Investigating A Multi-Domain Polyketide Synthase in Amphidinium carterae.

Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate Amphidinium carterae as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of A. carterae multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to lack an AT domain in toxin-producing species, indicating trans-acting domains, and was shown by Western blotting to be post-transcriptionally processed. These results demonstrate how toxin synthesis in dinoflagellates can be differentiated from fat synthesis despite common underlying pathway.

Multilevel human secondary lymphoid immune system compartmentalization revealed by complementary imaging approaches.

Secondary human lymphoid tissue immune reactions take place in a highly coordinated environment with compartmentalization representing a fundamental feature of this organization. In situ profiling methodologies are indispensable for the understanding of this compartmentalization. Here, we propose a complementary experimental approach aiming to reveal different aspects of this process. The analysis of human tonsils, using a combination of single cell phenotypic analysis based on flow cytometry and multiplex imaging and mass spectrometry-based methodologies, revealed a compartmentalized organization at the cellular and molecular levels. More specifically, the skewed distribution of highly specialized immune cell subsets and relevant soluble mediators was accompanied by a compartmentalized localization of several lipids across different anatomical areas of the tonsillar tissue. The performance of such combinatorial experimental approaches could lead to the identification of novel in situ interactions and molecular targets for the in vivo manipulation of lymphoid organ, particularly the germinal center, immune reactions.

Species-Specific Endotoxin Stimulus Determines Toll-Like Receptor 4- and Caspase 11-Mediated Pathway Activation Characteristics.

The innate immune system is the body's first line of defense against pathogens and its protection against infectious diseases. On the surface of host myeloid cells, Toll-like receptor 4 (TLR4) senses lipopolysaccharide (LPS), the major outer membrane component of Gram-negative bacteria. Intracellularly, LPS is recognized by caspase 11 through the noncanonical inflammasome to induce pyroptosis-an inflammatory form of lytic cell death. While TLR4-mediated signaling perturbations result in secretion of cytokines and chemokines that help clear infection and facilitate adaptive immunity, caspase 11-mediated pyroptosis leads to the release of damage-associated molecular patterns and inflammatory mediators. Although the core signaling events and many associated proteins in the TLR4 signaling pathway are known, the complex signaling events and protein networks within the noncanonical inflammasome pathway remain obscure. Moreover, there is mounting evidence for pathogen-specific innate immune tuning. We characterized the major LPS structures from two different pathogens, modeled their binding to the surface receptors, systematically examined macrophage inflammatory responses to these LPS molecules, and surveyed the temporal differences in global protein secretion resulting from TLR4 and caspase 11 activation in macrophages using mass spectrometry (MS)-based quantitative proteomics. This integrated strategy, spanning functional activity assays, top-down structural elucidation of endotoxins, and secretome analysis of stimulated macrophages, allowed us to identify crucial differences in TLR4- and caspase 11-mediated protein secretion in response to two Gram-negative bacterial endotoxins. IMPORTANCE Macrophages and monocytes are innate immune cells playing an important role in orchestrating the initial innate immune response to bacterial infection and the tissue damage. This response is facilitated by specific receptors on the cell surface and intracellularly. One of the bacterial molecules recognized is a Gram-negative bacteria cell wall component, lipopolysaccharide (LPS). The structure of LPS differs between different species. We have characterized the innate immune responses to the LPS molecules from two bacteria, Escherichia coli and Bordetella pertussis, administered either extracellularly or intracellularly, whose structures we first determined. We observed marked differences in the temporal dynamics and amounts of proteins secreted by the innate immune cells stimulated by any of these molecules and routes. This suggests that there is specificity in the first line of response to different Gram-negative bacteria that can be explored to tailor specific therapeutic interventions.

The tertiary oxime monoisonitrosoacetone penetrates the brain, reactivates inhibited acetylcholinesterase, and reduces mortality and morbidity following lethal sarin intoxication in guinea pigs.

The brain is a critical target for the toxic action of organophosphorus (OP) inhibitors of acetylcholinesterase (AChE) such as the nerve agent sarin. However, the available oxime antidote 2-PAM only reactivates OP-inhibited AChE in peripheral tissues. Monoisonitrosoacetone (MINA), a tertiary oxime, reportedly reactivates AChE in the central nervous system (CNS). The current study investigated whether MINA would be beneficial as a supplemental oxime treatment in preventing lethality and reducing morbidity following lethal sarin exposure, MINA supplement would improve AChE recovery in the body, and MINA would be detectable in the CNS. Guinea pigs were exposed to sarin and treated with atropine sulfate and 2-PAM at one minute. Additional 2-PAM or MINA was administered at 3, 5, 15, or 30 min after sarin exposure. Survival and morbidity were assessed at 2 and 24 h. AChE activity in brain and peripheral tissues was evaluated one hour after MINA and 2-PAM treatment. An in vivo microdialysis technique was used to determine partitioning of MINA into the brain. A liquid chromatography-tandem mass spectrometry method was developed for the analysis of MINA in microdialysates. MINA-treated animals exhibited significantly higher survival and lower morbidity compared to 2-PAM-treated animals. 2-PAM was significantly more effective in reactivating AChE in peripheral tissues, but only MINA reactivated AChE in the CNS. MINA was found in guinea pig brain microdialysate samples beginning at ~10 min after administration in a dose-related manner. The data strongly suggest that a centrally penetrating oxime could provide significant benefit as an adjunct to atropine and 2-PAM therapy for OP intoxication.

The Role of TLRs in Anti-cancer Immunity and Tumor Rejection.

In recent years, a lot of scientific interest has focused on cancer immunotherapy. Although chronic inflammation has been described as one of the hallmarks of cancer, acute inflammation can actually trigger the immune system to fight diseases, including cancer. Toll-like receptor (TLR) ligands have long been used as adjuvants for traditional vaccines and it seems they may also play a role enhancing efficiency of tumor immunotherapy. The aim of this perspective is to discuss the effects of TLR stimulation in cancer, expression of various TLRs in different types of tumors, and finally the role of TLRs in anti-cancer immunity and tumor rejection.

Multi-Omics Strategies Uncover Host-Pathogen Interactions.

With the success of the Human Genome Project, large-scale systemic projects became a reality that enabled rapid development of the systems biology field. Systems biology approaches to host-pathogen interactions have been instrumental in the discovery of some specifics of Gram-negative bacterial recognition, host signal transduction, and immune tolerance. However, further research, particularly using multi-omics approaches, is essential to untangle the genetic, immunologic, (post)transcriptional, (post)translational, and metabolic mechanisms underlying progression from infection to clearance of microbes. The key to understanding host-pathogen interactions lies in acquiring, analyzing, and modeling multimodal data obtained through integrative multi-omics experiments. In this article, we will discuss how multi-omics analyses are adding to our understanding of the molecular basis of host-pathogen interactions and systemic maladaptive immune response of the host to microbes and microbial products.

Pathogen Identification Direct From Polymicrobial Specimens Using Membrane Glycolipids.

With the increased prevalence of multidrug-resistant Gram-negative bacteria, the use of colistin and other last-line antimicrobials is being revisited clinically. As a result, there has been an emergence of colistin-resistant bacterial species, including Acinetobacter baumannii and Klebsiella pneumoniae. The rapid identification of such pathogens is vitally important for the effective treatment of patients. We previously demonstrated that mass spectrometry of bacterial glycolipids has the capacity to identify and detect colistin resistance in a variety of bacterial species. In this study, we present a machine learning paradigm that is capable of identifying A. baumannii, K. pneumoniae and their colistin-resistant forms using a manually curated dataset of lipid mass spectra from 48 additional Gram-positive and -negative organisms. We demonstrate that these classifiers detect A. baumannii and K. pneumoniae in isolate and polymicrobial specimens, establishing a framework to translate glycolipid mass spectra into pathogen identifications.

Rapid Food Product Analysis by Surface Acoustic Wave Nebulization Coupled Mass Spectrometry.

Rapid food product analysis is of great interest for quality control and assurance during the production process. Conventional quality control protocols require time and labor intensive sample preparation for analysis by state-of-the-art analytical methods. To reduce overall cost and facilitate rapid qualitative assessments, food products need to be tested with minimal sample preparation. We present a novel and simple method for assessing food product compositions by mass spectrometry using a novel surface acoustic wave nebulization method. This method provides significant advantages over conventional methods requiring no pumps, capillaries, or additional chemicals to enhance ionization for mass spectrometric analysis. In addition, the surface acoustic wave nebulization - mass spectrometry method is ideal for rapid analysis and to investigate certain compounds by using the mass spectra as a type of species-specific fingerprint analysis. We present for the first time surface acoustic wave nebulization generated mass spectra of a variety of fermented food products from a small selection of vinegars, wines, and beers.

Dataset describing the development, optimization and application of SRM/MRM based targeted proteomics strategy for quantification of potential biomarkers of EGFR TKI sensitivity.

The data presented here describes the use of targeted proteomic assays to quantify potential biomarkers of Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) sensitivity in lung adenocarcinoma and is related to the research article: "Quantitative targeted proteomic analysis of potential markers of tyrosine kinase inhibitor (TKI) sensitivity in EGFR mutated lung adenocarcinoma" [1]. This article describes the data associated with liquid chromatography coupled to multiple reaction monitoring (LC-MRM) method development which includes selection of an optimal transition list, retention time prediction and building of reverse calibration curves. Sample preparation and optimization which includes phosphotyrosine peptide enrichment via a combination of pan-phosphotyrosine antibodies is described. The dataset also consists of figures, tables and Excel files describing the quantitative results of testing these optimized methods in two lung adenocarcinoma cell lines with EGFR mutations.

Correction to: Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate.

In the preceding article "Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate" by Oyler et al., an error in the J5 E. coli LPS chemical structure (Figs. 2 and 4) was introduced and propagated into the final revision.

Quantitative targeted proteomic analysis of potential markers of tyrosine kinase inhibitor (TKI) sensitivity in EGFR mutated lung adenocarcinoma.

Lung cancer causes the highest mortality among all cancers. Patients harboring kinase domain mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors (TKIs), however, acquired resistance always develops. Moreover, 30-40% of patients with EGFR mutations exhibit primary resistance. Hence, there is an unmet need for additional biomarkers of TKI sensitivity that complement EGFR mutation testing and predict treatment response. We previously identified phosphopeptides whose phosphorylation is inhibited upon treatment with EGFR TKIs, erlotinib and afatinib in TKI sensitive cells, but not in resistant cells. These phosphosites are potential biomarkers of TKI sensitivity. Here, we sought to develop modified immuno-multiple reaction monitoring (immuno-MRM)-based quantitation assays for select phosphosites including EGFR-pY1197, pY1172, pY998, AHNAK-pY160, pY715, DAPP1-pY139, CAV1-pY14, INPPL1-pY1135, NEDD9-pY164, NF1-pY2579, and STAT5A-pY694. These sites were significantly hypophosphorylated by erlotinib and a 3rd generation EGFR TKI, osimertinib, in TKI-sensitive H3255 cells, which harbor the TKI-sensitizing EGFRL858R mutation. However, in H1975 cells, which harbor the TKI-resistant EGFRL858R/T790M mutant, osimertinib, but not erlotinib, could significantly inhibit phosphorylation of EGFR-pY-1197, STAT5A-pY694 and CAV1-pY14, suggesting these sites also predict response in TKI-resistant cells. We could further validate EGFR-pY-1197 as a biomarker of TKI sensitivity by developing a calibration curve-based modified immuno-MRM assay. SIGNIFICANCE: In this report, we have shown the development and optimization of MRM assays coupled with global phosphotyrosine enrichment (modified immuno-MRM) for a list of 11 phosphotyrosine peptides. Our optimized assays identified the targets reproducibly in biological samples with good selectivity. We also developed and characterized quantitation methods to determine endogenous abundance of these targets and correlated the results of the relative quantification with amounts estimated from the calibration curves. This approach represents a way to validate and verify biomarker candidates discovered from large-scale global phospho-proteomics analysis. The application of these modified immuno-MRM assays in lung adenocarcinoma cells provides proof-of concept for the feasibility of clinical applications. These assays may be used in prospective clinical studies of EGFR TKI treatment of EGFR mutant lung cancer to correlate treatment response and other clinical endpoints.

Top Down Tandem Mass Spectrometric Analysis of a Chemically Modified Rough-Type Lipopolysaccharide Vaccine Candidate.

Recent advances in lipopolysaccharide (LPS) biology have led to its use in drug discovery pipelines, including vaccine and vaccine adjuvant discovery. Desirable characteristics for LPS vaccine candidates include both the ability to produce a specific antibody titer in patients and a minimal host inflammatory response directed by the innate immune system. However, in-depth chemical characterization of most LPS extracts has not been performed; hence, biological activities of these extracts are unpredictable. Additionally, the most widely adopted workflow for LPS structure elucidation includes nonspecific chemical decomposition steps before analyses, making structures inferred and not necessarily biologically relevant. In this work, several different mass spectrometry workflows that have not been previously explored were employed to show proof-of-principle for top down LPS primary structure elucidation, specifically for a rough-type mutant (J5) E. coli-derived LPS component of a vaccine candidate. First, ion mobility filtered precursor ions were subjected to collision induced dissociation (CID) to define differences in native J5 LPS v. chemically detoxified J5 LPS (dLPS). Next, ultra-high mass resolving power, accurate mass spectrometry was employed for unequivocal precursor and product ion empirical formulae generation. Finally, MS3 analyses in an ion trap instrument showed that previous knowledge about dissociation of LPS components can be used to reconstruct and sequence LPS in a top down fashion. A structural rationale is also explained for differential inflammatory dose-response curves, in vitro, when HEK-Blue hTLR4 cells were administered increasing concentrations of native J5 LPS v. dLPS, which will be useful in future drug discovery efforts. Graphical Abstract ᅟ.

Sponge symbioses between Xestospongia deweerdtae and Plakortis spp. are not motivated by shared chemical defense against predators.

The recently described epizoic sponge-sponge symbioses between Xestospongia deweerdtae and two species of Plakortis present an unusual series of sponge interactions. Sponges from the genus Plakortis are fierce allelopathic competitors, rich in cytotoxic secondary metabolites, and yet X. deweerdtae flourishes as an epizoic encrustation on Plakortis deweerdtaephila and Plakortis symbiotica. Our objective in this study was to evaluate the hypothesis that X. deweerdtae grows epizoic to these two species of Plakortis due to a shared chemical defense against predators. We collected free-living individuals of X. deweerdtae and symbiotic pairs from a wide geographical range to generate crude organic extracts and a series of polarity fractions from sponge extract. We tested the deterrency of these extracts against three common coral reef predators: the bluehead wrasse, Thalassoma bifasciatum, the Caribbean sharpnose puffer, Canthigaster rostrata, and the white spotwrist hermit crab, Pagurus criniticornis. While the chemical defenses of P. deweerdtaephila and P. symbiotica are more potent than those of X. deweerdtae, all of the sponge species we tested significantly deterred feeding in all three generalist predators. The free-living form of X. deweerdtae is mostly defended across the region, with a few exceptions. The associated form of X. deweerdtae is always defended, and both species of Plakortis are very strongly defended, with puffers refusing to consume extract-treated pellets until the extract was diluted to 1/256× concentration. Using diode-array high performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry (LC-MS/IT-TOF), we found two secondary metabolites from P. deweerdtaephila, probably the cyclic endoperoxides plakinic acid I and plakinic acid K, in low concentrations in the associated-but not the free-living-form of X. deweerdtae, suggesting a possible translocation of defensive chemicals from the basibiont to the epibiont. Comparing the immense deterrency of Plakortis spp. extracts to the extracts of X. deweerdtae gives the impression that there may be some sharing of chemical defenses: one partner in the symbiosis is clearly more defended than the other and a small amount of its defensive chemistry may translocate to the partner. However, X. deweerdtae effectively deters predators with its own defensive chemistry. Multiple lines of evidence provide no support for the shared chemical defense hypothesis. Given the diversity of other potential food resources available to predators on coral reefs, it is improbable that the evolution of these specialized sponge-sponge symbioses has been driven by predation pressure.

Lipid A structural modifications in extreme conditions and identification of unique modifying enzymes to define the Toll-like receptor 4 structure-activity relationship.

Strategies utilizing Toll-like receptor 4 (TLR4) agonists for treatment of cancer, infectious diseases, and other targets report promising results. Potent TLR4 antagonists are also gaining attention as therapeutic leads. Though some principles for TLR4 modulation by lipid A have been described, a thorough understanding of the structure-activity relationship (SAR) is lacking. Only through a complete definition of lipid A-TLR4 SAR is it possible to predict TLR4 signaling effects of discrete lipid A structures, rendering them more pharmacologically relevant. A limited 'toolbox' of lipid A-modifying enzymes has been defined and is largely composed of enzymes from mesophile human and zoonotic pathogens. Expansion of this 'toolbox' will result from extending the search into lipid A biosynthesis and modification by bacteria living at the extremes. Here, we review the fundamentals of lipid A structure, advances in lipid A uses in TLR4 modulation, and the search for novel lipid A-modifying systems in extremophile bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Detection of Carbofuran-Protein Adducts in Serum of Occupationally Exposed Pesticide Factory Workers in Pakistan.

This study was conducted to investigate the protein adducts with pesticides in a cohort of 172 factory workers that were exposed to a mixture of pesticides. The 35 samples showing considerable variation in biochemical parameters, i.e., butyrylcholinestrase (BChE), serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), gamma-glutamyl transferase (GGT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP/ALKP), lactate dehydrogenase (LDH), creatine phosphokinase (CPK) enzymes, and controls were analyzed by reversed-phase nanoscale liquid chromatography tandem mass spectrometry (nLC-MS/MS) on an Orbitrap mass spectrometer employing a shotgun proteomics approach. Only protein adducts with carbofuran were found on serum proteins of these workers. These adducts were of carbofuran labeled lysine (Lys-142, Lys-183, Lys-287, and Lys-467), arginine (Arg-210, Arg-242, and Arg-256) from serum albumin, and serine (Ser-07, Ser-54, and Ser-150) from immunoglobulin proteins. The arginine residues (Arg-210, Arg-242, Arg-246, and Arg-434) from albumin were also found to be glycated in serum of workers showing a high level of glucose who also had glycated arginine (Arg-1120) modified with carbofuran in their tankyrase-1-binding protein. The number of tandem mass spectra of modified peptides increased with increasing time of exposure. This is the first report to demonstrate the presence of carbofuran-labeled albumin, immunoglobulin, and glycated arginine, which shows that lysine and arginine of human albumin and serine of immunoglobulin are covalently modified in the serum of workers that were occupationally exposed to carbofuran, and the modification is detectable by tandem mass spectrometry. These peptides modified with carbofuran can potentially be used as a biomarker of carbofuran exposure.

Rapid lipid a structure determination via surface acoustic wave nebulization and hierarchical tandem mass spectrometry algorithm.

RATIONALE: Surface acoustic wave nebulization (SAWN) is an easy to use sample transfer method for rapid mass spectrometric analysis. A new standing wave (SW) SAWN chip, with higher ionization efficiency than our previously reported design, is used for rapid analysis of lipids. METHODS: The crude, yet fast, Caroff protocol was used for lipid A extraction from Francisella novicida. SW-SAWN with a Waters Synapt G2S quadrupole time-of-flight (QTOF) mass spectrometer was used to generate lipid A ions. Quadrupole collision-induced dissociation (Q-CID) of lipid A at varying CID energies was used to approximate the ion trap MSn data required for our hierarchical tandem mass spectrometry (HiTMS) algorithm. Structural hypotheses can be obtained directly from the HiTMS algorithm to identify species-specific lipid A molecules. RESULTS: SW-SAWN successfully generated ions from lipid A extracted from Francisella novicida using the faster Caroff method. In addition, varying collision energies were used to generate tandem mass spectra similar to MS3 and MS4 spectra from an ion trap. The Q-CID spectra are compatible with our HiTMS algorithm and offer an improvement over lipid A tandem mass spectra acquired in an ion trap. CONCLUSIONS: Combining SW-SAWN and Q-CID enabled more structural assignments than previously reported in half the time. The ease of generating spectra by SAWN tandem MS in combination with HiTMS interpretation offers high-throughput lipid A structural analysis and thereby rapid detection of pathogens based on lipid fingerprinting. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation and computational characterization of the facilitated transport of Glc carbon C-1 oxime reactivators across a blood brain barrier model.

We are evaluating a facilitative transport strategy to move oximes across the blood brain barrier (BBB) to reactivate inhibited brain acetylcholinesterase (AChE). We selected glucose (Glc) transporters (GLUT) for this purpose as these transporters are highly represented in the BBB. Glc conjugates have successfully moved drugs across the BBB and previous work has shown that Glc-oximes (sugar-oximes, SOxs) can reduce the organophosphonate induced hypothermia response. We previously evaluated the reactivation potential of Glc carbon C-1 SOxs. Here we report the reactivation parameters for VX- and GB-inhibited human (Hu) AChE of the best SOx (13c) and our findings that the kinetics are similar to those of the parent oxime. Although crystals of Torpedo californica AChE were produced, neither soaked or co-crystallized experiments were successful at concentrations below 20mM 13c, and higher concentrations cracked the crystals. 13c was non-toxic to neuroblastoma and kidney cell lines at 12-18 mM, allowing high concentrations to be used in a BBB kidney cell model. The transfer of 13c from the donor side was asymmetric with the greatest loss of 13c from the apical- or luminal-treated side. There was no apparent transfer from the basolateral side. The 13cP(app) results indicate a 'low' transport efficiency; however, mass accounting revealed only a 20% recovery from the apical dose in which high concentrations were found in the cell lysate fraction. Molecular modeling of 13c through the GLUT-1 channel demonstrated that transport of 13c was more restricted than Glc. Selected sites were compared and the 13c binding energies were greater than two times those of Glc.