Hepatic glutamine synthetase controls N5-methylglutamine in homeostasis and cancer.

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a ß-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite.

Impact of spatial metabolomics on immune-microenvironment in oral cancer prognosis: a clinical report.

MALDI imaging for metabolites and immunohistochemistry for 38 immune markers was used to characterize the spatial biology of 2 primary oral tumours, one from a patient with an early recurrence (Tumour R), and the other from a patient with no recurrence 2 years after treatment completion (Tumour NR). Tumour R had an increased purine nucleotide metabolism in different regions of tumour and adenosine-mediated suppression of immune cells compared to Tumour NR. The differentially expressed markers in the different spatial locations in tumour R were CD33, CD163, TGF-ß, COX2, PD-L1, CD8 and CD20. These results suggest that altered tumour metabolomics concomitant with a modified immune microenvironment could be a potential marker of recurrence.

Connecting structure and function from organisms to molecules in small-animal symbioses through chemo-histo-tomography.

Our understanding of metabolic interactions between small symbiotic animals and bacteria or parasitic eukaryotes that reside within their bodies is extremely limited. This gap in knowledge originates from a methodological challenge, namely to connect histological changes in host tissues induced by beneficial and parasitic (micro)organisms to the underlying metabolites. We addressed this challenge and developed chemo-histo-tomography (CHEMHIST), a culture-independent approach to connect anatomic structure and metabolic function in millimeter-sized symbiotic animals. CHEMHIST combines chemical imaging of metabolites based on mass spectrometry imaging (MSI) and microanatomy-based micro-computed X-ray tomography (micro-CT) on the same animal. Both high-resolution MSI and micro-CT allowed us to correlate the distribution of metabolites to the same animal's three-dimensional (3D) histology down to submicrometer resolutions. Our protocol is compatible with tissue-specific DNA sequencing and fluorescence in situ hybridization for the taxonomic identification and localization of the associated micro(organisms). Building CHEMHIST upon in situ imaging, we sampled an earthworm from its natural habitat and created an interactive 3D model of its physical and chemical interactions with bacteria and parasitic nematodes in its tissues. Combining MSI and micro-CT, we present a methodological groundwork for connecting metabolic and anatomic phenotypes of small symbiotic animals that often represent keystone species for ecosystem functioning.

A Caged In-Source Laser-Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI-MS Imaging.

Insufficient vacuum stability of matrix chemicals is a major limitation in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of large tissue sample cohorts. Here, we designed and synthesized the photo-cleavable caged molecule 4,5-dimethoxy-2-nitrobenzyl-2,5-dihydroxyacetophenone (DMNB-2,5-DHAP) and employed it for lipid MALDI-MSI of mouse brain tissue sections. DMNB-2,5-DHAP is vacuum-stable in a high vacuum MALDI ion source for at least 72 h. Investigation of the uncaging process suggested that the built-in laser (355 nm) in the MALDI ion source promoted the in situ generation of 2,5-DHAP. A caging group is used for the first time in designing a MALDI matrix that is vacuum-stable, uncaged upon laser irradiation during the measurement process, and that boosts lipid ion intensity with MALDI-2 laser-induced postionization.

Structural amyloid plaque polymorphism is associated with distinct lipid accumulations revealed by trapped ion mobility mass spectrometry imaging.

Understanding of Alzheimer's disease (AD) pathophysiology requires molecular assessment of how key pathological factors, specifically amyloid ß (Aß) plaques, influence the surrounding microenvironment. Here, neuronal lipids have been implicated in Aß plaque pathology, though the lipid microenvironment in direct proximity to Aß plaques is still not fully resolved. A further challenge is the microenvironmental molecular heterogeneity, across structurally polymorphic Aß features, such as diffuse, immature, and mature, fibrillary aggregates, whose resolution requires the integration of advanced, multimodal chemical imaging tools. Herein, we used matrix-assisted laser desorption/ionization trapped ion mobility spectrometry time-of-flight based mass spectrometry imaging (MALDI TIMS TOF MSI) in combination with hyperspectral confocal microscopy to probe the lipidomic microenvironment associated with structural polymorphism of Aß plaques in transgenic Alzheimer's disease mice (tgAPPSWE ). Using on tissue and ex situ validation, TIMS MS/MS facilitated unambiguous identification of isobaric lipid species that showed plaque pathology-associated localizations. Integrated multivariate imaging data analysis revealed multiple, Aß plaque-enriched lipid patterns for gangliosides (GM), phosphoinositols (PI), phosphoethanolamines (PE), and phosphatidic acids (PA). Conversely, sulfatides (ST), cardiolipins (CL), and polyunsaturated fatty acid (PUFA)-conjugated phosphoserines (PS), and PE were depleted at plaques. Hyperspectral amyloid imaging further delineated the unique distribution of PA and PE species to mature plaque core regions, while PI, LPI, GM2 and GM3 lipids localized to immature Aß aggregates present within the periphery of Aß plaques. Finally, we followed AD pathology-associated lipid changes over time, identifying plaque- growth and maturation to be characterized by peripheral accumulation of PI (18:0/22:6). Together, these data demonstrate the potential of multimodal imaging approaches to overcome limitations associated with conventional advanced MS imaging applications. This allowed for the differentiation of both distinct lipid components in a complex micro-environment as well as their correlation to disease-relevant amyloid plaque polymorphs. Cover Image for this issue: https://doi.org/10.1111/jnc.15390.

Rapid visualization of lipopeptides and potential bioactive groups of compounds by combining ion mobility and MALDI imaging mass spectrometry.

Mass spectrometry imaging (MSI) has become a powerful method for mapping metabolite distribution in a tissue. Applied to bacterial colonies, MSI has a bright future, both for the discovery of new bioactive compounds and for a better understanding of bacterial antibiotic resistance mechanisms. Coupled with separation techniques such as ion mobility mass spectrometry (IM-MS), the identification of metabolites directly on the image is now possible and does not require additional analysis such as HPLC-MS/MS. In this article, we propose to apply a semi-targeted workflow for rapid IM-MSI data analysis focused on the search for bioactive compounds. First, chemically-related compounds showing a repetitive mass unit (i.e. lipids and lipopeptides) were targeted based on the Kendrick mass defect analysis. The detected groups of potentially bioactive compounds were then confirmed by fitting their measured ion moibilites to their measured m/z values. Using both their m/z and ion mobility values, the selected groups of compounds were identified using the available databases and finally their distribution was observed on the image. Using this workflow on a co-culture of bacteria, we were able to detect and localize bioactive compounds involved in the microbial interaction.

A new classification method for MALDI imaging mass spectrometry data acquired on formalin-fixed paraffin-embedded tissue samples.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) shows a high potential for applications in histopathological diagnosis, and in particular for supporting tumor typing and subtyping. The development of such applications requires the extraction of spectral fingerprints that are relevant for the given tissue and the identification of biomarkers associated with these spectral patterns. We propose a novel data analysis method based on the extraction of characteristic spectral patterns (CSPs) that allow automated generation of classification models for spectral data. Formalin-fixed paraffin embedded (FFPE) tissue samples from N=445 patients assembled on 12 tissue microarrays were analyzed. The method was applied to discriminate primary lung and pancreatic cancer, as well as adenocarcinoma and squamous cell carcinoma of the lung. A classification accuracy of 100% and 82.8%, resp., could be achieved on core level, assessed by cross-validation. The method outperformed the more conventional classification method based on the extraction of individual m/z values in the first application, while achieving a comparable accuracy in the second. LC-MS/MS peptide identification demonstrated that the spectral features present in selected CSPs correspond to peptides relevant for the respective classification. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Integration of 3D multimodal imaging data of a head and neck cancer and advanced feature recognition.

In the last years, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) became an imaging technique which has the potential to characterize complex tumor tissue. The combination with other modalities and with standard histology techniques was achieved by the use of image registration methods and enhances analysis possibilities. We analyzed an oral squamous cell carcinoma with up to 162 consecutive sections with MALDI MSI, hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) against CD31. Spatial segmentation maps of the MALDI MSI data were generated by similarity-based clustering of spectra. Next, the maps were overlaid with the H&E microscopy images and the results were interpreted by an experienced pathologist. Image registration was used to fuse both modalities and to build a three-dimensional (3D) model. To visualize structures below resolution of MALDI MSI, IHC was carried out for CD31 and results were embedded additionally. The integration of 3D MALDI MSI data with H&E and IHC images allows a correlation between histological and molecular information leading to a better understanding of the functional heterogeneity of tumors. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Two new isoforms of the human hepatoma-derived growth factor interact with components of the cytoskeleton.

Hepatoma-derived growth factor (HDGF) is involved in diverse, apparently unrelated processes, such as cell proliferation, apoptosis, DNA-repair, transcriptional control, ribosome biogenesis and cell migration. Most of the interactions of HDGF with diverse molecules has been assigned to the hath region of HDGF. In this study we describe two previously unknown HDGF isoforms, HDGF-B and HDGF-C, generated via alternative splicing with structurally unrelated N-terminal regions of their hath region, which is clearly different from the well described isoform, HDGF-A. In silico modeling revealed striking differences near the PHWP motif, an essential part of the binding site for glycosaminoglycans and DNA/RNA. This observation prompted the hypothesis that these isoforms would have distinct interaction patterns with correspondingly diverse roles on cellular processes. Indeed, we discovered specific associations of HDGF-B and HDGF-C with cytoskeleton elements, such as tubulin and dynein, suggesting previously unknown functions of HDGF in retrograde transport, site directed localization and/or cytoskeleton organization. In contrast, the main isoform HDGF-A does not interact directly with the cytoskeleton, but via RNA with messenger ribonucleoprotein (mRNP) complexes. In summary, the discovery of HDGF splice variants with their discrete binding activities and subcellular distributions opened new avenues for understanding its biological function and importance.

An approach to optimize sample preparation for MALDI imaging MS of FFPE sections using fractional factorial design of experiments.

A standardized workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI imaging MS) is a prerequisite for the routine use of this promising technology in clinical applications. We present an approach to develop standard operating procedures for MALDI imaging MS sample preparation of formalin-fixed and paraffin-embedded (FFPE) tissue sections based on a novel quantitative measure of dataset quality. To cover many parts of the complex workflow and simultaneously test several parameters, experiments were planned according to a fractional factorial design of experiments (DoE). The effect of ten different experiment parameters was investigated in two distinct DoE sets, each consisting of eight experiments. FFPE rat brain sections were used as standard material because of low biological variance. The mean peak intensity and a recently proposed spatial complexity measure were calculated for a list of 26 predefined peptides obtained by in silico digestion of five different proteins and served as quality criteria. A five-way analysis of variance (ANOVA) was applied on the final scores to retrieve a ranking of experiment parameters with increasing impact on data variance. Graphical abstract MALDI imaging experiments were planned according to fractional factorial design of experiments for the parameters under study. Selected peptide images were evaluated by the chosen quality metric (structure and intensity for a given peak list), and the calculated values were used as an input for the ANOVA. The parameters with the highest impact on the quality were deduced and SOPs recommended.

2D and 3D MALDI-imaging: conceptual strategies for visualization and data mining.

3D imaging has a significant impact on many challenges in life sciences, because biology is a 3-dimensional phenomenon. Current 3D imaging-technologies (various types MRI, PET, SPECT) are labeled, i.e. they trace the localization of a specific compound in the body. In contrast, 3D MALDI mass spectrometry-imaging (MALDI-MSI) is a label-free method imaging the spatial distribution of molecular compounds. It complements 3D imaging labeled methods, immunohistochemistry, and genetics-based methods. However, 3D MALDI-MSI cannot tap its full potential due to the lack of statistical methods for analysis and interpretation of large and complex 3D datasets. To overcome this, we established a complete and robust 3D MALDI-MSI pipeline combined with efficient computational data analysis methods for 3D edge preserving image denoising, 3D spatial segmentation as well as finding colocalized m/z values, which will be reviewed here in detail. Furthermore, we explain, why the integration and correlation of the MALDI imaging data with other imaging modalities allows to enhance the interpretation of the molecular data and provides visualization of molecular patterns that may otherwise not be apparent. Therefore, a 3D data acquisition workflow is described generating a set of 3 different dimensional images representing the same anatomies. First, an in-vitro MRI measurement is performed which results in a three-dimensional image modality representing the 3D structure of the measured object. After sectioning the 3D object into N consecutive slices, all N slices are scanned using an optical digital scanner, enabling for performing the MS measurements. Scanning the individual sections results into low-resolution images, which define the base coordinate system for the whole pipeline. The scanned images conclude the information from the spatial (MRI) and the mass spectrometric (MALDI-MSI) dimension and are used for the spatial three-dimensional reconstruction of the object performed by image registration techniques. Different strategies for automatic serial image registration applied to MS datasets are outlined in detail. The third image modality is histology driven, i.e. a digital scan of the histological stained slices in high-resolution. After fusion of reconstructed scan images and MRI the slice-related coordinates of the mass spectra can be propagated into 3D-space. After image registration of scan images and histological stained images, the anatomical information from histology is fused with the mass spectra from MALDI-MSI. As a result of the described pipeline we have a set of 3 dimensional images representing the same anatomies, i.e. the reconstructed slice scans, the spectral images as well as corresponding clustering results, and the acquired MRI. Great emphasis is put on the fact that the co-registered MRI providing anatomical details improves the interpretation of 3D MALDI images. The ability to relate mass spectrometry derived molecular information with in vivo and in vitro imaging has potentially important implications. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.

MALDI imaging mass spectrometry: discrimination of pathophysiological regions in traumatized skeletal muscle by characteristic peptide signatures.

Due to formation of fibrosis and the loss of contractile muscle tissue, severe muscle injuries often result in insufficient healing marked by a significant reduction of muscle force and motor activity. Our previous studies demonstrated that the local transplantation of mesenchymal stromal cells into an injured skeletal muscle of the rat improves the functional outcome of the healing process. Since, due to the lack of sufficient markers, the accurate discrimination of pathophysiological regions in injured skeletal muscle is inadequate, underlying mechanisms of the beneficial effects of mesenchymal stromal cell transplantation on primary trauma and trauma adjacent muscle area remain elusive. For discrimination of these pathophysiological regions, formalin-fixed injured skeletal muscle tissue was analyzed by MALDI imaging MS. By using two computational evaluation strategies, a supervised approach (ClinProTools) and unsupervised segmentation (SCiLS Lab), characteristic m/z species could be assigned to primary trauma and trauma adjacent muscle regions. Using "bottom-up" MS for protein identification and validation of results by immunohistochemistry, we could identify two proteins, skeletal muscle alpha actin and carbonic anhydrase III, which discriminate between the secondary damage on adjacent tissue and the primary traumatized muscle area. Our results underscore the high potential of MALDI imaging MS to describe the spatial characteristics of pathophysiological changes in muscle.

Deep topographic proteomics of a human brain tumour.

The spatial organisation of cellular protein expression profiles within tissue determines cellular function and is key to understanding disease pathology. To define molecular phenotypes in the spatial context of tissue, there is a need for unbiased, quantitative technology capable of mapping proteomes within tissue structures. Here, we present a workflow for spatially-resolved, quantitative proteomics of tissue that generates maps of protein abundance across tissue slices derived from a human atypical teratoid-rhabdoid tumour at three spatial resolutions, the highest being 40 µm, to reveal distinct abundance patterns of thousands of proteins. We employ spatially-aware algorithms that do not require prior knowledge of the fine tissue structure to detect proteins and pathways with spatial abundance patterns and correlate proteins in the context of tissue heterogeneity and cellular features such as extracellular matrix or proximity to blood vessels. We identify PYGL, ASPH and CD45 as spatial markers for tumour boundary and reveal immune response-driven, spatially-organised protein networks of the extracellular tumour matrix. Overall, we demonstrate spatially-aware deep proteo-phenotyping of tissue heterogeneity, to re-define understanding tissue biology and pathology at the molecular level.

Multiplexed neuropeptide mapping in ant brains integrating microtomography and three-dimensional mass spectrometry imaging.

Neuropeptides are important regulators of animal physiology and behavior. Hitherto the gold standard for the localization of neuropeptides have been immunohistochemical methods that require the synthesis of antibody panels, while another limiting factor has been the brain's opacity for subsequent in situ light or fluorescence microscopy. To address these limitations, we explored the integration of high-resolution mass spectrometry imaging (MSI) with microtomography for a multiplexed mapping of neuropeptides in two evolutionary distant ant species, Atta sexdens and Lasius niger. For analyzing the spatial distribution of chemically diverse peptide molecules across the brain in each species, the acquisition of serial mass spectrometry images was essential. As a result, we have comparatively mapped the three-dimensional (3D) distributions of eight conserved neuropeptides throughout the brain microanatomy. We demonstrate that integrating the 3D MSI data into high-resolution anatomy models can be critical for studying organs with high plasticity such as brains of social insects. Several peptides, like the tachykinin-related peptides (TK) 1 and 4, were widely distributed in many brain areas of both ant species, whereas others, for instance myosuppressin, were restricted to specific regions only. Also, we detected differences at the species level; many peptides were identified in the optic lobe of L. niger, but only one peptide (ITG-like) was found in this region in A. sexdens. Building upon MS imaging studies on neuropeptides in invertebrate model systems, our approach leverages correlative MSI and computed microtomography for investigating fundamental neurobiological processes by visualizing the unbiased 3D neurochemistry in its complex anatomic environment.

Exploring three-dimensional matrix-assisted laser desorption/ionization imaging mass spectrometry data: three-dimensional spatial segmentation of mouse kidney.

Three-dimensional (3D) imaging has a significant impact on many challenges of life sciences. Three-dimensional matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an emerging label-free bioanalytical technique capturing the spatial distribution of hundreds of molecular compounds in 3D by providing a MALDI mass spectrum for each spatial point of a 3D sample. Currently, 3D MALDI-IMS cannot tap its full potential due to the lack efficient computational methods for constructing, processing, and visualizing large and complex 3D MALDI-IMS data. We present a new pipeline of efficient computational methods, which enables analysis and interpretation of a 3D MALDI-IMS data set. Construction of a MALDI-IMS data set was done according to the state-of-the-art protocols and involved sample preparation, spectra acquisition, spectra preprocessing, and registration of serial sections. For analysis and interpretation of 3D MALDI-IMS data, we applied the spatial segmentation approach which is well-accepted in analysis of two-dimensional (2D) MALDI-IMS data. In line with 2D data analysis, we used edge-preserving 3D image denoising prior to segmentation to reduce strong and chaotic spectrum-to-spectrum variation. For segmentation, we used an efficient clustering method, called bisecting k-means, which is optimized for hierarchical clustering of a large 3D MALDI-IMS data set. Using the proposed pipeline, we analyzed a central part of a mouse kidney using 33 serial sections of 3.5 µm thickness after the PAXgene tissue fixation and paraffin embedding. For each serial section, a 2D MALDI-IMS data set was acquired following the standard protocols with the high spatial resolution of 50 µm. Altogether, 512 495 mass spectra were acquired that corresponds to approximately 50 gigabytes of data. After registration of serial sections into a 3D data set, our computational pipeline allowed us to reveal the 3D kidney anatomical structure based on mass spectrometry data only. Finally, automated analysis discovered molecular masses colocalized with major anatomical regions. In the same way, the proposed pipeline can be used for analysis and interpretation of any 3D MALDI-IMS data set in particular of pathological cases.

In situ isobaric lipid mapping by MALDI-ion mobility separation-mass spectrometry imaging.

The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high-resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids.

Characterization of the DraT/DraG system for posttranslational regulation of nitrogenase in the endophytic betaproteobacterium Azoarcus sp. strain BH72.

DraT/DraG-mediated posttranslational regulation of the nitrogenase Fe protein by ADP-ribosylation has been described for a few diazotrophic bacteria belonging to the class Alphaproteobacteria. Here we present for the first time the DraT/DraG system of a betaproteobacterium, Azoarcus sp. strain BH72, a diazotrophic grass endophyte. Its genome harbors one draT ortholog and two physically unlinked genes coding for ADP-ribosylhydrolases. Northern blot analysis revealed cotranscription of draT with two genes encoding hypothetical proteins. Furthermore, draT and draG2 were expressed under all studied conditions, whereas draG1 expression was nitrogen regulated. By using Western blot analysis of deletion mutants and nitrogenase assays in vivo, we demonstrated that DraT is required for the nitrogenase Fe protein modification but not for the physiological inactivation of nitrogenase activity. A second mechanism responsible for nitrogenase inactivation must operate in this bacterium, which is independent of DraT. Fe protein demodification was dependent mainly on DraG1, corroborating the assumption from phylogenetic analysis that DraG2 might be mostly involved in processes other than the posttranslational regulation of nitrogenase. Nitrogenase in vivo reactivation was impaired in a draG1 mutant and a mutant lacking both draG alleles after anaerobiosis shifts and subsequent adjustment to microaerobic conditions, suggesting that modified dinitrogenase reductase was inactive. Our results demonstrate that the DraT/DraG system, despite some differences, is functionally conserved in diazotrophic proteobacteria.

Targeted Feature Extraction in MALDI Mass Spectrometry Imaging to Discriminate Proteomic Profiles of Breast and Ovarian Cancer.

PURPOSE: To develop a mass spectrometry imaging (MSI) based workflow for extracting m/z values related to putative protein biomarkers and using these for reliable tumor classification. EXPERIMENTAL DESIGN: Given a list of putative breast and ovarian cancer biomarker proteins, a set of related m/z values are extracted from heterogeneous MSI datasets derived from formalin-fixed paraffin-embedded tissue material. Based on these features, a linear discriminant analysis classification model is trained to discriminate the two tumor types. RESULTS: It is shown that the discriminative power of classification models based on the extracted features is increased compared to the automatic training approach, especially when classifiers are applied to spectral data acquired under different conditions (instrument, preparation, laboratory). CONCLUSIONS AND CLINICAL RELEVANCE: Robust classification models not confounded by technical variation between MSI measurements are obtained. This supports the assumption that the classification of the respective tumor types is based on biological rather than technical differences, and that the selected features are related to the proteomic profiles of the tumor types under consideration.

Neratinib protects pancreatic beta cells in diabetes.

The loss of functional insulin-producing ß-cells is a hallmark of diabetes. Mammalian sterile 20-like kinase 1 (MST1) is a key regulator of pancreatic ß-cell death and dysfunction; its deficiency restores functional ß-cells and normoglycemia. The identification of MST1 inhibitors represents a promising approach for a ß-cell-protective diabetes therapy. Here, we identify neratinib, an FDA-approved drug targeting HER2/EGFR dual kinases, as a potent MST1 inhibitor, which improves ß-cell survival under multiple diabetogenic conditions in human islets and INS-1E cells. In a pre-clinical study, neratinib attenuates hyperglycemia and improves ß-cell function, survival and ß-cell mass in type 1 (streptozotocin) and type 2 (obese Leprdb/db) diabetic mouse models. In summary, neratinib is a previously unrecognized inhibitor of MST1 and represents a potential ß-cell-protective drug with proof-of-concept in vitro in human islets and in vivo in rodent models of both type 1 and type 2 diabetes.

Molecular and chemical dialogues in bacteria-protozoa interactions.

Protozoan predation of bacteria can significantly affect soil microbial community composition and ecosystem functioning. Bacteria possess diverse defense strategies to resist or evade protozoan predation. For soil-dwelling Pseudomonas species, several secondary metabolites were proposed to provide protection against different protozoan genera. By combining whole-genome transcriptome analyses with (live) imaging mass spectrometry (IMS), we observed multiple changes in the molecular and chemical dialogues between Pseudomonas fluorescens and the protist Naegleria americana. Lipopeptide (LP) biosynthesis was induced in Pseudomonas upon protozoan grazing and LP accumulation transitioned from homogeneous distributions across bacterial colonies to site-specific accumulation at the bacteria-protist interface. Also putrescine biosynthesis was upregulated in P. fluorescens upon predation. We demonstrated that putrescine induces protozoan trophozoite encystment and adversely affects cyst viability. This multifaceted study provides new insights in common and strain-specific responses in bacteria-protozoa interactions, including responses that contribute to bacterial survival in highly competitive soil and rhizosphere environments.