitor complexes. The determination of the above parameters relies on the changes in the fluorescence emission intensity (λ(ex)=325 nm, λ(em)=400 nm) of c-SAHA due to its competitive binding against other HDAC inhibitors, and such determination neither requires employment of polarization accessories nor is dependent on the fluorescence energy transfer from the enzyme's tryptophan residues to the probe. Our highly sensitive and robust analytical protocol presented here is applicable to most of the HDAC isozymes, and it can be easily adopted in a high-throughput mode for screening the HDAC inhibitors as well as for quantitatively determining their K(d) and k(off) values.

Low-Temperature Biosurfactants from Polar Microbes.

Surfactants, both synthetic and natural, are used in a wide range of industrial applications, including the degradation of petroleum hydrocarbons. Organisms from extreme environments are well-adapted to the harsh conditions and represent an exciting avenue of discovery of naturally occurring biosurfactants, yet microorganisms from cold environments have been largely overlooked for their biotechnological potential as biosurfactant producers. In this study, four cold-adapted bacterial isolates from Antarctica are investigated for their ability to produce biosurfactants. Here we report on the physical properties and chemical structure of biosurfactants from the genera Janthinobacterium, Psychrobacter, and Serratia. These organisms were able to grow on diesel, motor oil, and crude oil at 4 °C. Putative identification showed the presence of sophorolipids and rhamnolipids. Emulsion index test (E24) activity ranged from 36.4-66.7%. Oil displacement tests were comparable to 0.1-1.0% sodium dodecyl sulfate (SDS) solutions. Data presented herein are the first report of organisms of the genus Janthinobacterium to produce biosurfactants and their metabolic capabilities to degrade diverse petroleum hydrocarbons. The organisms' ability to produce biosurfactants and grow on different hydrocarbons as their sole carbon and energy source at low temperatures (4 °C) makes them suitable candidates for the exploration of hydrocarbon bioremediation in low-temperature environments.

Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis.

BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.

The Role of Mass Spectrometry in Structural Studies of Flavin-Based Electron Bifurcating Enzymes.

For decades, biologists and biochemists have taken advantage of atomic resolution structural models of proteins from X-ray crystallography, nuclear magnetic resonance spectroscopy, and more recently cryo-electron microscopy. However, not all proteins relent to structural analyses using these approaches, and as the depth of knowledge increases, additional data elucidating a mechanistic understanding of protein function is desired. Flavin-based electron bifurcating enzymes, which are responsible for producing high energy compounds through the simultaneous endergonic and exergonic reduction of two intercellular electron carriers (i.e., NAD+ and ferredoxin) are one class of proteins that have challenged structural biologists and in which there is great interest to understand the mechanism behind electron gating. A limited number of X-ray crystallography projects have been successful; however, it is clear that to understand how these enzymes function, techniques that can reveal detailed in solution information about protein structure, dynamics, and interactions involved in the bifurcating reaction are needed. In this review, we cover a general set of mass spectrometry-based techniques that, combined with protein modeling, are capable of providing information on both protein structure and dynamics. Techniques discussed include surface labeling, covalent cross-linking, native mass spectrometry, and hydrogen/deuterium exchange. We cover how biophysical data can be used to validate computationally generated protein models and develop mechanistic explanations for regulation and performance of enzymes and protein complexes. Our focus will be on flavin-based electron bifurcating enzymes, but the broad applicability of the techniques will be showcased.

The inhibition, reactivation and mechanism of VX-, sarin-, fluoro-VX and fluoro-sarin surrogates following their interaction with HuAChE and HuBuChE.

In this study, the mechanisms of HuAChE and HuBChE inhibition by Me-P(O) (OPNP) (OR) [PNP = p-nitrophenyl; R = CH2CH3, CH2CH2F, OCH(CH3)2, OCH(CH3) (CH2F)] representing surrogates and fluoro-surrogates of VX and sarin were studied by in vitro kinetics and mass spectrometry. The in vitro measures showed that the VX- and fluoro-VX surrogates were relatively strong inhibitors of HuAChE and HuBChE (ki ∼ 105-106 M-1min-1) and underwent spontaneous and 2-PAM-mediated reactivation within 30 min. The sarin surrogates were weaker inhibitors of HuAChE and HuBChE (ki ∼ 104-105 M-1min-1), and in general did not undergo spontaneous reactivation, although HuAChE adducts were partially reactivatable at 18 h using 2-PAM. The mechanism of HuAChE and HuBChE inhibition by the surrogates was determined by Q-TOF and MALDI-TOF mass spectral analyses. The surrogate-adducted proteins were trypsin digested and the active site-containing peptide bearing the OP-modified serine identified by Q-TOF as triply- and quadruply-charged ions representing the respective increase in mass of the attached OP moiety. Correspondingly, monoisotopic ions of the tryptic peptides representing the mass increase of the OP-adducted peptide was identified by MALDI-TOF. The mass spectrometry analyses validated the identity of the OP moiety attached to HuAChE or HuBChE as MeP(O) (OR)-O-serine peptides (loss of the PNP leaving group) via mechanisms consistent with those found with chemical warfare agents. MALDI-TOF MS analyses of the VX-modified peptides versus time showed a steady reduction in adduct versus parent peptide (reactivation), whereas the sarin-surrogate-modified peptides remained largely intact over the course of the experiment (24 h). Overall, the presence of a fluorine atom on the surrogate modestly altered the rate constants of inhibition and reactivation, however, the mechanism of inhibition (ejection of PNP group) did not change.

Angiotensin converting enzyme versus angiotensin converting enzyme-2 selectivity of MLN-4760 and DX600 in human and murine bone marrow-derived cells.

Angiotensin-converting enzymes, ACE and ACE2, are key members of renin angiotensin system. Activation of ACE2/Ang-(1-7) pathway enhances cardiovascular protective functions of bone marrow-derived stem/progenitor cells. The current study evaluated the selectivity of ACE2 inhibitors, MLN-4760 and DX-600, and ACE and ACE2 activities in human (hu) and murine (mu) bone marrow cells. Assays were carried out in hu and mu mononuclear cells (MNCs) and huCD34(+) cells or mu-lineage-depleted (muLin(-)) cells, human-recombinant (rh) enzymes, and mu-heart with enzyme-specific substrates. ACE or ACE2 inhibition by racemic MLN-4760, its isomers MLN-4760-A and MLN-4760-B, DX600 and captopril were characterized. MLN-4760-B is relatively less efficacious and less-selective than the racemate or MLN-4760-A at hu-rhACE2, and all three of them inhibited 43% rhACE. In huMNCs, MLN-4760-B detected 63% ACE2 with 28-fold selectivity over ACE. In huCD34(+) cells, MLN-4760-B detected 38% of ACE2 activity with 63-fold selectivity. In mu-heart and muMNCs, isomer B was 100- and 228-fold selective for ACE2, respectively. In muLin(-) cells, MLN-4760-B detected 25% ACE2 activity with a pIC50 of 6.3. The racemic mixture and MLN-4760-A showed lower efficacy and poor selectivity for ACE2 in MNCs and mu-heart. ACE activity detected by captopril was 32% and 19%, respectively, in huCD34(+) and muLin(-) cells. DX600 was less efficacious, and more selective for ACE2 compared to MLN-4760-B in all samples tested. These results suggest that MLN-4760-B is a better antagonist of ACE2 than DX600 at 10 µm concentration in human and murine bone marrow cells, and that these cells express more functional ACE2 than ACE.

Highly diastereoselective palladium-catalyzed indium-mediated allylation of chiral hydrazones.

The general and efficient palladium-catalyzed indium-mediated allylation of chiral hydrazones was accomplished with excellent yield (72-92%) and diastereoselectivity (up to 99:1). The development of this reaction and the substrate scope are described. The conversion was found to be proportional to the phosphine concentration, which provided insight into the mechanism and competing pathways of the redox transmetalation process.