Tracking Inhibition of Human Small C-Terminal Domain Phosphatase 1 Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.

Optimized 13C-TrEnDi Enhances the Sensitivity of Plasmenyl Ether Glycerophospholipids and Demonstrates Compatibility with Other Derivatization Strategies.

The identification and quantitation of plasmalogen glycerophospholipids is challenging due to their isobaric overlap with plasmanyl ether-linked glycerophospholipids, susceptibility to acid degradation, and their typically low abundance in biological samples. Trimethylation enhancement using diazomethane (TrEnDi) can be used to significantly enhance the signal of glycerophospholipids through the creation of quaternary ammonium groups producing fixed positive charges using 13C-diazomethane in complex lipid extracts. Although TrEnDi requires a strong acid for complete methylation, we report an optimized protocol using 10 mM HBF4 with the subsequent addition of a buffer solution that prevents acidic hydrolysis of plasmalogen species and enables the benefits of TrEnDi to be realized for this class of lipids. These optimized conditions were applied to aliquots of bovine liver extract (BLE) to achieve permethylation of plasmalogen lipids within a complex mixture. Treating aliquots of unmodified and TrEnDi-derivatized BLE samples with 80% formic acid and comparing their liquid chromatography mass spectrometry (LCMS) results to analogous samples not treated with formic acid, enabled the identification of 29 plasmalogen species. On average, methylated plasmalogen species from BLE demonstrated 2.81-fold and 28.1-fold sensitivity gains over unmodified counterparts for phosphatidylcholine and phosphatidylethanolamine plasmalogen species, respectively. Furthermore, the compatibility of employing 13C-TrEnDi and a previously reported iodoacetalization strategy was demonstrated to effectively identify plasmenyl-ether lipids in complex biological extracts at greater levels of sensitivity. Overall, we detail an optimized 13C-TrEnDi derivatization strategy that enables the analysis of plasmalogen glycerophospholipids with no undesired cleavage of radyl groups, boosting their sensitivity in LCMS and LCMS/MS analyses.

Neuronal ageing is promoted by the decay of the microtubule cytoskeleton.

Natural ageing is accompanied by a decline in motor, sensory, and cognitive functions, all impacting quality of life. Ageing is also the predominant risk factor for many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. We need to therefore gain a better understanding of the cellular and physiological processes underlying age-related neuronal decay. However, gaining this understanding is a slow process due to the large amount of time required to age mammalian or vertebrate animal models. Here, we introduce a new cellular model within the Drosophila brain, in which we report classical ageing hallmarks previously observed in the primate brain. These hallmarks include axonal swellings, cytoskeletal decay, a reduction in axonal calibre, and morphological changes arising at synaptic terminals. In the fly brain, these changes begin to occur within a few weeks, ideal to study the underlying mechanisms of ageing. We discovered that the decay of the neuronal microtubule (MT) cytoskeleton precedes the onset of other ageing hallmarks. We showed that the MT-binding factors Tau, EB1, and Shot/MACF1, are necessary for MT maintenance in axons and synapses, and that their functional loss during ageing triggers MT bundle decay, followed by a decline in axons and synaptic terminals. Furthermore, genetic manipulations that improve MT networks slowed down the onset of neuronal ageing hallmarks and confer aged specimens the ability to outperform age-matched controls. Our work suggests that MT networks are a key lesion site in ageing neurons and therefore the MT cytoskeleton offers a promising target to improve neuronal decay in advanced age.

A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion.

Fluorescence resonance energy transfer (FRET) reporters are commonly used in the final stages of nucleic acid amplification tests to indicate the presence of nucleic acid targets, where fluorescence is restored by nucleases that cleave the FRET reporters. However, the need for dual labelling and purification during manufacturing contributes to the high cost of FRET reporters. Here we demonstrate a low-cost silver nanocluster reporter that does not rely on FRET as the on/off switching mechanism, but rather on a cluster transformation process that leads to fluorescence color change upon nuclease digestion. Notably, a 90 nm red shift in emission is observed upon reporter cleavage, a result unattainable by a simple donor-quencher FRET reporter. Electrospray ionization-mass spectrometry results suggest that the stoichiometric change of the silver nanoclusters from Ag13 (in the intact DNA host) to Ag10 (in the fragments) is probably responsible for the emission colour change observed after reporter digestion. Our results demonstrate that DNA-templated silver nanocluster probes can be versatile reporters for detecting nuclease activities and provide insights into the interactions between nucleases and metallo-DNA nanomaterials.

Usability of ENTRUST as an Assessment Tool for Entrustable Professional Activities (EPAs): A Mixed Methods Analysis.

OBJECTIVE: As the American Board of Surgery transitions to a competency-based model of surgical education centered upon entrustable professional activities (EPAs), there is a growing need for objective tools to determine readiness for entrustment. This study evaluates the usability of ENTRUST, an innovative virtual patient simulation platform to assess surgical trainees' decision-making skills in preoperative, intra-operative, and post-operative settings. DESIGN: This is a mixed-methods analysis of the usability of the ENTRUST platform. Quantitative data was collected using the system usability scale (SUS) and Likert responses. Analysis was performed with descriptive statistics, bivariate analysis, and multivariable linear regression. Qualitative analysis of open-ended responses was performed using the Nielsen-Shneiderman Heuristics framework. SETTING: This study was conducted at an academic institution in a proctored exam setting. PARTICIPANTS: The analysis includes n = 47 (PGY 1-5) surgical residents who completed an online usability survey following the ENTRUST Inguinal Hernia EPA Assessment. RESULTS: The ENTRUST platform had a median SUS score of 82.5. On bivariate and multivariate analyses, there were no significant differences between usability based on demographic characteristics (all p > 0.05), and SUS score was independent of ENTRUST performance (r = 0.198, p = 0.18). Most participants agreed that the clinical workup of the patient was engaging (91.5%) and felt realistic (85.1%). The most frequent heuristics represented in the qualitative analysis included feedback, visibility, match, and control. Additional themes of educational value, enjoyment, and ease-of-use highlighted participants' perspectives on the usability of ENTRUST. CONCLUSIONS: ENTRUST demonstrates high usability in this population. Usability was independent of ENTRUST score performance and there were no differences in usability identified in this analysis based on demographic subgroups. Qualitative analysis highlighted the acceptability of ENTRUST and will inform ongoing development of the platform. The ENTRUST platform holds potential as a tool for the assessment of EPAs in surgical residency programs.

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as "C16" are likely associated with pathogenicity. Presented here are the results of CRISPR-Cas9 gene-editing experiments disrupting the polyketide synthase and terpene synthase genes associated with the C16 biosynthetic gene cluster in F. graminearum. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products of the C16 biosynthetic gene cluster as the decalin-containing diterpenoid pyrones, FDDP-D and FDDP-E. Both decalin-containing diterpenoid pyrones were confirmed to be produced in wheat heads challenged with F. graminearum in growth chamber trials. The extent to which the F. graminearum C16 biosynthetic gene cluster is dispersed within the genus Fusarium is discussed along with a proposed role of the FDDPs as pathogen virulence factors.

Correlation of Performance on ENTRUST and Traditional Oral Objective Structured Clinical Examination for High-Stakes Assessment in the College of Surgeons of East, Central, and Southern Africa.

BACKGROUND: To address the global need for accessible evidence-based tools for competency-based education, we developed ENTRUST, an innovative online virtual patient simulation platform to author and securely deploy case scenarios to assess surgical decision-making competence. STUDY DESIGN: In partnership with the College of Surgeons of East, Central, and Southern Africa, ENTRUST was piloted during the Membership of the College of Surgeons (MCS) 2021 examination. Examinees (n = 110) completed the traditional 11-station oral objective structured clinical examinations (OSCEs), followed by 3 ENTRUST cases, authored to query similar clinical content of 3 corresponding OSCE cases. ENTRUST scores were analyzed for associations with MCS Examination outcome using independent sample t tests. Correlation of ENTRUST scores to MCS Examination Percentage and OSCE station scores was calculated with Pearson correlations. Bivariate and multivariate analyses were performed to evaluate predictors of performance. RESULTS: ENTRUST performance was significantly higher in examinees who passed the MCS examination compared with those who failed (p < 0.001). The ENTRUST score was positively correlated with MCS Examination Percentage (p < 0.001) and combined OSCE station scores (p < 0.001). On multivariate analysis, there was a strong association between MCS Examination Percentage and ENTRUST Grand Total Score (p < 0.001), Simulation Total Score (p = 0.018), and Question Total Score (p < 0.001). Age was a negative predictor for ENTRUST Grand Total and Simulation Total Score, but not for Question Total Score. Sex, native language status, and intended specialty were not associated with performance on ENTRUST. CONCLUSIONS: This study demonstrates feasibility and initial validity evidence for the use of ENTRUST in a high-stakes examination context for assessment of surgical decision-making. ENTRUST holds potential as an accessible learning and assessment platform for surgical trainees worldwide.

Improved Chromatography and MS-Based Detection of Glyphosate and Aminomethylphosphonic Acid Using iTrEnDi.

Glyphosate (GLY), a synthetic, nonselective systemic herbicide that is particularly effective against perennial weeds, is the most used weedkiller in the world. There are growing concerns over GLY accumulation in the environment and the attendant human health-associated risks, and despite increased attention in the media, GLY and its breakdown product aminomethylphosphonic acid (AMPA) remain elusive to many analytical strategies. Chemical derivatization coupled with high-performance liquid chromatography-mass spectrometry (HPLC-MS) addresses the challenge of quantifying low levels of GLY and AMPA in complex samples. Here we demonstrate the use of in situ trimethylation enhancement using diazomethane (iTrEnDi) to derivatize GLY and AMPA into permethylated products ([GLYTr]+ and [AMPATr]+, respectively) prior to analysis via HPLC-MS. iTrEnDi produced quantitative yields and resulted in a 12-340-fold increases in HPLC-MS-based sensitivity for [GLYTr]+ and [AMPATr]+, respectively, compared with underivatized counterparts. The limits of detection of derivatized compounds were found to be 0.99 ng/L for [GLYTr]+ and 1.30 ng/L for [AMPATr]+, demonstrating significant sensitivity improvements compared to previously established derivatization techniques. iTrEnDi is compatible with the direct derivatization of Roundup formulations. Finally, as proof of principle, a simple aqueous extraction followed by iTrEnDi enabled the detection of [GLYTr]+ and [AMPATr]+ on the exterior of field-grown soybeans that were sprayed with Roundup. Overall, iTrEnDi ameliorates issues relating to low proton affinity and chromatographic retention, boosting HPLC-MS-based sensitivity and enabling the elucidation of elusive analytes such as GLY and AMPA within agricultural systems.

Untargeted metabolomics screening reveals unique secondary metabolite production from Alternaria section Alternaria.

Alternaria section Alternaria is comprised of many species that infect a broad diversity of important crop plants and cause post-harvest spoilage. Alternaria section Alternaria species, such as A. alternata and A. arborescens, are prolific producers of secondary metabolites that act as virulence factors of disease and are mycotoxins that accumulate in infected tissues-metabolites that can vary in their spectrum of production between individuals from the same fungal species. Untargeted metabolomics profiling of secondary metabolite production using mass spectrometry is an effective means to detect phenotypic anomalies in secondary metabolism within a species. Secondary metabolite phenotypes from 36 Alternaria section Alternaria isolates were constructed to observe frequency of production patterns. A clear and unique mass feature pattern was observed for three of the strains that were linked with the production of the dehydrocurvularin family of toxins and associated detoxification products. Examination of corresponding genomes revealed the presence of the dehydrocurvularin biosynthesis gene cluster associated with a sub-telomeric accessory region. A comparison of sequence similarity and occurrences of the dehydrocurvularin biosynthetic gene cluster within Pleosporalean fungi is presented and discussed.

Structural Characterization and Quantitation of Ether-Linked Glycerophospholipids in Peroxisome Biogenesis Disorder Tissue by Ultraviolet Photodissociation Mass Spectrometry.

The biological impact of ether glycerophospholipids (GP) in peroxisomal disorders and other diseases makes them significant targets as biomarkers for diagnostic assays or deciphering pathology of the disorders. Ether lipids include both plasmanyl and plasmenyl lipids, which each contain an ether or a vinyl ether bond at the sn-1 linkage position, respectively. This linkage, in contrast to traditional diacyl GPs, precludes their detailed characterization by mass spectrometry via traditional collisional-based MS/MS techniques. Additionally, the isomeric nature of plasmanyl and plasmenyl pairs of ether lipids introduces a further level of complexity that impedes analysis of these species. Here, we utilize 213 nm ultraviolet photodissociation mass spectrometry (UVPD-MS) for detailed characterization of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) plasmenyl and plasmanyl lipids in mouse brain tissue. 213 nm UVPD-MS enables the successful differentiation of these four ether lipid subtypes for the first time. We couple this UVPD-MS methodology to reversed-phase liquid chromatography (RPLC) for characterization and relative quantitation of ether lipids from normal and diseased (Pex7 deficiency modeling the peroxisome biogenesis disorder, RCDP) mouse brain tissue, highlighting the ability to pinpoint specific structural features of ether lipids that are important for monitoring aberrant lipid metabolism in peroxisomal disorders.

Kinetic, ESI-CID-MS and Computational Studies of π-Allyliridium C,O-Benzoate-Catalyzed Allylic Amination: Understanding the Effect of Cesium Ion.

The mechanism of π-allyliridium C,O-benzoate-catalyzed allylic amination was studied by (a) reaction progress kinetic analysis (RPKA), (b) tandem ESI-MS analysis, and (c) computational studies involving density functional theory (DFT) calculations. Reaction progress kinetic analysis (RPKA) reveals a zero-order dependence on allyl acetate, first-order dependence on catalyst and fractional-order dependence on amine. These data corroborate rapid ionization of the allylic acetate followed by turnover limiting C-N bond formation. To illuminate the origins of the 0.4 kinetic order dependence on amine, ESI-MS analyses of quaternary ammonium-labelled piperazine with multistage collision induced dissociation (CID) were conducted that corroborate intervention of cesium-bridged amine dimers that dissociate to form monomeric cesium amide nucleophiles. Computational data align with RPKA and ESI-CID-MS analyses and suggest early transition states mitigate the impact of steric factors, thus enabling formation of highly substituted C-N bonds with complete levels of branched regioselectivity. Specifically, trans-effects of the iridium complex facilitate nucleophilic attack at the more substituted allyl terminus trans to phosphorus with enantioselectivity governed by steric repulsions between the chiral bisphosphine ligand and the π-allyl of a dominant diastereomer of the stereogenic-at-metal complex. Beyond defining aspects of the mechanism of π-allyliridium C,O-benzoate-catalyzed allylic amination, these data reveal that a key feature of cesium carbonate not only lies in its enhanced basicity, but also its capacity for Lewis-acid enhanced Brønsted acidification of amines.

Quantitative High-Field NMR- and Mass Spectrometry-Based Fatty Acid Sequencing Reveals Internal Structure in Ru-Catalyzed Deuteration of Docosahexaenoic Acid.

Ru-based catalysis results in highly unsaturated fatty acid (HUFA) ethyl esters (EE) deuterated to various extents. The products carry 2H (D) mainly at their bis-allylic positions, where they are resistant to autoxidation compared to natural HUFA and are promising as neurological and retinal drugs. We characterized the extent of deuteration at each allylic position of docosa-4,7,10,13,16,19-hexaenoic acid deuterated to completion at bis-allylic and allylic positions (D-DHA) by two-dimensional (2D) and high-field (600 and 950 MHz) NMR. In separate experiments, the kinetics of docosahexaenoic acid (DHA) EE deuteration was evaluated using Paternò-Büchi (PB) reaction tandem mass spectrometry (MS/MS) analysis, enabling deuteration to be quantitatively characterized for isotopologues (D0-D14 DHA) at each internal allylic position. NMR analysis shows that the net deuteration of the isotopologue mixture is about 94% at the bis-allylic positions, and less than 1% remained as the protiated -CH2-. MS analysis shows that deuteration kinetics follow an increasing curve at bis-allylic positions with higher rate for internal bis-allylic positions. Percent D of bis-allylic positions increases linearly from D1 to D9 in which all internal bis-allylic positions (C9, C12, C15) deuterate uniformly and more rapidly than external bis-allylic positions (C6, C18). The mono-allylic positions near the methyl end (C21) show a steep increase of D only after the D10 isotopologue has been deuterated to >90%, while the mono-allylic position near the carboxyl position, C3, deuterates last and least. These data establish detailed methods for the characterization of Ru-catalyzed deuteration of HUFA as well as the phenomenological reaction kinetics as net product is formed.

Massively Parallel Selection of NanoCluster Beacons.

NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here, a high-throughput selection method is reported that takes advantage of repurposed next-generation-sequencing chips to screen the activation fluorescence of ≈40 000 activator sequences. It is found that the nucleobases at positions 7-12 of the 18-nucleotide-long activator are critical to creating bright NCBs and positions 4-6 and 2-4 are hotspots to generate yellow-orange and red POTs, respectively. Based on these findings, a "zipper-bag" model is proposed that can explain how these hotspots facilitate the formation of distinct silver cluster chromophores and alter their chemical yields. Combining high-throughput screening with machine-learning algorithms, a pipeline is established to design bright and multicolor NCBs in silico.

Enhancing the Signal-to-Noise of Diagnostic Fragment Ions of Unsaturated Glycerophospholipids via Precursor Exclusion Ultraviolet Photodissociation Mass Spectrometry (PEx-UVPD-MS).

Understanding and elucidating the diverse structures and functions of lipids has motivated the development of many innovative tandem mass spectrometry (MS/MS) strategies. Higher-energy activation methods, such as ultraviolet photodissociation (UVPD), generate unique fragment ions from glycerophospholipids that can be used to perform in-depth structural analysis and facilitate the deconvolution of isomeric lipid structures in complex samples. Although detailed characterization is central to the correlation of lipid structure to biological function, it is often impeded by the lack of sufficient instrument sensitivity for highly bioactive but low-abundance phospholipids. Here, we present precursor exclusion (PEx) UVPD, a simple yet powerful technique to enhance the signal-to-noise (S/N) of informative low-abundance fragment ions produced from UVPD of glycerophospholipids. Through the exclusion of the large population of undissociated precursor ions with an MS3 strategy, the S/N of diagnostic fragment ions from PC 18:0/18:2(9Z, 12Z) increased up to an average of 13x for PEx-UVPD compared to UVPD alone. These enhancements were extended to complex mixtures of lipids from bovine liver extract to confidently identify 35 unique structures using liquid chromatography PEx-UVPD. This methodology has the potential to advance lipidomics research by offering deeper structure elucidation and confident identification of biologically active lipids.

Enhanced Ion Mobility Separation and Characterization of Isomeric Phosphatidylcholines Using Absorption Mode Fourier Transform Multiplexing and Ultraviolet Photodissociation Mass Spectrometry.

The structural diversity of phospholipids plays a critical role in cellular membrane dynamics, energy storage, and cellular signaling. Despite its importance, the extent of this diversity has only recently come into focus, largely owing to advances in separation science and mass spectrometry methodology and instrumentation. Characterization of glycerophospholipid (GP) isomers differing only in their acyl chain configurations and locations of carbon-carbon double bonds (C═C) remains challenging due to the need for both effective separation of isomers and advanced tandem mass spectrometry (MS/MS) technologies capable of double-bond localization. Drift tube ion mobility spectrometry (DTIMS) coupled with MS can provide both fast separation and accurate determination of collision cross section (CCS) of molecules but typically lacks the resolving power needed to separate phospholipid isomers. Ultraviolet photodissociation (UVPD) can provide unambiguous double-bond localization but is challenging to implement on the timescales of modern commercial drift tube time-of-flight mass spectrometers. Here, we present a novel method for coupling DTIMS with a UVPD-enabled Orbitrap mass spectrometer using absorption mode Fourier transform multiplexing that affords simultaneous localization of double bonds and accurate CCS measurements even when isomers cannot be fully resolved in the mobility dimension. This method is demonstrated on two- and three-component mixtures and shown to provide CCS measurements that differ from those obtained by individual analysis of each component by less than 1%.

iTrEnDi: In Situ Trimethylation Enhancement Using Diazomethane: Improved and Expanded Glycerophospholipid and Sphingolipid Analyses via a Microscale Autonomous Derivatization Platform.

Trimethylation enhancement using diazomethane (TrEnDi) is a derivatization technique that significantly enhances the signal intensity of glycerophospholipid species in mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses. Here, we describe a novel apparatus that is able to conduct in situ TrEnDi (iTrEnDi) by generating and immediately reacting small amounts of gaseous diazoalkane with analyte molecules. iTrEnDi allows complete and rapid methylation of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and sphingomyelin (SM) in a safe manner by removing any need for direct handling of dangerous diazoalkane solutions. iTrEnDi-modified PC ([PCTr]+) and PE ([PETr]+) showed similar sensitivity enhancements and fragmentation patterns compared to our previously reported methodology. iTrEnDi yielded dimethylated PA ([PATr]), which exhibited dramatically improved chromatographic behavior and a 14-fold increase in liquid chromatography MS (LCMS) sensitivity compared to unmodified PA. In comparison to in-solution-based TrEnDi, iTrEnDi demonstrated a modest decrease in sensitivity, likely due to analyte losses during handling. However, the enhanced safety benefits of iTrEnDi coupled with its ease of use and capacity for automation, as well as its accommodation of more-reactive diazoalkane species, vastly improve the accessibility and utility of this derivatization technique. Finally, as a proof of concept, iTrEnDi was used to produce diazoethane (DZE), a more-reactive diazoalkane than diazomethane. Reaction between DZE and PC yielded ethylated [PCTr]+, which fragmented via MS/MS to produce a high-intensity characteristic fragment ion, enabling a novel and highly sensitive precursor ion scan.

Activation of biosynthetic gene clusters by the global transcriptional regulator TRI6 in Fusarium graminearum.

In F. graminearum, the transcription factor TRI6 positively regulates the trichothecene biosynthetic gene cluster (BGC) leading to the production of the secondary metabolite 15-acetyl deoxynivalenol. Secondary metabolites are not essential for survival, instead, they enable the pathogen to successfully infect its host. F. graminearum has the potential to produce a diverse array of secondary metabolites (SMs). However, given high functional specificity and energetic cost, most of these clusters remain silent, unless the organism is subjected to an environment conducive to SM production. Alternatively, secondary metabolite gene clusters (SMCs) can be activated by genetically manipulating their activators or repressors. In this study, a combination of transcriptomic and metabolomics analyses with a deletion and overexpressor mutants of TRI6 was used to establish the role of TRI6 in the regulation of several BGCs in F. graminearum. Evidence for direct and indirect regulation of BGCs by TRI6 was obtained by chromatin immunoprecipitation and yeast two-hybrid experiments. The results showed that the trichothecene genes are under direct control, while the gramillin gene cluster is indirectly controlled by TRI6 through its interaction with the pathway-specific transcription factor GRA2.

Coupling Headgroup and Alkene Specific Solution Modifications with Gas-Phase Ion/Ion Reactions for Sensitive Glycerophospholipid Identification and Characterization.

Shotgun lipidomics provides sensitive and fast lipid identification without the need for chromatographic separation. Challenges faced by shotgun analysis of glycerophospholipids (GPs) include the lack of signal uniformity across GP classes and the inability to determine the carbon-carbon double bond (C═C) location within the fatty acyl chains of an unsaturated species. Two distinct derivatization strategies were employed to both enhance the ionization of GPs, via trimethylation enhancement using 13C-diazomethane (13C-TrEnDi), as well as determine location of double bonds within fatty acyl chains, employing an in-solution photochemical reaction with acetone (via the Paternò-Büchi reaction). The modified GPs were then subjected to positive ion mode ionization via electrospray ionization, producing uniform ionization efficiencies for different classes of GP species. The GPs were charge inverted via gas-phase ion/ion reactions and sequentially fragmented using ion trap collision-induced dissociation (CID). The CID of the species led to fragmentation producing diagnostic ions indicative of C═C bond location. The approach enabled enhanced ionization and the identification of phosphatidylcholine and phosphatidylethanolamine species at the C═C level in a bovine lipid extract.

A multidimensional systems biology analysis of cellular senescence in aging and disease.

BACKGROUND: Cellular senescence, a permanent state of replicative arrest in otherwise proliferating cells, is a hallmark of aging and has been linked to aging-related diseases. Many genes play a role in cellular senescence, yet a comprehensive understanding of its pathways is still lacking. RESULTS: We develop CellAge (http://genomics.senescence.info/cells), a manually curated database of 279 human genes driving cellular senescence, and perform various integrative analyses. Genes inducing cellular senescence tend to be overexpressed with age in human tissues and are significantly overrepresented in anti-longevity and tumor-suppressor genes, while genes inhibiting cellular senescence overlap with pro-longevity and oncogenes. Furthermore, cellular senescence genes are strongly conserved in mammals but not in invertebrates. We also build cellular senescence protein-protein interaction and co-expression networks. Clusters in the networks are enriched for cell cycle and immunological processes. Network topological parameters also reveal novel potential cellular senescence regulators. Using siRNAs, we observe that all 26 candidates tested induce at least one marker of senescence with 13 genes (C9orf40, CDC25A, CDCA4, CKAP2, GTF3C4, HAUS4, IMMT, MCM7, MTHFD2, MYBL2, NEK2, NIPA2, and TCEB3) decreasing cell number, activating p16/p21, and undergoing morphological changes that resemble cellular senescence. CONCLUSIONS: Overall, our work provides a benchmark resource for researchers to study cellular senescence, and our systems biology analyses reveal new insights and gene regulators of cellular senescence.

Trimethylation Enhancement Using 13C-Diazomethane: Gas-Phase Charge Inversion of Modified Phospholipid Cations for Enhanced Structural Characterization.

Methylation of phospholipids (PL) leads to increased uniformity in positive electrospray ionization (ESI) efficiencies across the various PL subclasses. This effect is realized in the approach referred to as "trimethylation enhancement using 13C-diazomethane" (13C-TrEnDi), which results in the methyl esterification of all acidic sites and the conversion of amines to quaternary ammonium sites. Collision-induced dissociation (CID) of these cationic modified lipids enables class identification by forming distinctive headgroup fragments based on the number of 13C atoms incorporated during derivatization. However, there are no distinctive fragment ions in positive mode that provide fatty acyl information for any of the modified lipids. Gas-phase ion/ion reactions of 13C-TrEnDi-modified phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), and sphingomyelin (SM) cations with dicarboxylate anions are shown to charge-invert the positively charged phospholipids to the negative mode. An electrostatically bound complex anion is shown to fragment predominantly via a novel headgroup dication transfer to the reagent anion. Fragmentation of the resulting anionic product yields fatty acyl information, in the case of the glycerophospholipids (PE, PS, and PC), via ester bond cleavage. Analogous information is obtained from modified SM lipid anions via amide bond cleavage. Fragmentation of the anions generated from charge inversion of the 13C-TrEnDi-modified phospholipids was also found to yield lipid class information without having to perform CID in positive mode. The combination of 13C-TrEnDi modification of lipid mixtures with charge inversion to the negative-ion mode retains the advantages of uniform ionization efficiency in the positive-ion mode with the additional structural information available in the negative-ion mode without requiring the lipids to be ionized directly in both ionization modes.