Step-by-Step Approach to Build Multiple Reaction Monitoring (MRM) Profiling Instrument Acquisition Methods for Class-based Lipid Exploratory Analysis by Mass Spectrometry.

Multiple reaction monitoring (MRM) profiling is a strategy for the exploratory analysis of small molecules and lipids by direct sample injection, ie, without the use of chromatographic separation. It is based on instrument methods that comprise a list of ion transitions (MRMs), in which the precursor ion is the expected ionized m/z of the lipid at its species level, ie, the description of lipid class and number of carbon and double bonds in the fatty acid chain(s), and the product ion is a fragment expected for the lipid class or for the fatty acid neutral loss. The Lipid Maps database is expanding constantly, and therefore the MRM-profiling methods associated with this database need to be continuously updated. Here, we provide a comprehensive overview and the key references for the MRM-profiling methodology and workflow, followed by a step-by-step approach to build MRM-profiling instrument acquisition methods for class-based lipid exploratory analysis based on the Lipid Maps database. The detailed workflow includes (1) importing the list of lipids from the database; (2) for a given class, combining isomeric lipids described at full structural level into 1 entry to obtain the neutral mass at species level; (3) attributing the standard Lipid Maps abbreviated nomenclature for the lipid at its species level; (4) predicting the ionized precursor ions; and (5) adding the expected product ion. We also describe how to simulate the precursor ion for the suspect screening of modified lipids using lipid oxidation and their expected product ions as an example. After determining the MRMs, information about collision energy, dwell time, and other instrument parameters are added to finalize the acquisition method. As an example of final method output, we describe the format for Agilent MassHunter v.B.06 and provide the parameters in which optimization can be performed by lipid class using one or more lipid standards.

Suspect screening of exogenous compounds using multiple reaction screening (MRM) profiling in human urine samples.

Thousands of chemical compounds produced by industry are dispersed in the human environment widely enough to reach the world population, and the introduction of new chemicals constantly occurs. As new synthetic molecules emerge, rapid analytical workflows for screening possible presence of exogenous compounds in biofluids can be useful as a first pass analysis to detect chemical exposure and guide the development and application of more elaborate LC-MS/MS methods for quantification. In this study, a suspect screening workflow using the multiple reaction monitoring (MRM) profiling method is proposed as a first pass exploratory technique to survey selected exogenous molecules in human urine samples. The workflow was applied to investigate 12 human urine samples using 310 MRMs related to the chemical functionalities of 87 exogenous compounds present in the METLIN database and reported in the literature. A total of 11 MRMs associated with five different compounds were detected in the samples. Product ion scans for the precursor ions of the selected MRMs were acquired as a further identification step for these chemicals. The suspect screening results suggested the presence of five exogenous compounds in the human urine samples analyzed, namely metformin, metoprolol, acetaminophen, paraxanthine and acrylamide. LC-MS/MS was applied as a last step to confirm these results, and the presence of four out of the five targets selected by MRM profiling were corroborated, indicating that this workflow can support the selection of suspect compounds to screen complex samples and guide more time-consuming and specific quantification analyses.

Shedding Light on the Dynamic Role of the "Target of Rapamycin" Kinase in the Fast-Growing C4 Species Setaria viridis, a Suitable Model for Biomass Crops.

The Target of Rapamycin (TOR) kinase pathway integrates energy and nutrient availability into metabolism promoting growth in eukaryotes. The overall higher efficiency on nutrient use translated into faster growth rates in C4 grass plants led to the investigation of differential transcriptional and metabolic responses to short-term chemical TOR complex (TORC) suppression in the model Setaria viridis. In addition to previously described responses to TORC inhibition (i.e., general growth arrest, translational repression, and primary metabolism reprogramming) in Arabidopsis thaliana (C3), the magnitude of changes was smaller in S. viridis, particularly regarding nutrient use efficiency and C allocation and partitioning that promote biosynthetic growth. Besides photosynthetic differences, S. viridis and A. thaliana present several specificities that classify them into distinct lineages, which also contribute to the observed alterations mediated by TOR. Indeed, cell wall metabolism seems to be distinctly regulated according to each cell wall type, as synthesis of non-pectic polysaccharides were affected in S. viridis, whilst assembly and structure in A. thaliana. Our results indicate that the metabolic network needed to achieve faster growth seems to be less stringently controlled by TORC in S. viridis.

A novel experimental workflow to determine the impact of storage parameters on the mass spectrometric profiling and assessment of representative phosphatidylethanolamine lipids in mouse tissues.

Evaluation of signaling lipids is essential for measuring biological processes. There is a lack of experimental data regarding the proper storage of extracts for signaling lipid analysis, potentially impacting the procedures that can lead to accurate and reproducible evaluation. In this study, the importance of pre-analytical conditions for analyzing ion transitions for phosphatidylethanolamines (PEs), an abundant signaling phospholipid, was systematically assessed. A novel workflow was utilized involving an MRM-based experimental approach followed by statistical analysis. Specifically, lipids were extracted from the brain, heart, lungs, and serum of C57BL/6 mice. Extract subsets were resuspended in organic solvents prior to storage in various temperature conditions. Mass spectrometry analysis by multiple reaction monitoring (MRM) profiling was performed at four time points (1 day, 2 weeks, 2 months, or 6 months) to measure relative amounts of PEs in distinct lipid extract aliquots. We introduce an innovative statistical workflow to measure the changes in relative amounts of PEs in the profiles over time to determine lipid extract storage conditions in which fewer profile changes occur. Results demonstrated that time is the most significant factor affecting the changes in lipid samples, with temperature and solvent having comparatively minor effects. We conclude that for lipid extracts obtained by Bligh & Dyer extraction, storage at - 80.0 °C without solvent for less than 2 weeks before analysis is ideal. By considering the data generated by this study, lipid extract storage practices may be optimized and standardized, enhancing the validity and reproducibility of lipid assessments.

Elucidating mechanisms involved in flavor generation of dry-aged beef loins using metabolomics approach.

The present study was conducted to identify flavor-related chemical compounds and to elucidate beef flavor development in response to dry-aging. Paired grass-fed beef loins (n = 18) were obtained at 7 d postmortem, cut into two sections and assigned to 3 aging methods: conventional dry-aging (DA), vacuum packaged wet-aging (WA) and dry-aging in a bag (DW) for 28 days. Following aging, samples were analyzed for UPLC-MS metabolomics, volatile, fatty acid profiling, and consumer sensory comment analysis. Greater number of proteins and nucleotides derived metabolites were liberated in dry-aged samples compared to WA (P < 0.05). In particular, the liberation of gammaglutmayl peptides and glutamine metabolites through the glutathione metabolism were identified. While fatty acid profile was not affected by treatments (P > 0.05), higher concentrations of volatile compounds were found in the dry-aged (P < 0.05). Dry-aging process decreased the presence of terpenoid and steroid lipid group, which could possibly result in reducing undesirable flavor of grass-fed beef.

Multiple reaction monitoring profiling as an analytical strategy to investigate lipids in extracellular vesicles.

Extracellular vesicles (EVs) convey information used in cell-to-cell interactions. Lipid analysis of EVs remains challenging because of small sample amounts available. Lipid discovery using traditional mass spectrometry platforms based on liquid chromatography and high mass resolution typically employs milligram sample amounts. We report a simple workflow for lipid profiling of EVs based on multiple reaction monitoring (MRM) profiling that uses microgram amounts of sample. After liquid-liquid extraction, individual EV samples were injected directly into the electrospray ionization (ESI) ion source at low flow rates (10 µl/min) and screened for 197 MRM transitions chosen to be a characteristic of several classes of lipids. This choice was based on a discovery experiment, which applied 1,419 MRMs associated with multiple lipid classes to a representative pooled sample. EVs isolated from 12 samples of human lymphocytes and 16 replicates from six different rat cells lines contained an estimated amount of total lipids of 326 to 805 µg. Samples showed profiles that included phosphatidylcholine (PC), sphingomyelin (SM), cholesteryl ester (CE), and ceramide (Cer) lipids, as well as acylcarnitines. The lipid profiles of human lymphocyte EVs were distinguishable using principal component and cluster analysis in terms of prior antibody and drug exposure. Lipid profiles of rat cell lines EV's were distinguishable by their tissue of origin.

Aldol Reactions of Biorenewable Triacetic Acid Lactone Precursor Evaluated Using Desorption Electrospray Ionization Mass Spectrometry High-Throughput Experimentation and Validated by Continuous Flow Synthesis.

Desorption electrospray ionization-mass spectrometry (DESI-MS) was used as a high-throughput experimentation (HTE) tool to rapidly identify derivatives of the biobased platform molecule triacetic acid lactone (TAL). TAL is a platform molecule capable of conversion to a wide range of useful commodity chemicals, agrochemicals, and advanced pharmaceutical intermediates. In the present study, a diverse family of aldol reaction mixtures were prepared in high-density microtiter plates with a liquid handling robot, then printed with a pin tool onto a PTFE surface for analysis by DESI-MS. Our DESI-MS results indicate that aldol products of TAL were obtained for each substrate tested, in good agreement with previously reported TAL reactivity. These HTE experiments also revealed solvent-dependent reactivity trends that facilitated reaction scale up. Our findings suggest that DESI-MS analysis can rapidly inform the selection of optimal reaction conditions from a wide variety of conditions for scale up using continuous synthesis conditions.

High-Throughput Experimentation and Continuous Flow Evaluation of Nucleophilic Aromatic Substitution Reactions.

Nucleophilic aromatic substitution (SNAr) reactions were optimized using high-throughput experimentation techniques for execution under flow conditions. A total of 3072 unique reactions were evaluated with an analysis time of ∼3.5 s per reaction using a system that combines a liquid handling robot for reaction mixture preparation with desorption electrospray ionization (DESI) mass spectrometry (MS) for analysis. The reactions were performed in bulk microtiter arrays with and without incubation. In-house developed software was used to process the data and generate heat maps of the results. This information was then used to select the most promising conditions for continuous synthesis under microfluidic reactor conditions. Our results show that this HTE approach provides robust guidance for narrowing the range of conditions needed for optimization of SNAr reactions.

High Throughput Experimentation Using DESI-MS to Guide Continuous-Flow Synthesis.

We demonstrate the use of accelerated reactions with desorption electrospray ionization mass spectrometry (DESI-MS) as a tool for predicting the outcome of microfluidic reactions. DESI-MS was employed as a high throughput experimentation tool to provide qualitative predictions of reaction outcomes, so that vast regions of chemical reactivity space may be more rapidly explored and areas of optimal efficiency identified. This work is part of a larger effort to accelerate reaction optimization to enable the rapid development of continuous-flow syntheses of small molecules in high yield. In order to build confidence in this approach, however, it is necessary to establish a robust predictive connection between reactions performed under analogous DESI-MS, batch, and microfluidic reaction conditions. In the present work, we explore the potential of high throughput DESI-MS experiments to identify trends in reactivity based on chemical structure, solvent, temperature, and stoichiometry that are consistent across these platforms. N-alkylation reactions were used as the test case due to their ease of reactant and product detection by electrospray ionization mass spectrometry (ESI-MS) and their great importance in API synthesis. While DESI-MS narrowed the scope of possibilities for reaction selection among some parameters such as solvent, others like stoichiometry and temperature still required further optimization under continuous synthesis conditions. DESI-MS high throughput experimentation (HTE) reaction evaluation significantly reduced the search space for flow chemistry optimization, thus representing a significant savings in time and materials to achieve a desired transformation with high efficiency.

Lipidome profiles of postnatal day 2 vaginal swabs reflect fat composition of gilt's postnatal diet.

We hypothesized that postnatal development of the vagina is impacted by early nutritional environment. Our objective was to determine if lipid profiles of vaginal swabs were different between postnatal gilts suckled by sow or fed milk replacer the first 48 h after birth, with or without a lard-based fat supplement. Gilts (>1.3 kg) were selected at birth across 8 litters and assigned to one of four treatments: 1) suckled by sow (S, n = 8); 2) suckled by sow plus administration of a fat supplement (SF, n = 5); 3) bottle-fed solely milk replacer (B, n = 8); or 4) bottle-fed solely milk replacer plus administration of a fat supplement (BF, n = 7). At 48 h postnatal, vaginal swabs of gilts were taken with a cytology brush, and lipids were extracted for analysis using multiple reaction monitoring (MRM)-profiling. Lipids extracted from serum collected at 48 h from gilts, milk collected at 24 h from sows, and milk replacer were also analyzed with MRM-profiling. Receiver operating characteristic curve analysis found 18 lipids recovered from vaginal swabs that highly distinguished between S and B gilts [area-under-the-curve (AUC) > 0.9], including phosphatidylethanolamine with 34 carbons and four unsaturations in the fatty acyl residues [PE (34:4)]. Twelve lipids from vaginal swabs highly correlated (r > 0.6; p < 0.01) with nutrition source. Lipids with greater abundance in milk replacer drove association. For example, mean intensity of PE (34:4) was 149-fold higher in milk replacer than colostrum. Consequently, PE (34:4) was found to have 1.6- and 2.12-fold higher levels in serum and vaginal swab samples (p < 0.001), respectively, of B gilts as compared to S gilts. Findings support that vaginal swabs can be used to noninvasively study effects of perinatal nutrition on tissue composition.

Multiple Reaction Monitoring Profiling (MRM-Profiling) of Lipids To Distinguish Strain-Level Differences in Microbial Resistance in Escherichia coli.

The worldwide increase in antimicrobial resistance is due to antibiotic overuse in agriculture and overprescription in medicine. For appropriate and timely patient support, faster diagnosis of antimicrobial resistance is required. Current methods for bacterial identification rely on genomics and proteomics and use comparisons with databases of known strains, but the diagnostic value of metabolites and lipids has not been explored significantly. Standard mass spectrometry/chromatography methods involve multiple dilutions during sample preparation and separation. To increase the amount of chemical information acquired and the speed of analysis of lipids, multiple reaction monitoring profiling (MRM-Profiling) has been applied. The MRM-Profiling workflow includes a discovery stage and a screening stage. The discovery stage employs precursor (PREC) ion and neutral loss (NL) scans to screen representative pooled samples for functional groups associated with particular lipid classes. The information from the first stage is organized in precursor/product ion pairs, or MRMs, and the screening stage rapidly interrogates individual samples for these MRMs. In this study, we performed MRM-Profiling of lipid extracts from four different strains of Escherichia coli cultured with amoxicillin or amoxicillin/clavulanate, a ß-lactam and ß-lactamase inhibitor, respectively. t tests, analysis of variance and receiver operating characteristic (ROC) curves were used to determine the significance of each MRM. Principal component analysis was applied to distinguish different strains cultured under conditions that allowed or disallowed development of bacterial resistance. The results demonstrate that MRM-Profiling distinguishes the lipid profiles of resistant and nonresistant E. coli strains.

Comparison of the RNA Content of Extracellular Vesicles Derived from Paracoccidioidesbrasiliensis and Paracoccidioides lutzii.

Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis. We have previously characterized the <200-nt RNA sub-populations contained in fungal extracellular vesicles (EVs) from P. brasiliensis Pb18 and other pathogenic fungi. We have presently used the RNA-seq strategy to compare the <200- and >200-nt RNA fractions contained in EVs isolated from culture supernatants of P. brasiliensis Pb18, Pb3, and P. lutzii Pb01. Shared mRNA sequences were related to protein modification, translation, and DNA metabolism/biogenesis, while those related to transport and oxidation-reduction were exclusive to Pb01. The presence of functional full-length mRNAs was validated by in vitro translation. Among small non-coding (nc)RNA, 15 were common to all samples; small nucleolar (sno)RNAs were enriched in P. brasiliensis EVs, whereas for P. lutzii there were similar proportions of snoRNA, rRNA, and tRNA. Putative exonic sRNAs were highly abundant in Pb18 EVs. We also found sRNA sequences bearing incomplete microRNA structures mapping to exons. RNA-seq data suggest that extracellular fractions containing Pb18 EVs can modulate the transcriptome of murine monocyte-derived dendritic cells in a transwell system. Considering that sRNA classes are involved in transcription/translation modulation, our general results may indicate that differences in virulence among fungal isolates can be related to their distinct EV-RNA content.

An Integrative Proteomic/Lipidomic Analysis of the Gold Nanoparticle Biocorona in Healthy and Obese Conditions.

Introduction: When nanoparticles (NPs) enter a physiological environment, a coating of biomolecules or biocorona (BC) forms on the surface. Formation of the NP-BC is dependent on NP properties, the physiological environment, and time. The BC influences NP properties and biological interactions such as cellular internalization, immune responses, biodistribution, and others, leading to pharmacological and toxicological consequences. To date, examination of the NP-BC has focused primarily on protein components and healthy conditions. Therefore, we evaluated the protein and lipid content of BCs that formed on physicochemically distinct gold nanoparticles (AuNPs) under healthy and obese conditions. A comprehensive understanding of the NP-BC is necessary for the translation of in vitro toxicity assessments to clinical applications. Materials and Methods: AuNPs with two coatings (poly-N-vinylpyrrolidone [PVP] or citrate) and diameters (20 or 100 nm) were incubated in pooled human serum, and an integrated proteomic/lipidomic approach was used to evaluate BC composition. Macrophages were utilized to evaluate differential immune responses due to variations in the AuNP-BC. Results: AuNPs form distinct BCs based on physicochemical properties and the surrounding environment, with the obese BC containing more proteins and fewer lipids than the healthy BC. Differential macrophage inflammatory responses were observed based on AuNP properties and BC composition. Discussion and Conclusion: Overall, these findings demonstrate that AuNP size and coating, as well as physiological environment, influence the protein and lipid composition of the BC, which impacts cellular responses following exposure. These findings demonstrate that incorporation of BCs representing distinct physiological conditions may enhance the translatability of nanosafety in vitro studies.

Comparison of the RNA Content of Extracellular Vesicles Derived from Paracoccidioides brasiliensis and Paracoccidioides lutzii

Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis. We have previously characterized the <200-nt RNA sub-populations contained in fungal extracellular vesicles (EVs) from P. brasiliensis Pb18 and other pathogenic fungi. We have presently used the RNA-seq strategy to compare the <200- and >200-nt RNA fractions contained in EVs isolated from culture supernatants of P. brasiliensis Pb18, Pb3, and P. lutzii Pb01. Shared mRNA sequences were related toprotein modification, translation, and DNA metabolism/biogenesis, while those related to transport and oxidation-reduction were exclusive to Pb01. The presence of functional full-length mRNAs was validated by in vitro translation. Among small non-coding (nc)RNA, 15 were common to all samples; small nucleolar (sno)RNAs were enriched in P. brasiliensis EVs, whereas for P. lutzii there were similar proportions of snoRNA, rRNA, and tRNA. Putative exonic sRNAs were highly abundant in Pb18 EVs. We also found sRNA sequences bearing incomplete microRNA structures mapping to exons. RNA-seq data suggest that extracellular fractions containing Pb18 EVs can modulate the transcriptome of murine monocyte-derived dendritic cells in a transwell system. Considering that sRNA classes are involved in transcription/translation modulation, our general results may indicate that differences in virulence among fungal isolates can be related to their distinct EV-RNA content

Comparison of the RNA content of extracellular vesicles derived from Paracoccidioides brasiliensis and Paracoccidioides lutzii

Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis. We have previously characterized the <200-nt RNA sub-populations contained in fungal extracellular vesicles (EVs) from P. brasiliensis Pb18 and other pathogenic fungi. We have presently used the RNA-seq strategy to compare the <200- and >200-nt RNA fractions contained in EVs isolated from culture supernatants of P. brasiliensis Pb18, Pb3, and P. lutzii Pb01. Shared mRNA sequences were related toprotein modification, translation, and DNA metabolism/biogenesis, while those related to transport and oxidation-reduction were exclusive to Pb01. The presence of functional full-length mRNAs was validated by in vitro translation. Among small non-coding (nc)RNA, 15 were common to all samples; small nucleolar (sno)RNAs were enriched in P. brasiliensis EVs, whereas for P. lutzii there were similar proportions of snoRNA, rRNA, and tRNA. Putative exonic sRNAs were highly abundant in Pb18 EVs. We also found sRNA sequences bearing incomplete microRNA structures mapping to exons. RNA-seq data suggest that extracellular fractions containing Pb18 EVs can modulate the transcriptome of murine monocyte-derived dendritic cells in a transwell system. Considering that sRNA classes are involved in transcription/translation modulation, our general results may indicate that differences in virulence among fungal isolates can be related to their distinct EV-RNA content

Embryonic atrazine exposure elicits proteomic, behavioral, and brain abnormalities with developmental time specific gene expression signatures.

Atrazine (ATZ), the second most commonly used herbicide in the United States, is an endocrine disrupting chemical linked to cancer and a common drinking water contaminant. This study further investigates ATZ-related developmental toxicity by testing the following hypotheses in zebrafish: the effects of embryonic ATZ exposure are dependent on timing of exposure; embryonic ATZ exposure alters brain development and function; and embryonic ATZ exposure changes protein abundance in carcinogenesis-related pathways. After exposing embryos to 0, 0.3, 3, or 30 parts per billion (ppb) ATZ, we monitored the expression of cytochrome P450 family 17 subfamily A member 1 (cyp17a1), glyoxalase I (glo1), ring finger protein 14 (rnf14), salt inducible kinase 2 (sik2), tetratricopeptide domain 3 (ttc3), and tumor protein D52 like 1 (tpd52l1) at multiple embryonic time points to determine normal expression and if ATZ exposure altered expression. Only cyp17a1 had normal dynamic expression, but ttc3 and tpd52l1 had ATZ-related expression changes before 72 h. Larvae exposed to 0.3 ppb ATZ had increased brain length, while larvae exposed to 30 ppb ATZ were hypoactive. Proteomic analysis identified altered protein abundance in pathways related to cellular function, neurodevelopment, and genital-tract cancer. The results indicate embryonic ATZ toxicity involves interactions of multiple pathways. SIGNIFICANCE: This is the first report of proteomic alterations following embryonic exposure to atrazine, an environmentally persistent pesticide and common water contaminant. Although the transcriptomic alterations in larval zebrafish with embryonic atrazine exposure have been reported, neither the time at which gene expression changes occur nor the resulting proteomic changes have been investigated. This study seeks to address these knowledge gaps by evaluating atrazine's effect on gene expression through multiple time points during embryogenesis, and correlating changes in gene expression to pathological alterations in brain length and functional changes in behavior. Finally, pathway analysis of the proteomic alterations identifies connections between the molecular changes and functional outcomes associated with embryonic atrazine exposure.

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes.

Trypanosoma cruzi is the etiologic agent of Chagas' disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas' disease.

High throughput reaction screening using desorption electrospray ionization mass spectrometry.

We report the high throughput analysis of reaction mixture arrays using methods and data handling routines that were originally developed for biological tissue imaging. Desorption electrospray ionization (DESI) mass spectrometry (MS) is applied in a continuous on-line process at rates that approach 104 reactions per h at area densities of up to 1 spot per mm2 (6144 spots per standard microtiter plate) with the sprayer moving at ca. 104 microns per s. Data are analyzed automatically by MS using in-house software to create ion images of selected reagents and products as intensity plots in standard array format. Amine alkylation reactions were used to optimize the system performance on PTFE membrane substrates using methanol as the DESI spray/analysis solvent. Reaction times can be <100 µs when reaction acceleration occurs in microdroplets, enabling the rapid screening of processes like N-alkylation and Suzuki coupling reactions as reported herein. Products and by-products were confirmed by on-line MS/MS upon rescanning of the array.

Development and Characterization of an Endotoxemia Model in Zebra Fish.

Endotoxemia is a condition in which endotoxins enter the blood stream and cause systemic and sometimes lethal inflammation. Zebra fish provides a genetically tractable model organism for studying innate immunity, with additional advantages in live imaging and drug discovery. However, a bona fide endotoxemia model has not been established in zebra fish. Here, we have developed an acute endotoxemia model in zebra fish by injecting a single dose of LPS directly into the circulation. Hallmarks of human acute endotoxemia, including systemic inflammation, extensive tissue damage, circulation blockade, immune cell mobilization, and emergency hematopoiesis, were recapitulated in this model. Knocking out the adaptor protein Myd88 inhibited systemic inflammation and improved zebra fish survival. In addition, similar alternations of pathways with human acute endotoxemia were detected using global proteomic profiling and MetaCore™ pathway enrichment analysis. Furthermore, treating zebra fish with a protein tyrosine phosphatase nonreceptor type 11 (Shp2) inhibitor decreased systemic inflammation, immune mobilization, tissue damage, and improved survival in the endotoxemia model. Together, we have established and characterized the phenotypic and gene expression changes of a zebra fish endotoxemia model, which is amenable to genetic and pharmacological discoveries that can ultimately lead to a better mechanistic understanding of the dynamics and interplay of the innate immune system.

A carbohydrate moiety of secreted stage-specific glycoprotein 4 participates in host cell invasion by Trypanosoma cruzi extracellular amastigotes

Trypanosoma cruzi is the etiologic agent of Chagas' disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas' disease.