Gallein potentiates isoniazid's ability to suppress Mycobacterium tuberculosis growth.

Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), can be difficult to treat because of drug tolerance. Increased intracellular polyphosphate (polyP) in Mtb enhances tolerance to antibiotics, and capsular polyP in Neisseria gonorrhoeae potentiates resistance to antimicrobials. The mechanism by which bacteria utilize polyP to adapt to antimicrobial pressure is not known. In this study, we found that Mtb adapts to the TB frontline antibiotic isoniazid (INH) by enhancing the accumulation of cellular, extracellular, and cell surface polyP. Gallein, a broad-spectrum inhibitor of the polyphosphate kinase that synthesizes polyP, prevents this INH-induced increase in extracellular and cell surface polyP levels. Gallein and INH work synergistically to attenuate Mtb's ability to grow in in vitro culture and within human macrophages. Mtb when exposed to INH, and in the presence of INH, gallein inhibits cell envelope formation in most but not all Mtb cells. Metabolomics indicated that INH or gallein have a modest impact on levels of Mtb metabolites, but when used in combination, they significantly reduce levels of metabolites involved in cell envelope synthesis and amino acid, carbohydrate, and nucleoside metabolism, revealing a synergistic effect. These data suggest that gallein represents a promising avenue to potentiate the treatment of TB.

Differences between human male and female neutrophils in mRNA, translation efficiency, protein, and phosphoprotein profiles.

Background Human males and females show differences in the incidence of neutrophil-associated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and reactive arthritis, and differences in neutrophil physiological responses such as a faster response to the chemorepellent SLIGKV. Little is known about the basis of sex-based differences in human neutrophils. Methods Starting with human neutrophils from healthy donors, we used RNA-seq to examine total mRNA profiles, mRNAs not associated with ribosomes and thus not being translated, mRNAs in monosomes, and mRNAs in polysomes and thus heavily translated. We used mass spectrometry systems to identify proteins and phosphoproteins. Results There were sex-based differences in the translation of 24 mRNAs. There were 132 proteins with higher levels in male neutrophils; these tended to be associated with RNA regulation, ribosome, and phosphoinositide signaling pathways, whereas 30 proteins with higher levels in female neutrophils were associated with metabolic processes, proteosomes, and phosphatase regulatory proteins. Male neutrophils had increased phosphorylation of 32 proteins. After exposure to SLIGKV, male neutrophils showed a faster response in terms of protein phosphorylation compared to female neutrophils. Conclusions Male neutrophils have higher levels of proteins and higher phosphorylation of proteins associated with RNA processing and signaling pathways, while female neutrophils have higher levels of proteins associated with metabolism and proteolytic pathways. This suggests that male neutrophils might be more ready to adapt to a new environment, and female neutrophils might be more effective at responding to pathogens. This may contribute to the observed sex-based differences in neutrophil behavior and neutrophil-associated disease incidence and severity.

PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway.

The directed movement of eukaryotic cells is crucial for processes such as embryogenesis and immune cell trafficking. The enzyme Phosphatase and tensin homolog (PTEN) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. Dictyostelium discoideum cells require both PTEN and the PTEN-like phosphatase CnrN to locally inhibit Ras activation to induce biased movement of cells away from the secreted chemorepellent protein AprA. Both PTEN and CnrN decrease basal levels of PI(3,4,5)P 3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P 2 levels, decrease PI(3,4,5)P 3 levels, inhibit proliferation, decrease myosin II phosphorylation, and increase filopod sizes. AprA causes PTEN, similar to CnrN, to localize to the side of the cell towards AprA in an AprA gradient. However, PTEN and CnrN also have distinct roles in some signaling pathways. PTEN, but not CnrN, decreases basal levels of PI(4,5)P 2 , AprA requires PTEN, but not CnrN, to induce cell roundness, and CnrN and PTEN have different effects on the number of filopods and pseudopods, and the sizes of filopods. Together, our results suggest that CnrN and PTEN play unique roles in D. discoideum signaling pathways, and possibly dephosphorylate PI(3,4,5)P 3 in different membrane domains, to mediate chemorepulsion away from AprA.

Gallein and isoniazid act synergistically to attenuate Mycobacterium tuberculosis growth in human macrophages.

Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB), can be difficult to treat because of drug resistance. Increased intracellular polyphosphate (polyP) in Mtb enhances resistance to antibiotics, and capsular polyP in Neisseria gonorrhoeae potentiates resistance to antimicrobials. The mechanism by which bacteria utilize polyP to adapt to antimicrobial pressure is not known. In this study, we found that Mtb adapts to the TB frontline antibiotic isoniazid (INH) by enhancing the accumulation of cellular, extracellular, and cell surface polyP. Gallein, a broad-spectrum inhibitor of the polyphosphate kinase that synthesizes polyP, prevents this INH-induced increase in extracellular and cell surface polyP levels. Gallein and INH work synergistically to attenuate Mtb's ability to grow in in vitro culture and within human macrophages. Mtb when exposed to INH, and in the presence of INH, gallein inhibits cell envelope formation in most but not all Mtb cells. Metabolomics indicated that INH or gallein have a modest impact on levels of Mtb metabolites, but when used in combination, they significantly reduce levels of metabolites involved in cell envelope synthesis and amino acid, carbohydrate, and nucleoside metabolism, revealing a synergistic effect. These data suggest that gallein represents a promising avenue to potentiate the treatment of TB.

Challenges and Opportunities for Commercializing Technologies in the Pulmonary Arena: An Official American Thoracic Society Report.

"Translational medicine" has been a buzzword for over two decades. The concept was intended to be lofty, to reflect a new "bench-to-bedside" approach to basic and clinical research that would bridge fields, close gaps, accelerate innovation, and shorten the time and effort it takes to bring novel technologies from basic discovery to clinical application. Has this approach been successful and lived up to its promise? Despite incredible scientific advances and innovations developed within academia, successful clinical translation into real-world solutions has been difficult. This has been particularly challenging within the pulmonary field, because there have been fewer U.S. Food and Drug Administration-approved drugs and higher failure rates for pulmonary therapies than with other common disease areas. The American Thoracic Society convened a working group with the goal of identifying major challenges related to the commercialization of technologies within the pulmonary space and opportunities to enhance this process. A survey was developed and administered to 164 participants within the pulmonary arena. This report provides a summary of these survey results. Importantly, this report identifies a number of poorly recognized challenges that exist in pulmonary academic settings, which likely contribute to diminished efficiency of commercialization efforts, ultimately hindering the rate of successful clinical translation. Because many innovations are initially developed in academic settings, this is a global public health issue that impacts the entire American Thoracic Society community. This report also summarizes key resources and opportunities and provides recommendations to enhance successful commercialization of pulmonary technologies.

Polyphosphate uses mTOR, pyrophosphate, and Rho GTPase components to potentiate bacterial survival in Dictyostelium.

IMPORTANCE: Although most bacteria are quickly killed after phagocytosis by a eukaryotic cell, some pathogenic bacteria escape death after phagocytosis. Pathogenic Mycobacterium species secrete polyP, and the polyP is necessary for the bacteria to prevent their killing after phagocytosis. Conversely, exogenous polyP prevents the killing of ingested bacteria that are normally killed after phagocytosis by human macrophages and the eukaryotic microbe Dictyostelium discoideum. This suggests the possibility that in these cells, a signal transduction pathway is used to sense polyP and prevent killing of ingested bacteria. In this report, we identify key components of the polyP signal transduction pathway in D. discoideum. In cells lacking these components, polyP is unable to inhibit killing of ingested bacteria. The pathway components have orthologs in human cells, and an exciting possibility is that pharmacologically blocking this pathway in human macrophages would cause them to kill ingested pathogens such as Mycobacterium tuberculosis.

A phosphatidylinositol phosphate kinase inhibits Ras activation and regulates chemorepulsion in Dictyostelium discoideum.

During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of cells away from a stimulus) remains poorly understood. Proliferating Dictyostelium discoideum cells secrete a chemorepellent protein called AprA. Examining existing knockout strains, we previously identified proteins required for AprA-induced chemorepulsion, and a genetic screen suggested that the enzyme phosphatidylinositol phosphate kinase A (PIPkinA, also known as Pik6) might also be needed for chemorepulsion. Here, we show that cells lacking PIPkinA are not repelled by AprA, and that this phenotype is rescued by expression of PIPkinA. To bias cell movement, AprA inhibits Ras activation at the side of the cell closest to the source of AprA, and we find that PIPkinA is required for AprA to inhibit Ras activation. PIPkinA decreases levels of phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], and possibly because of these effects, potentiates phagocytosis and inhibits cell proliferation. Cells lacking PIPkinA show normal AprA binding, suggesting that PIPkinA regulates chemorepulsion at a step between the AprA receptor and AprA inhibition of Ras activation.

Starvation Induces Extracellular Accumulation of Polyphosphate in Dictyostelium discoideum to Inhibit Macropinocytosis, Phagocytosis, and Exocytosis.

Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved D. discoideum cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity; this effect requires GrlD and Ppk1, but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, and to both decrease energy expenditures and retain nutrients.

Starvation induces extracellular accumulation of polyphosphate in Dictyostelium discoideum to inhibit macropinocytosis, phagocytosis, and exocytosis.

Dictyostelium discoideum is a soil-dwelling unicellular eukaryote that accumulates extracellular polyphosphate (polyP). At high cell densities, when the cells are about to overgrow their food supply and starve, the corresponding high extracellular concentrations of polyP allow the cells to preemptively anticipate starvation, inhibit proliferation, and prime themselves to begin development. In this report, we show that starved D. discoideum cells accumulate cell surface and extracellular polyP. Starvation reduces macropinocytosis, exocytosis, and phagocytosis, and we find that these effects require the G protein-coupled polyP receptor (GrlD) and two enzymes, Polyphosphate kinase 1 (Ppk1), which is required for synthesizing intracellular polyP, cell surface polyP, and some of the extracellular polyP, and Inositol hexakisphosphate kinase (I6kA), which is required for cell surface polyP and polyP binding to cells, and some of the extracellular polyP. PolyP reduces membrane fluidity, and we find that starvation reduces membrane fluidity, and this effect requires GrlD and Ppk1 but not I6kA. Together, these data suggest that in starved cells, extracellular polyP decreases membrane fluidity, possibly as a protective measure. In the starved cells, sensing polyP appears to decrease energy expenditure from ingestion, and decrease exocytosis, to both decrease energy expenditures and retain nutrients.

The mRNA-binding protein DDX3 mediates TGF-ß1 upregulation of translation and promotes pulmonary fibrosis.

Pulmonary fibrosis is potentiated by a positive feedback loop involving the extracellular sialidase enzyme neuraminidase 3 (NEU3) causing release of active TGF-ß1 and TGF-ß1 upregulating NEU3 by increasing translation without affecting mRNA levels. In this report, we elucidate the TGF-ß1 upregulation of the translation mechanism. In human lung fibroblasts, TGF-ß1 increased levels of proteins, including NEU3, by increasing translation of the encoding mRNAs without significantly affecting levels of these mRNAs. A total of 180 of these mRNAs shared a common 20-nucleotide motif. Deletion of this motif from NEU3 mRNA eliminated the TGF-ß1 upregulation of NEU3 translation, while insertion of this motif in 2 mRNAs insensitive to TGF-ß1 caused TGF-ß1 to upregulate their translation. RNA-binding proteins including DEAD box helicase 3, X-linked (DDX3), bind the RNA motif, and TGF-ß1 regulates their protein levels and/or binding to the motif. We found that DDX3 was upregulated in the fibrotic lesions in patients with pulmonary fibrosis, and inhibiting DDX3 in fibroblasts reduced TGF-ß1 upregulation of NEU3 levels. In the mouse bleomycin model of pulmonary fibrosis, injections of the DDX3 inhibitor RK-33 potentiated survival and reduced lung inflammation, fibrosis, and tissue levels of DDX3, TGF-ß1, and NEU3. These results suggest that inhibiting an mRNA-binding protein that mediates TGF-ß1 upregulation of translation can reduce pulmonary fibrosis.

Changes in lung sialidases in male and female mice after bleomycin aspiration.

Aim of the study: Sialidases, also called neuraminidases, are enzymes that cleave terminal sialic acids from glycoconjugates. In humans and mice, lung fibrosis is associated with desialylation of glycoconjugates and upregulation of sialidases. There are four mammalian sialidases, and it is unclear when the four mammalian sialidases are elevated over the course of inflammatory and fibrotic responses, whether tissue resident and inflammatory cells express different sialidases, and if sialidases are differentially expressed in male and females. Materials and Methods: To determine the time course of sialidase expression and the identity of sialidase expressing cells, we used the bleomycin model of pulmonary fibrosis in mice to examine levels of sialidases during inflammation (days 3 - 10) and fibrosis (days 10 - 21). Results: Bleomycin aspiration increased sialidase NEU1 at days 14 and 21 in male mice and day 10 in female mice. NEU2 levels increased at day 7 in male and day 10 in female mice. NEU3 appears to have a biphasic response in male mice with increased levels at day 7 and then at days 14 and 21, whereas in female mice NEU3 levels increased over 21 days. In control mice, the sialidases were mainly expressed by EpCAM positive epithelial cells, but after bleomycin, epithelial cells, CD45 positive immune cells, and alveolar cells expressed NEU1, NEU2, and NEU3. Sialidase expression was higher in male compared to female mice. There was little expression of NEU4 in murine lung tissue. Conclusions: These results suggest that sialidases are dynamically expressed following bleomycin, that sialidases are differentially expressed in male and females, and that of the four sialidases only NEU3 upregulation is associated with fibrosis in both male and female mice.

The sialidase NEU3 promotes pulmonary fibrosis in mice.

BACKGROUND: Sialic acid is often the distal sugar on glycoconjugates, and sialidases are enzymes that remove this sugar. In fibrotic lesions in human and mouse lungs, there is extensive desialylation of glycoconjugates, and upregulation of sialidases including the extracellular sialidase NEU3. In the bleomycin model of pulmonary fibrosis, mice lacking NEU3 (Neu3-/-) showed strongly attenuated bleomycin-induced weight loss, lung damage, inflammation, and fibrosis. This indicates that NEU3 is necessary for the full spectrum of bleomycin-induced pulmonary fibrosis. METHODS: To determine if NEU3 is sufficient to induce pulmonary fibrosis, recombinant murine NEU3 and a mutated inactive recombinant murine NEU3 protein were produced. Mice were given recombinant NEU3 proteins by oropharyngeal aspiration, either alone or 10 days after bleomycin challenge. Over the course of 21 days, mice were assessed for weight change, and after euthanasia, bronchoalveolar lavage fluid cells and lung tissue were assessed for inflammation and fibrosis. RESULTS: Aspiration of recombinant murine NEU3 caused inflammation and fibrosis in the lungs, while inactive NEU3 caused inflammation but not fibrosis. Mice were also treated with recombinant murine NEU3 starting 10 days after bleomycin. In male but not female mice, recombinant murine NEU3 increased inflammation and fibrosis. Inactive NEU3 did not enhance bleomycin-induced lung fibrosis. CONCLUSION: These results suggest that NEU3 is sufficient to induce fibrosis in the lungs, that aspiration of NEU3 has a greater effect on male mice, and that this effect is mediated by NEU3's enzymic activity.

Dictyostelium discoideum cells retain nutrients when the cells are about to outgrow their food source.

Dictyostelium discoideum is a unicellular eukaryote that eats bacteria, and eventually outgrows the bacteria. D. discoideum cells accumulate extracellular polyphosphate (polyP), and the polyP concentration increases as the local cell density increases. At high cell densities, the correspondingly high extracellular polyP concentrations allow cells to sense that they are about to outgrow their food supply and starve, causing the D. discoideum cells to inhibit their proliferation. In this report, we show that high extracellular polyP inhibits exocytosis of undigested or partially digested nutrients. PolyP decreases plasma membrane recycling and apparent cell membrane fluidity, and this requires the G protein-coupled polyP receptor GrlD, the polyphosphate kinase Ppk1 and the inositol hexakisphosphate kinase I6kA. PolyP alters protein contents in detergent-insoluble crude cytoskeletons, but does not significantly affect random cell motility, cell speed or F-actin levels. Together, these data suggest that D. discoideum cells use polyP as a signal to sense their local cell density and reduce cell membrane fluidity and membrane recycling, perhaps as a mechanism to retain ingested food when the cells are about to starve. This article has an associated First Person interview with the first author of the paper.

The extracellular sialidase NEU3 primes neutrophils.

Some extracellular glycoconjugates have sialic acid as the terminal sugar, and sialidases are enzymes that remove this sugar. Mammals have 4 sialidases and can be elevated in inflammation and fibrosis. In this report, we show that incubation of human neutrophils with the extracellular human sialidase NEU3, but not NEU1, NEU2 or NEU4, induces human male and female neutrophils to change from a round to a more amoeboid morphology, causes the primed human neutrophil markers CD11b, CD18, and CD66a to localize to the cell cortex, and decreases the localization of the unprimed human neutrophil markers CD43 and CD62-L at the cell cortex. NEU3, but not the other 3 sialidases, also causes human male and female neutrophils to increase their F-actin content. Human neutrophils treated with NEU3 show a decrease in cortical levels of Sambucus nigra lectin staining and an increase in cortical levels of peanut agglutinin staining, indicating a NEU3-induced desialylation. The inhibition of NEU3 by the NEU3 inhibitor 2-acetylpyridine attenuated the NEU3 effect on neutrophil morphology, indicating that the effect of NEU3 is dependent on its enzymatic activity. Together, these results indicate that NEU3 can prime human male and female neutrophils, and that NEU3 is a potential regulator of inflammation.

Sex-Based Differences in Human Neutrophil Chemorepulsion.

A considerable amount is known about how eukaryotic cells move toward an attractant, and the mechanisms are conserved from Dictyostelium discoideum to human neutrophils. Relatively little is known about chemorepulsion, where cells move away from a repellent signal. We previously identified pathways mediating chemorepulsion in Dictyostelium, and here we show that these pathways, including Ras, Rac, protein kinase C, PTEN, and ERK1 and 2, are required for human neutrophil chemorepulsion, and, as with Dictyostelium chemorepulsion, PI3K and phospholipase C are not necessary, suggesting that eukaryotic chemorepulsion mechanisms are conserved. Surprisingly, there were differences between male and female neutrophils. Inhibition of Rho-associated kinases or Cdc42 caused male neutrophils to be more repelled by a chemorepellent and female neutrophils to be attracted to the chemorepellent. In the presence of a chemorepellent, compared with male neutrophils, female neutrophils showed a reduced percentage of repelled neutrophils, greater persistence of movement, more adhesion, less accumulation of PI(3,4,5)P3, and less polymerization of actin. Five proteins associated with chemorepulsion pathways are differentially abundant, with three of the five showing sex dimorphism in protein localization in unstimulated male and female neutrophils. Together, this indicates a fundamental difference in a motility mechanism in the innate immune system in men and women.

Identification of novel proteins in the Dictyostelium discoideum chemorepulsion pathway using REMI.

Chemorepulsion, the biased migration of a cell away from a signal, is essential for many biological processes and the ability to manipulate chemorepulsion could lead to new therapeutics for a variety of diseases. However, little is known about eukaryotic cell chemorepulsion. Utilizing the model organism Dictyostelium discoideum, we previously identified an endogenous chemorepellent protein secreted by D. discoideum cells called AprA, and proteins involved in the AprA-induced chemorepulsion pathway including the G protein-coupled receptor GrlH, G beta and G protein alpha 8 protein subunits, protein kinase A, components of the mammalian target of rapamycin complex 2 (mTORC2), phospholipase A, PTEN and a PTEN-like phosphatase (CnrN), a retinoblastoma orthologue (RblA), extracellular signal-regulated kinase 1 (Erk1), p-21 activated protein kinase D (PakD), and the Ras proteins RasC and RasG. In this report, we used a genetic screen to identify 17 additional proteins involved in the AprA-induced chemorepulsion pathway .

Inhibitors of the Sialidase NEU3 as Potential Therapeutics for Fibrosis.

Fibrosing diseases are a major medical problem, and are associated with more deaths per year than cancer in the US. Sialidases are enzymes that remove the sugar sialic acid from glycoconjugates. In this review, we describe efforts to inhibit fibrosis by inhibiting sialidases, and describe the following rationale for considering sialidases to be a potential target to inhibit fibrosis. First, sialidases are upregulated in fibrotic lesions in humans and in a mouse model of pulmonary fibrosis. Second, the extracellular sialidase NEU3 appears to be both necessary and sufficient for pulmonary fibrosis in mice. Third, there exist at least three mechanistic ways in which NEU3 potentiates fibrosis, with two of them being positive feedback loops where a profibrotic cytokine upregulates NEU3, and the upregulated NEU3 then upregulates the profibrotic cytokine. Fourth, a variety of NEU3 inhibitors block pulmonary fibrosis in a mouse model. Finally, the high sialidase levels in a fibrotic lesion cause an easily observed desialylation of serum proteins, and in a mouse model, sialidase inhibitors that stop fibrosis reverse the serum protein desialylation. This then indicates that serum protein sialylation is a potential surrogate biomarker for the effect of sialidase inhibitors, which would facilitate clinical trials to test the exciting possibility that sialidase inhibitors could be used as therapeutics for fibrosis.

A chemorepellent inhibits local Ras activation to inhibit pseudopod formation to bias cell movement away from the chemorepellent.

The ability of cells to sense chemical gradients is essential during development, morphogenesis, and immune responses. Although much is known about chemoattraction, chemorepulsion remains poorly understood. Proliferating Dictyostelium cells secrete a chemorepellent protein called AprA. AprA prevents pseudopod formation at the region of the cell closest to the source of AprA, causing the random movement of cells to be biased away from the AprA. Activation of Ras proteins in a localized sector of a cell cortex helps to induce pseudopod formation, and Ras proteins are needed for AprA chemorepulsion. Here we show that AprA locally inhibits Ras cortical activation through the G protein-coupled receptor GrlH, the G protein subunits Gß and Gα8, Ras protein RasG, protein kinase B, the p21-activated kinase PakD, and the extracellular signal-regulated kinase Erk1. Diffusion calculations and experiments indicate that in a colony of cells, high extracellular concentrations of AprA in the center can globally inhibit Ras activation, while a gradient of AprA that naturally forms at the edge of the colony allows cells to activate Ras at sectors of the cell other than the sector of the cell closest to the center of the colony, effectively inducing both repulsion from the colony and cell differentiation. Together, these results suggest that a pathway that inhibits local Ras activation can mediate chemorepulsion.

Submicron 3-D mass spectrometry imaging reveals an asymmetric molecular distribution on chemotaxing cells.

Background: Dictyostelium discoideum is a ~10 µm diameter unicellular eukaryote that lives on soil surfaces. When starved, D. discoideum cells aggregate into streams of cells in a process called chemotaxis. In this report, we studied D. discoideum cells during chemotaxis using 3D - mass spectrometry imaging (3D-MSI). Methods: The 3D-MSI consisted of the sequential generation of 2D molecular maps using burst alignment coupled to delayed extraction time-of flight secondary ion mass spectrometry (TOF-SIMS) combined with a soft sputtering beam to access the different layers. Results: Molecular maps with sub-cellular high spatial resolution (~300 nm) indicated the presence of ions at m/z = 221 and 236 at the front and sides, but reduced levels at the back, of cells moving toward of aggregation streams. The 3D-MSI also detected an ion at m/z = 240 at the edges and back, but reduced levels at the front, of aggregating cells. Other ions showed an even distribution across the cells. Conclusions: Together, these results demonstrate the utility of sub-micron MSI to study eukaryotic chemotaxis.

Using Dictyostelium to Develop Therapeutics for Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) involves damage to lungs causing an influx of neutrophils from the blood into the lung airspaces, and the neutrophils causing further damage, which attracts more neutrophils in a vicious cycle. There are ∼190,000 cases of ARDS per year in the US, and because of the lack of therapeutics, the mortality rate is ∼40%. Repelling neutrophils out of the lung airspaces, or simply preventing neutrophil entry, is a potential therapeutic. In this minireview, we discuss how our lab noticed that a protein called AprA secreted by growing Dictyostelium cells functions as a repellent for Dictyostelium cells, causing cells to move away from a source of AprA. We then found that AprA has structural similarity to a human secreted protein called dipeptidyl peptidase IV (DPPIV), and that DPPIV is a repellent for human neutrophils. In animal models of ARDS, inhalation of DPPIV or DPPIV mimetics blocks neutrophil influx into the lungs. To move DPPIV or DPPIV mimetics into the clinic, we need to know how this repulsion works to understand possible drug interactions and side effects. Combining biochemistry and genetics in Dictyostelium to elucidate the AprA signal transduction pathway, followed by drug studies in human neutrophils to determine similarities and differences between neutrophil and Dictyostelium chemorepulsion, will hopefully lead to the safe use of DPPIV or DPPIV mimetics in the clinic.