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Background: Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in-part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1β signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 inflammasome and associated signaling components in neutrophils, the serum levels of IL-1β, the effector molecule in granulopoiesis was not impacted by MI suggesting that IL-1β is not released systemically. We hypothesize that IL-1β is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. Methods: Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the LAD artery, and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in non-infarcted parabionts. Finally, utilizing multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1β secretion and granulopoiesis. Cardiac function was assessed by echocardiography. Results: MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the non-infarcted parabionts. Prior priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed neutrophils secrete IL-1β through formation of gasdermin D pores and, promote granulopoiesis. Pharmacological and/ or genetic strategies aimed at inhibition of neutrophil homing or release of IL-1β in the BM markedly suppressed MI-induced granulopoiesis and, improved cardiac function. Conclusions: Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (e.g., IL-1β) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1β release

Myocardial infarction (MI)-induced heart failure is one of the leading causes of death in the modern world. This is in part due to abnormal cardiac remodeling, instigated by a large influx of neutrophils immediately following MI. These activated neutrophils in the heart release a plethora of proteolytic enzymes and exacerbate tissue injury leading to cardiac failure and death. The role of neutrophils during MI is complex with the intensity and duration of their residency dictating the ensuing remodeling process. We have previously shown that the recruitment of neutrophils to heart post-MI is mirrored by an increased proliferation of progenitors (granulopoiesis) in the bone marrow (BM). However, the increase in the number of neutrophils at the infarct begins much earlier (< 6 hours post MI) than the peak granulopoiesis (24 hours post-MI) in the BM. These finding suggest that sources other than granulopoiesis may contribute heavily to the initial neutrophil burden of the heart. One such source is the marginated pool. In the circulation, the marginated pool of neutrophils represents the same size as that of the circulating pool. We hypothesize that “ demargination of neutrophils from the vascular wall contribute to the neutrophil burden of the heart particularly during the early hours after MI ”. To test this hypothesis, we created a mouse model of MI by permanent ligation of the left anterior descending coronary artery and studied the impact of early recruitment of neutrophil to ischemic heart. We found that the initial wave of neutrophils recruited to the ischemic heart were exclusively sourced from the vasculature and not granulopoiesis in the BM/ Spleen. The neutrophils that were recruited to the heart exhibit distinct hallmarks of demargination including decreased F-actin, CD62L and increased Adam17 expression. This early recruitment of neutrophils was orchestrated by catecholamine stress as strategies aimed at inhibition of catecholamine biosynthesis or blockade of β adrenergic receptors (β-AR) significantly reduced neutrophil burden and improved cardiac function. Interestingly, this protective effect was lost when neutrophil recruitment was suppressed over a longer period. In conclusion we show that while short-term blockade of β-AR signaling suppresses the early neutrophil swarming, suppression beyond the reparative phase (> 3 days post-MI) adversely impact the cardiac function. Together these data suggest that catecholamine stress induced-demargination constitute the major source of neutrophils during the early hours of MI. Understanding the precise signaling mechanisms that drive demargination and optimizing the right dose/duration of a specific β-AR blocker may help suppress the initial onslaught of neutrophils without compromising their overall beneficial function during the reparative phase of inflammation.

Pyrolysis oil (PO) is an inexpensive biofuel produced via the fast pyrolysis of lignocellulosic biomass. However, PO is unstable and has low energy density, making it unsuitable as a fuel in conven...

Matrix-assisted laser desorption/ionization (MALDI)-MS imaging has been utilized to image a variety of biomolecules, including neuropeptides. Washing a tissue section is an effective way to eliminate interfering background and improve detection of low concentration target analyte molecules; however, many previous methods have not been tested for neuropeptide analysis via MALDI-MS imaging. Using crustacean as a neurological model organism, we developed a new, simple washing procedure and applied this method to characterize neuropeptide changes due to hypoxia stress. By using a 10 second 50:50 EtOH:H(2)O wash, neuropeptide coverage was improved by 1.15-fold, while normalized signal intensities were increased by 5.28-fold.Specifically, hypoxia and hypercapnia stress conditions were investigated due to their environmental relevance to marine invertebrates. Many neuropeptides, including RFamides, pyrokinin, and cardioactive peptides, showed distinct up- and down-regulation for specific neuropeptide isoforms. Since crustacean neuropeptides are homologous to those found in humans, results from these studies can be applied to understand potential roles of neuropeptides involved in medical hypoxia and hypercapnia.

Site-specific O-glycoproteome mapping in complex biological systems provides a molecular basis for understanding the structure-function relationships of glycoproteins and their roles in physiological and pathological processes. Previous O-glycoproteome analysis in cerebrospinal fluid (CSF) focused on sialylated glycoforms, while missing information on other glycosylation types. In order to achieve an unbiased O-glycosylation profile, we developed an integrated strategy combining universal boronic acid enrichment, high-pH fractionation, and electron-transfer and higher-energy collision dissociation (EThcD) for enhanced intact O-glycopeptide analysis. We applied this strategy to analyze the O-glycoproteome in CSF, resulting in the identification of 308 O-glycopeptides from 110 O-glycoproteins, covering both sialylated and nonsialylated glycoforms. To our knowledge, this is the largest data set of O-glycoproteins and O-glycosites reported for CSF to date. We also developed a peptidomics workflow that utilized the EThcD and a three-step database searching strategy for comprehensive PTM analysis of endogenous peptides, including N-glycosylation, O-glycosylation, and other common peptide PTMs. Interestingly, among the 1411 endogenous peptides identified, 89 were O-glycosylated, and only one N-glycosylated peptide was found, indicating that CSF endogenous peptides were predominantly O-glycosylated. Analyses of the O-glycoproteome and endogenous peptidome PTMs were also conducted in the CSF of MCI and AD patients to provide a landscape of glycosylation patterns in different disease states. Our results showed a decreasing trend in fucosylation and an increasing trend of endogenous peptide O-glycosylation, which may play an important role in AD progression.

Children with kidney disease and primary hypertension may be more vulnerable to COVID-19. We examined COVID-19 vaccine hesitancy among parents of children with chronic kidney disease or hypertension.Sequential explanatory mixed-methods design; survey followed by in-depth interviews.Parents of children aged 18 years with kidney disease or primary hypertension within a large pediatric practice.Parental attitudes toward general childhood and influenza vaccines assessed by the Vaccine Hesitancy Scale. Kidney disease classification, demographic and socioeconomic factors, experiences with COVID-19, COVID-19 mitigation activities and self-efficacy, and sources of vaccine information.Willingness to vaccinate child against COVID-19.Analysis of variance (ANOVA) test to compare parental attitudes toward general childhood and influenza vaccination with attitudes toward COVID-19 vaccination. Multinomial logistic regression to assess predictors of willingness to vaccinate against COVID-19. Thematic analysis of interview data to characterize influences on parental attitudes.Of the participants, 207 parents completed the survey (39% of approached): 75 (36%) were willing, 80 (39%) unsure, and 52 (25%) unwilling to vaccinate their child against COVID-19. Hesitancy toward general childhood and influenza vaccines was highest among the unwilling group (P 0.001). More highly educated parents more likely to be willing to vaccinate their children, while Black race was associated with being more likely to be unwilling. Rushed COVID-19 vaccine development as well as fear of serious and unknown long-term side effects were themes that differed across the parental groups that were willing, unsure, or unwilling to vaccinate their children. Although doctors and health care teams are trusted sources of vaccine information, perceptions of benefit versus harm and experiences with doctors differed among these 3 groups. The need for additional information on COVID-19 vaccines was greatest among those unwilling or unsure about vaccinating.Generalizability may be limited.Two-thirds of parents of children with kidney disease or hypertension were unsure or unwilling to vaccinate their child against COVID-19. Higher hesitancy toward routine childhood and influenza vaccination was associated with hesitancy toward COVID-19 vaccines. Enhanced communication of vaccine information relevant to kidney patients in an accessible manner should be examined as a means to reduce vaccine hesitancy.Children with kidney disease or hypertension may do worse with COVID-19. As there are now effective vaccines to protect children from COVID-19, we wanted to find out what parents think about COVID-19 vaccines and what influences their attitudes. We surveyed and then interviewed parents of children who had received a kidney transplant, were receiving maintenance dialysis, had chronic kidney disease, or had hypertension. We found that two-thirds of parents were hesitant to vaccinate their children. Their reasons varied, but the key issues included the need for information pertinent to their child and a consistent message from doctors and other health care providers. These findings may inform an effective vaccine campaign to protect children with kidney disease and hypertension.

The body’s inflammatory response involves a series of processes that are necessary for the immune system to mitigate threats from invading pathogens. Leukocyte migration is a crucial process in both homeostatic and inflammatory states. The mechanisms involved in immune cell recruitment to the site of inflammation are numerous and require several cascades and cues of activation. Immune cells have multiple origins and can be recruited from primary and secondary lymphoid, as well as reservoir organs within the body to generate an immune response to certain stimuli. However, no matter the origin, an important aspect of any inflammatory response is the web of networks that facilitates immune cell trafficking. The vasculature is an important organ for this trafficking, especially during an inflammatory response, mainly because it allows cells to migrate towards the source of insult/injury and serves as a reservoir for leukocytes and granulocytes under steady state conditions. One of the most active and vital leukocytes in the immune system’s arsenal are neutrophils. Neutrophils exist under two forms in the vasculature: a marginated pool that is attached to the vessel walls, and a demarginated pool that freely circulates within the blood stream. In this review, we seek to present the current consensus on the mechanisms involved in leukocyte margination and demargination, with a focus on the role of neutrophil migration patterns during physio-pathological conditions, in particular diabetes and cardiovascular disease.

Myocardial infarction (MI) induces a rapid and robust inflammatory response characterized by infiltration of different leukocyte cell types to the infarcted heart. Neutrophils are the first cells to arrive at the infarct where they release a plethora of proteolytic enzymes, exacerbate tissue injury, expand infarct size and promote cardiac dysfunction/ failure. We previously have shown that granulopoiesis (in the bone marrow, BM) is the major source of neutrophils during MI. However, the increase in the number of neutrophils at the infarct begins much earlier than the peak response in the BM (~ 24 hours) suggesting that sources other than granulopoiesis may contribute to the overall neutrophil pool. Because the marginated pool of neutrophils represents the same size of circulating pool, we hypothesized that demargination of neutrophils from the vascular wall contribute to the neutrophil burden in the heart, particularly during the early hours after MI.Using a mouse model of the permanent ligation of the left anterior descending coronary artery, BM ablation of hematopoietic stem cells, flow cytometry and BM transplant techniques, we found that the first wave of neutrophils recruited to the ischemic heart are exclusively sourced from the vasculature and not granulopoiesis in the BM/ spleen. The neutrophils recruited at the heart bore all hallmarks of demargination induced by dexamethasone including decreased F-actin, CD62L, and increased Adam17 expression. The recruitment of neutrophils is orchestrated by catecholamine stress as experimental strategies aimed at blockade of β1 and β2 adrenergic receptors (AR) reduced neutrophil burden in the ischemic heart. Interestingly, despite a decrease in neutrophil burden, the cardiac function did not improve but rather declined. However, short-term inhibition (6-12 hours post-MI) of β AR system either by receptor blockade (propranolol) or inhibition of catecholamine synthesis (AMPT) not only reduced neutrophil burden but also significantly improved cardiac function. Together these data suggest that catecholamine stress induced-demargination constitute the major source of neutrophils during the early hours post-MI. Pharmacological strategies aimed at suppressing the initial onslaught of neutrophils on the ischemic heart may represent a novel and viable approach towards a better resolution of the injury. Currently, studies are underway to define the signaling mechanisms that drive demargination and, to optimize the dose/ duration of a specific β receptor blocker to achieve better functional outcomes.

Myocardial infarction (MI) provokes a massive systemic inflammatory response characterized by enhanced infiltration of neutrophils to the ischemic heart via granulopoiesis in the bone marrow. We recently discovered that S100A8/A9 is released by infiltrating neutrophils in the infarct to induce granulopoiesis by interacting with TLR4 on naïve neutrophils, priming the NLPR3-inflammasome for release of IL-1β which then leads to increased granulopoiesis. However, the mechanism of S100A8/A9 release from the infiltrating-neutrophils remain unclear. We hypothesized that neutrophils release S100A8/A9 via NETosis, a form of cell-death that involves extrusion of decondensed chromatin along with its entire granular content including S100A8/A9. To validate this hypothesis we found that neutrophils sorted from mouse heart 24 hours post-MI were enriched for genetic signature for an Nox-independent NETosis, dominated by Padi4. Furthermore, we found a robust increase in citrullination of histone moieties, colocalization of S100A8/A9 with H3Cit, and increased S100A8/A9 levels in the heart as early as 6 hours after MI. Depletion of Padi4 in the mouse myeloid cells by bone marrow transplantation studies was also associated with suppressed granulopoiesis, fewer neutrophils in the blood and heart, and reduced cardiac and serum S100A8/A9 after MI. To validate these findings in humans, we measured various markers of NETosis and found a marked increase in serum double-stranded DNA, MPO (myeloperoxidase) elastase, which paralleled S100A8/A9 levels in STEMI patients from the time of admission through post-revascularization by percutaneous coronary intervention. Taken together we propose that interventions to reduce NETosis and/or S100A8/A9 release particularly during the acute inflammatory phase could represent a departure from the status quo in managing post-MI inflammation and the subsequent heart failure.

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Biomass conversion to alcohols using supercritical methanol depolymerization and hydrodeoxygenation (SCM-DHO) with CuMgAl mixed metal oxide is a promising process for biofuel production. We demonstrate how maple wood can be converted at high weight loadings and product concentrations in a batch and a semi-continuous reactor to a mixture of C2–C10 linear and cyclic alcohols. Maple wood was solubilized semi-continuously in supercritical methanol and then converted to a mixture of C2–C9 alcohols and aromatics over a packed bed of CuMgAlOx catalyst. Up to 95 wt% of maple wood can be solubilized in the methanol by using four temperature holds at 190, 230, 300, and 330 °C. Lignin was solubilized at 190 and 230 °C to a mixture of monomers, dimers, and trimers while hemicellulose and cellulose solubilized at 300 and 330 °C to a mixture of oligomeric sugars and liquefaction products. The hemicellulose, cellulose, and lignin were converted to C2–C10 alcohol fuel precursors over a packed bed of CuMgAlOx catalyst with 70–80% carbon yield of the entire maple wood. The methanol reforming activity of the catalyst decreased by 25% over four beds of biomass, which corresponds to 5 turnovers for the catalyst, but was regenerable after calcination and reduction. In batch reactions, maple wood was converted at 10 wt% in methanol with 93% carbon yield to liquid products. The product concentration can be increased to 20 wt% by partially replacing the methanol with liquid products. The yield of alcohols in the semi-continuous reactor was approximately 30% lower than in batch reactions likely due to degradation of lignin and cellulose during solubilization. These results show that solubilization of whole biomass can be separated from catalytic conversion of the intermediates while still achieving a high yield of products. However, close contact of the catalyst and the biomass during solubilization is critical to achieve the highest yields and concentration of products.

Supercritical methanol depolymerization and hydrodeoxygenation (SCM-DHDO) is a process to produce fuel from biomass with supercritical methanol and CuMgAlOx catalyst. Previous research has shown that SCM-DHDO can convert cellulose, lignin and whole biomass feedstock in a single reaction step. In this paper, we show that SCM-DHDO can convert pyrolytic lignin derived from pyrolysis oil at approximately 50% carbon yield to an upgraded oil product. The upgraded oil mainly contains lignin monomers and dimers while an additional 50% of the carbon was converted into undesired products including methanol, insoluble pyrolytic lignin and coke. We demonstrated that after a 4 h SCM-DHDO reaction the pyrolytic lignin molecular weight did not significantly change, but the ketones, acids and aldehydes in the pyrolytic lignin were completely converted. Increasing the reaction time of SCM-DHDO reaction from 2 to 10 h increased the lignin monomer and dimer yield. The reduced CuAlMgOx catalyst hydrogenated 57.4% of the aromatic carbons to aliphatic carbons and removed 64% of the oxygen. However, the product oil could still not be distilled because of the thermal instability of the upgraded pyrolytic lignin.

Neutrophils, the most abundant of all leucocytes and the first cells to arrive at the sites of sterile inflammation/injury act as a double-edged sword. On one hand, they inflict a significant collateral damage to the tissues and on the other hand, they help facilitate wound healing by a number of mechanisms. Recent studies have drastically changed the perception of neutrophils from being simple one-dimensional cells with an unrestrained mode of action to a cell type that display maturity and complex behaviour. It is now recognized that neutrophils are transcriptionally active and respond to plethora of signals by deploying a wide variety of cargo to influence the activity of other cells in the vicinity. Neutrophils can regulate macrophage behaviour, display innate immune memory, and play a major role in the resolution of inflammation in a context-dependent manner. In this review, we provide an update on the factors that regulate neutrophil production and the emerging dichotomous role of neutrophils in the context of cardiovascular diseases, particularly in atherosclerosis and the ensuing complications, myocardial infarction, and heart failure. Deciphering the complex behaviour of neutrophils during inflammation and resolution may provide novel insights and in turn facilitate the development of potential therapeutic strategies to manage cardiovascular disease.

Neuropeptides are low abundance signaling molecules that modulate almost every physiological process, and dysregulation of neuropeptides is implicated in disease pathology. Mass spectrometry (MS) imaging is becoming increasingly useful for studying neuropeptides as new sample preparation methods for improving neuropeptide detection are developed. In particular, proper tissue washes prior to MS imaging have shown to be quick and effective strategies for increasing the number of detectable neuropeptides. Treating tissues with solvents could result in either gain or loss of detection of analytes, and characterization of these wash effects is important for studies targeting sub-classes of neuropeptides. In this communication, we apply aqueous tissue washes that contain sodium phosphate salts, including 10% neutral buffered formalin (NBF), on crustacean brain tissues. Our optimized method resulted in complementary identification of neuropeptides between washed and unwashed tissues, indicating that our wash protocol may be used to increase total neuropeptide identifications. Finally, we show that identical neuropeptides were detected between tissues treated with 10% NBF and an aqueous 1% w/v sodium phosphate solution (composition of 10% NBF without formaldehyde), suggesting that utilizing a salt solution wash affects neuropeptide detection and formaldehyde does not affect neuropeptide detection when our wash protocol is performed.

As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF. In the present study, we have developed an N-glycoproteomic approach that combines enhanced N-glycopeptide sequential enrichment by hydrophilic interaction chromatography (HILIC) and boronic acid enrichment with electron transfer and higher-energy collision dissociation (EThcD) for large-scale intact N-glycopeptide analysis. The application of the developed approach to the analyses of human CSF samples enabled identifications of a total of 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins. To our knowledge, this is the largest site-specific N-glycoproteome dataset reported for CSF to date. Such dataset provides molecular basis for a better understanding of the structure–function relationships of glycoproteins and their roles in CNS-related physiological and pathological processes. As accumulating evidence suggests that defects in glycosylation are involved in Alzheimer's disease (AD) pathogenesis, in the present study, a comparative in-depth N-glycoproteomic analysis was conducted for CSF samples from healthy control and AD patients, which yielded a comparable N-glycoproteome coverage but a distinct expression pattern for different categories of glycoforms, such as decreased fucosylation in AD CSF samples. Altered glycosylation patterns were detected for a number of N-glycoproteins including alpha-1-antichymotrypsin, ephrin-A3 and carnosinase CN1 etc., which serve as potentially interesting targets for further glycosylation-based AD study and may eventually lead to molecular elucidation of the role of glycosylation in AD progression., Graphical abstract, Highlights • Efficient N-glycopeptide sequential enrichment by HILIC and boronic acid enrichment. • Site-specific intact N-glycopeptide characterization using EThcD. • In-depth site-specific N-glycoproteome analysis in human CSF. • Mapping the landscape of glycosylation patterns in Alzheimer's disease., In Brief An exploratory glycosylation-based biomarker study has been conducted for in-depth mapping of an overall glycosylation landscape and site-specific alteration in glycoproteome collected from cerebrospinal fluids (CSF) in healthy control and Alzheimer’s disease (AD) subjects. The comparison will shed light on the glycoproteome profile, dominant glycosylation differences and similarities, and some of the interesting glycoprotein candidates with specific glycosylation pattern alterations in AD.

The system-wide site-specific analysis of intact glycopeptides is crucial for understanding the exact functional relevance of protein glycosylation. A dedicated workflow with the capability to simultaneously characterize and quantify intact glycopeptides in a site-specific and high-throughput manner is essential to reveal specific glycosylation alteration patterns in complex biological systems. In this study, an enhanced, dedicated, large-scale site-specific quantitative N-glycoproteomics workflow has been established, which includes improved specific extraction of membrane-bound glycoproteins using the filter aided sample preparation (FASP) method, enhanced enrichment of N-glycopeptides using sequential hydrophilic interaction liquid chromatography (HILIC) and multi-lectin affinity (MLA) enrichment, site-specific N-glycopeptide characterization enabled by EThcD, relative quantitation utilizing isobaric N,N-dimethyl leucine (DiLeu) tags and automated FDR-based large-scale data analysis by Byonic. For the first time, our study shows that HILIC complements to a very large extent to MLA enrichment with only 20% overlapping in enriching intact N-glycopeptides. When applying the developed workflow to site-specific N-glycoproteome study in PANC1 cells, we were able to identify 1067 intact N-glycopeptides, representing 311 glycosylation sites and 88 glycan compositions from 205 glycoproteins. We further applied this approach to study the glycosylation alterations in PKM2 knockout cells vs. parental breast cancer cells and revealed altered N-glycoprotein/N-glycopeptide patterns and very different glycosylation microheterogeneity for different types of glycans. To obtain a more comprehensive map of glycoprotein alterations, N-glycopeptides after treatment with PNGase F were also analyzed. A total of 484 deglycosylated peptides were quantified, among which 81 deglycosylated peptides from 70 glycoproteins showed significant changes. KEGG pathway analysis revealed that the PI3K/Akt signaling pathway was highly enriched, which provided evidence to support the previous finding that PKM2 knockdown cancer cells rely on activation of Akt for their survival. With glycosylation being one of the most important signaling modulators, our results provide additional evidence that signaling pathways are closely regulated by metabolism.

Mass spectrometry imaging (MSI) is a powerful tool that enables untargeted investigations into the spatial distribution of molecular species in a variety of samples. It has the capability to image thousands of molecules, such as metabolites, lipids, peptides, proteins, and glycans, in a single experiment without labeling. The combination of information gained from mass spectrometry (MS) and visualization of spatial distributions in thin sample sections makes this a valuable chemical analysis tool useful for biological specimen characterization. After minimal but careful sample preparation, the general setup of an MSI experiment involves defining an (x, y) grid over the surface of the sample, with the grid area chosen by the user. The mass spectrometer then ionizes the molecules on the surface of the sample and collects a mass spectrum at each pixel on the section, with the resulting spatial resolution defined by the pixel size. After collecting the spectra, computational software can be used to select an ...

Mass spectrometry-based stable isotope labeling provides the advantages of multiplexing capability and accurate quantification but requires tailored bioinformatics tools for data analysis. Despite the rapid advancements in analytical methodology, it is often challenging to analyze stable isotope labeling-based metabolomics data, particularly for isobaric labeling using MS/MS reporter ions for quantification. We report Metandem, a novel online software tool for isobaric labeling-based metabolomics, freely available at http://metandem.com/web/. Metandem provides a comprehensive data analysis pipeline integrating feature extraction, metabolite quantification, metabolite identification, batch processing of multiple data files, online parameter optimization for custom datasets, data normalization, and statistical analysis. Systematic evaluation of the Metandem tool was demonstrated on UPLC-MS/MS, nanoLC-MS/MS, CE-MS/MS and MALDI-MS platforms, via duplex, 4-plex, 10-plex, and 12-plex isobaric labeling experiments and the application to various biological samples.

Restenosis, or renarrowing of the arterial lumen, is a common recurrent disease following balloon angioplasty and stenting treatments for cardiovascular disease. A major technical barrier for deciphering restenotic mechanisms is the dynamic, spatial profiling of bioactive lipids in the arterial wall, especially in small animals. Here, applying matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI), we conducted the first lipidomic study of temporal-spatial profiling in a small animal model of angioplasty-induced restenosis. Cross sections were collected 3, 7, and 14 days after balloon angioplasty of rat carotid arteries. MALDI-MSI analyses showed that diacylglycerols (DAGs), signaling lipids associated with restenosis, and lysophosphatidylcholines (LysoPCs), whose function was uncharacterized in restenosis, dramatically increased at postangioplasty day 7 and day 14 in the neointimal layer of balloon-injured arteries compared to uninjured controls. In contrast, sphingomyelins (SMs) did not increase, but rather decreased at day 3, day 7, and day 14 in injured arteries versus the uninjured control arteries. These results revealed previously unexplored distinct temporal-spatial lipid dynamics in the restenotic arterial wall. Additionally, we employed time-of-flight secondary ion mass spectrometry (TOF-SIMS) tandem MS imaging for both molecular identification and imaging at high spatial resolution. These imaging modalities provide powerful tools for unraveling novel mechanisms of restenosis involving lipids or small signaling molecules.

Interferon signaling is upregulated in dermatomyositis and thought to play a role in pathogenesis. An interferon gene signature in peripheral blood of dermatomyositis patients correlates with skin disease. However, studies have not analyzed how interferon signaling differs across dermatomyositis subtypes or with multiple organ system involvement. We hypothesized that strength and clinical utility of the dermatomyositis blood interferon signature depends on autoantibody subtype and clinical factors. Utilizing RNA sequencing of 377 blood samples derived from a cohort of 205 clinically phenotyped dermatomyositis patients, we found that blood interferon score is significantly elevated in the anti-MDA5 subtype compared to other subtypes (average 16.12, P

We recently developed a novel amine-reactive mass-defect-based chemical tag, dimethyl pyrimidinyl ornithine (DiPyrO), for quantitative proteomic analysis at the MS(1) level. In this work, we further extend the application of the DiPyrO tag, which provides amine group reactivity, optical detection capability, and improved electrospray sensitivity, to quantify N-linked glycans enzymatically released from glycoproteins in the glycosylamine form. Duplex DiPyrO tags that differ in mass by 45.3 mDa were used to label the glycosylamine moieties of freshly released N-glycosylamines from glycoprotein standards and human serum proteins. We demonstrate that both MALDI-LTQ-Orbitrap and nano-HILIC LC/MS/MS Fusion Lumos Orbitrap platforms are capable of resolving the singly or multiply charged N-glycans labeled with mass-defect DiPyrO tags. Dynamic range of quantification, based on MS(1) peak intensities, was evaluated across 2 orders of magnitude. With optimized N-glycan release conditions, glycosylamine labeling conditions, and MS acquisition parameters, the N-glycan profiles and abundances in human serum proteins of cancer patients before and after chemotherapy were compared. Moreover, this study also opens a door for using well-developed amine-reactive tags for relative quantification of glycans, which could be widely applied.

Pancreatic cancer has the worst prognosis of all cancers (5-year survival rate of 7%). Patients that are eligible for surgery often receive adjuvant chemotherapy to improve survival. There is a critical need for a tool to match individual patients with optimal drugs for their cancer. The goal of this work is to validate Optical Metabolic Imaging (OMI) of patient-derived pancreatic tumor organoids as a high-throughput predictive drug screen for patients. Three-dimensional organoids were successfully generated from surgically resected pancreatic tumors. These organoids were treated with the patient’s prescribed adjuvant therapy, and early metabolic changes were measured using multiphoton fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzymes NAD(P)H and FAD. Changes at the single-cell level were quantified using the OMI Index, a linear combination of the optical redox ratio (ratio of the fluorescence intensities of NAD(P)H to FAD), and the mean NAD(P)H and FAD fluorescence lifetimes. Population density modeling on the OMI Index was used to evaluate cell-level heterogeneities in drug responses. Additionally, mass spectrometry imaging (MSI) was used to map metabolites in organoids. Combining multiphoton OMI images and MSI images provides a detailed map of the complex metabolic changes that occur in response to treatment, which may be used to identify additional drug targets. Patient survival data after surgery was used as validation of drug response in organoids. This platform shows promise for predicting long-term response to therapy in pancreatic cancer patients.

Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography–tandem mass spectrometry (nanoRPLC–MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. The duplex mdDiLeu reagents were custom-synthesized with a mass difference of 20.5 mDa, arising from the subtle variation in nuclear binding energy between the two DiLeu isotopologues. Optimal MS resolving powers were determined to be 240K for labeled peptides and 120K for labeled metabolites on the Orbitrap Fusion Lumos instrument. The mdDiLeu labeling does not suffer from precursor interference and dynamic range compression, providing excellent accuracy for MS1-centric quantification. Quantitative information is only revealed at high MS resolution without increasing spectrum complexity and overlapping isotope distribution. Chromatographic performance of polar metabolites was dramatically improved by mdDiLeu labeling with modified hydrophobicity, enhanced ionization efficiency, and picomole levels of detection limits. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells.

Patient-derived 3D organoids show great promise for understanding patient heterogeneity and chemotherapy response in human-derived tissue. The combination of organoid culture techniques with mass spectrometry imaging provides a label-free methodology for characterizing drug penetration, patient-specific response, and drug biotransformation. However, current methods used to grow tumor organoids employ extracellular matrices that can produce small molecule background signal during mass spectrometry imaging analysis. Here, we develop a method to isolate 3D human tumor organoids out of a Matrigel extracellular matrix into gelatin mass spectrometry compatible microarrays for high-throughput mass spectrometry imaging analysis. The alignment of multiple organoids in the same z-axis is essential for sectioning organoids together and for maintaining reproducible sample preparation on a single glass slide for up to hundreds of organoids. This method successfully removes organoids from extracellular matrix interference and provides an organized array for high-throughput imaging analysis to easily identify organoids by eye for area selection and further analysis. With this method, mass spectrometry imaging can be readily applied to organoid systems for preclinical drug development and personalized medicine research initiatives.

Background: Clinical responses to checkpoint inhibitors in gastrointestinal cancers have largely been limited to MSI-high disease. Proteolysis of an immunosuppressive proteoglycan versican (VCAN) by ADAMTS proteases correlates with enhanced CD8+ T cell infiltration in colorectal cancer independent of mismatch repair status. Here we investigate the potential of this biomarker in pancreatic ductal adenocarcinoma (PDAC) as VCAN is abundant VCAN in this disease. Methods: KPC mice were used to generate mouse PDAC tissue and organotypic spheroids. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissues across 131 patients. IHC was used to determine levels of VCAN, an immunostimulatory fragment versikine (Vkine), and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were derived from BALB/c mice. VCAN-specific ADAMTS protease expression profiles were compared between antitumor (M1) and protumor (M2) mouse macrophages, human pancreatic stellate cells (PSCs) and patient-derived PDAC-organotypic spheroids. Results: IHC analysis of normal murine pancreas demonstrated low VCAN and Vkine staining. VCAN is abundant in metaplastic/neoplastic KPC mouse pancreatic tissues. Rare VCAN proteolytic predominant areas (samples with both low VCAN and high Vkine) in KPC tumors had increased tumor-infiltrating CD8+ T cells/ high-powered field compared to weak areas (17 vs 4, p Citation Format: Philip B. Emmerich, Chelsie Sievers, Hanna Rainiero, Rosabella Pitera, Connor Maloney, Susan Payne, Mitchell Depke, Cheri Pasch, Linda Clipson, Jillian K. Johnson, Kristina Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Stromal cell driven proteolysis of versican is limited by epithelial cells in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1909.

Background: Pancreatic ductal adenocarcinoma (PDAC) recently became the third-deadliest cancer due to resistance to chemotherapy and immunotherapies. The tumor microenvironment (TME) in PDAC is thought to contribute to this resistance, with up to 80% of the tumor bulk consisting of stroma. We investigated the role of two major stromal cell types, pancreatic stellate cells (PSCs) and macrophages, in the production of the immunosuppressive proteoglycan versican (VCAN) as a precursor to the identification of novel mechanisms that may enhance therapeutic response. Methods: B6 KPC mice were utilized as a murine model of PDAC. A human PDAC tissue microarray (TMA) was developed representing normal and neoplastic pancreatic tissue across 131 patients. Immunohistochemistry (IHC) was used to determine levels of VCAN and CD8+ T cells. Bone marrow-derived macrophages (BMDMs) were differentiated from BALB/c mouse femurs and polarized to M1 (antitumor) or M2 (protumor) status. Additionally, PDAC organotypic spheroids were derived from both human and murine tissues whereas PSCs were derived solely from human tissue. Results: IHC analysis of KPC tumors revealed elevated levels of stromal VCAN compared to normal pancreatic tissue (p Conclusions: The accumulation of VCAN is common in PDAC and correlates with CD8+ T cell exclusion. Epithelial and stromal components are responsible for VCAN production. VCAN deserves further investigation as a target for therapeutic interventions for PDAC. Citation Format: Hanna R. Rainiero, Philip B. Emmerich, Chelsie K. Sievers, Connor J. Maloney, Rosabella T. Pitera, Susan N. Payne, Mitchell G. Depke, Cheri A. Pasch, Linda Clipson, Jillian K. Johnson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican production is driven by both epithelial and stromal cells in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1904.

Pancreatic cancer has the worst prognosis of all cancers (5-year survival rate of 7%). There is a critical need for a tool to match individual patients with optimal drugs for their cancer. The goal of this work is to validate Optical Metabolic Imaging (OMI) of patient-derived pancreatic tumor organoids as a high-throughput predictive drug screen for patients. Drug induced changes in cell metabolism precede changes in tumor volume and thus present an earlier marker of treatment response. OMI is sensitive to cellular metabolism because it measures the intrinsic fluorescent properties of NAD(P)H and FAD, coenzymes of metabolic reactions. OMI endpoints include the optical redox ratio (the fluorescence intensity of NAD(P)H divided by the fluorescence intensity of FAD), and the fluorescence lifetimes of NAD(P)H and FAD. The redox ratio reflects the cellular redox balance, and the fluorescence lifetimes report on the binding activity of these coenzymes. OMI, performed using high-resolution multiphoton microscopy, has the unique ability to non-invasively monitor metabolism in living, intact 3D samples on the single-cell level, and can thus quantify inter-cellular heterogeneity in drug response. OMI also allows for high-throughput screening of potential cancer drugs and drug combinations on patient biopsy samples cultured ex vivo. These patient-specific organoids are grown in a 3D matrix that mimics the natural tumor environment. Drug response can be quantified separately in tumor cells and tumor-associated fibroblasts. Organoids were successfully generated from 9 of 11 surgically resected pancreatic tumors and 2 of 2 pancreatic intraepithelial neoplasias. These organoids were treated with a panel of standard and experimental therapies, and early metabolic changes were measured using OMI. Changes at the single-cell level were quantified using the OMI Index, a linear combination of the optical redox ratio and the mean NAD(P)H and FAD fluorescence lifetimes. Patient survival data after surgery and drug treatment was compared to drug response in organoids to that same treatment. Early results have focused on the standard gemcitabine + 5-fluorouracil (G+F) adjuvant treatment. Organoids generated from a patient whose CA19-9 biomarker levels increased while on G+F adjuvant therapy contained a subpopulation of 68% of the cells which were resistant to G+F therapy. Moreover, no G+F resistant cell subpopulations were found in organoids generated from a patient with no signs of disease recurrence over 14 months of G+F adjuvant therapy. Follow-up with the remaining patients is on-going. The ability of OMI to capture heterogeneous cell-level drug responses shows promise for predicting treatment response of individual pancreatic cancer patients. Citation Format: Joe T. Sharick, Tongcheng Qian, Jillian K. Johnson, Lingjun Li, Cheri A. Pasch, Dustin A. Deming, Alexander A. Parikh, Melissa C. Skala. Personalized medicine in pancreatic cancer using cellular-resolution optical metabolic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4100.