The novel bacteriocin BacYB1 produced by Leuconostoc mesenteroides YB1: From recent analytical characterization to biocontrol Verticillium dahliae and Agrobacterium tumefaciens.

Biocontrol of phytopathogens involving the use of bioactive compounds produced by lactic acid bacteria (LAB), is a promising approach to manage many diseases in agriculture. In this study, a lactic acid bacterium designated YB1 was isolated from fermented olives and selected for its antagonistic activity against Verticillium dahliae (V. dahliae) and Agrobacterium tumefaciens (A. tumefaciens). Based on the 16S rRNA gene nucleotide sequence analysis (1565 pb, accession number: OR714267), the new isolate YB1 bacterium was assigned as Leuconostoc mesenteroides YB1 (OR714267) strain. This bacterium produces an active peptide "bacteriocin" called BacYB1, which was purified in four steps. Matrix-assisted lasers desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) based approach was performed to identify and characterize BacYB1. The exact mass was 5470.75 Da, and the analysis of the N-terminal sequence (VTRASGASTPPGTASPFKTL) of BacYB1 revealed no significant similarity to currently available antimicrobial peptides. The BacYB1 displayed a bactericidal mode of action against A. tumefaciens. The potentiel role of BacYB1 to supress the growth of A. tumefaciens was confirmed by live-dead cells viability assay. In pot experiments, the biocontrol efficacy of BacYB1 against V. dahliae wilt on young olive trees was studied. The percentage of dead plants (PDP) and the final mean symptomes severity (FMS) of plants articifialy infected by V. dahliae and treated with the pre-purified peptide BacYB1 (preventive and curative treatments) were significantly inferior to untreated plants. Biochemical analysis of leaves of the plants has shown that polyophenols contents were highly detected in plants infected by V. dahliae and the highest contents of chlorophyl a, b and total chlorophyll were recorded in plants treated with the combination of BacYB1 with the biofertilisant Humivital. BacYB1 presents a promising alternative for the control of Verticillium wilt and crown gall diseases.

Microarrays, Enzymatic Assays, and MALDI-MS for Determining Specific Alterations to Mitochondrial Electron Transport Chain Activity, ROS Formation, and Lipid Composition in a Monkey Model of Parkinson's Disease.

Multiple evidences suggest that mitochondrial dysfunction is implicated in the pathogenesis of Parkinson's disease via the selective cell death of dopaminergic neurons, such as that which occurs after prolonged exposure to the mitochondrial electron transport chain (ETC) complex I inhibitor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP). However, the effects of chronic MPTP on the ETC complexes and on enzymes of lipid metabolism have not yet been thoroughly determined. To face these questions, the enzymatic activities of ETC complexes and the lipidomic profile of MPTP-treated non-human primate samples were determined using cell membrane microarrays from different brain areas and tissues. MPTP treatment induced an increase in complex II activity in the olfactory bulb, putamen, caudate, and substantia nigra, where a decrease in complex IV activity was observed. The lipidomic profile was also altered in these areas, with a reduction in the phosphatidylserine (38:1) content being especially relevant. Thus, MPTP treatment not only modulates ETC enzymes, but also seems to alter other mitochondrial enzymes that regulate the lipid metabolism. Moreover, these results show that a combination of cell membrane microarrays, enzymatic assays, and MALDI-MS provides a powerful tool for identifying and validating new therapeutic targets that might accelerate the drug discovery process.

Novel Combined Enzymatic Approach to Analyze Nonsialylated N-Linked Glycans through MALDI Imaging Mass Spectrometry.

Alterations to N-glycan expression are relevant to the progression of various diseases, particularly cancer. In many cases, specific N-glycan structural features such as sialylation, fucosylation, and branching are of specific interest. A novel MALDI imaging mass spectrometry workflow has been recently developed to analyze these features of N-glycosylation through the utilization of endoglycosidase enzymes to cleave N-glycans from associated glycoproteins. Enzymes that have previously been utilized to cleave N-glycans include peptide-N-glycosidase F (PNGase F) to target N-glycans indiscriminately and endoglycosidase F3 (Endo F3) to target core fucosylated N-glycans. In addition to these endoglycosidases, additional N-glycan cleaving enzymes could be used to target specific structural features. Sialidases, also termed neuraminidases, are a family of enzymes that remove terminal sialic acids from glycoconjugates. This work aims to utilize sialidase, in conjunction with PNGase F/Endo F3, to enzymatically remove sialic acids from N-glycans in an effort to increase sensitivity for nonsialylated N-glycan MALDI-IMS peaks. Improving detection of nonsialylated N-glycans allows for a more thorough analysis of specific structural features such as fucosylation or branching, particularly of low abundant structures. Sialidase utilization in MALDI-IMS dramatically increases sensitivity and increases on-tissue endoglycosidase efficiency, making it a very useful companion technique to specifically detect nonsialylated N-glycans.

Spatial N-glycomics of the human aortic valve in development and pediatric endstage congenital aortic valve stenosis.

Congenital aortic valve stenosis (AS) progresses as an obstructive narrowing of the aortic orifice due to deregulated extracellular matrix (ECM) production by aortic valve (AV) leaflets and leads to heart failure with no effective therapies. Changes in glycoprotein and proteoglycan distribution are a hallmark of AS, yet valvular carbohydrate content remains virtually uncharacterized at the molecular level. While almost all glycoproteins clinically linked to stenotic valvular modeling contain multiple sites for N-glycosylation, there are very few reports aimed at understanding how N-glycosylation contributes to the valve structure in disease. Here, we tested for spatial localization of N-glycan structures within pediatric congenital aortic valve stenosis. The study was done on valvular tissues 0-17 years of age with de-identified clinical data reporting pre-operative valve function spanning normal development, aortic valve insufficiency (AVI), and pediatric endstage AS. High mass accuracy imaging mass spectrometry (IMS) was used to localize N-glycan profiles in the AV structure. RNA-Seq was used to identify regulation of N-glycan related enzymes. The N-glycome was found to be spatially localized in the normal aortic valve, aligning with fibrosa, spongiosa or ventricularis. In AVI diagnosed tissue, N-glycans localized to hypertrophic commissures with increases in pauci-mannose structures. In all valve types, sialic acid (N-acetylneuraminic acid) N-glycans were the most abundant N-glycan group. Three sialylated N-glycans showed common elevation in AS independent of age. On-tissue chemical methods optimized for valvular tissue determined that aortic valve tissue sialylation shows both α2,6 and α2,3 linkages. Specialized enzymatic strategies demonstrated that core fucosylation is the primary fucose configuration and localizes to the normal fibrosa with disparate patterning in AS. This study identifies that the human aortic valve structure is spatially defined by N-glycomic signaling and may generate new research directions for the treatment of human aortic valve disease.

Brain region-specific amyloid plaque-associated myelin lipid loss, APOE deposition and disruption of the myelin sheath in familial Alzheimer's disease mice.

There is emerging evidence that amyloid beta (Aß) aggregates forming neuritic plaques lead to impairment of the lipid-rich myelin sheath and glia. In this study, we examined focal myelin lipid alterations and the disruption of the myelin sheath associated with amyloid plaques in a widely used familial Alzheimer's disease (AD) mouse model; 5xFAD. This AD mouse model has Aß42 peptide-rich plaque deposition in the brain parenchyma. Matrix-assisted laser desorption/ionization imaging mass spectrometry of coronal brain tissue sections revealed focal Aß plaque-associated depletion of multiple myelin-associated lipid species including sulfatides, galactosylceramides, and specific plasmalogen phopshatidylethanolamines in the hippocampus, cortex, and on the edges of corpus callosum. Certain phosphatidylcholines abundant in myelin were also depleted in amyloid plaques on the edges of corpus callosum. Further, lysophosphatidylethanolamines and lysophosphatidylcholines, implicated in neuroinflammation, were found to accumulate in amyloid plaques. Double staining of the consecutive sections with fluoromyelin and amyloid-specific antibody revealed amyloid plaque-associated myelin sheath disruption on the edges of the corpus callosum which is specifically correlated with plaque-associated myelin lipid loss only in this region. Further, apolipoprotein E, which is implicated in depletion of sulfatides in AD brain, is deposited in all the Aß plaques which suggest apolipoprotein E might mediate sulfatide depletion as a consequence of an immune response to Aß deposition. This high-spatial resolution matrix-assisted laser desorption/ionization imaging mass spectrometry study in combination with (immuno) fluorescence staining of 5xFAD mouse brain provides new understanding of morphological, molecular and immune signatures of Aß plaque pathology-associated myelin lipid loss and myelin degeneration in a brain region-specific manner. Read the Editorial Highlight for this article on page 7.

Zonal regulation of collagen-type proteins and posttranslational modifications in prostatic benign and cancer tissues by imaging mass spectrometry.

BACKGROUND: The emergence of reactive stroma is a hallmark of prostate cancer (PCa) progression and a potential source for prognostic and diagnostic markers of PCa. Collagen is a main component of reactive stroma and changes systematically and quantitatively to reflect the course of PCa, yet has remained undefined due to a lack of tools that can define collagen protein structure. Here we use a novel collagen-targeting proteomics approach to investigate zonal regulation of collagen-type proteins in PCa prostatectomies. METHODS: Prostatectomies from nine patients were divided into zones containing 0%, 5%, 20%, 70% to 80% glandular tissue and 0%, 5%, 25%, 70% by mass of PCa tumor following the McNeal model. Tissue sections from zones were graded by a pathologist for Gleason score, percent tumor present, percent prostatic intraepithelial neoplasia and/or inflammation (INF). High-resolution accurate mass collagen targeting proteomics was done on a select subset of tissue sections from patient-matched tumor or nontumor zones. Imaging mass spectrometry was used to investigate collagen-type regulation corresponding to pathologist-defined regions. RESULTS: Complex collagen proteomes were detected from all zones. COL17A and COL27A increased in zones of INF compared with zones with tumor present. COL3A1, COL4A5, and COL8A2 consistently increased in zones with tumor content, independent of tumor size. Collagen hydroxylation of proline (HYP) was altered in tumor zones compared with zones with INF and no tumor. COL3A1 and COL5A1 showed significant changes in HYP peptide ratios within tumor compared with zones of INF (2.59 ± 0.29, P value: .015; 3.75 ± 0.96 P value .036, respectively). By imaging mass spectrometry COL3A1 showed defined localization and regulation to tumor pathology. COL1A1 and COL1A2 showed gradient regulation corresponding to PCa pathology across zones. Pathologist-defined tumor regions showed significant increases in COL1A1 HYP modifications compared with COL1A2 HYP modifications. Certain COL1A1 and COL1A2 peptides could discriminate between pathologist-defined tumor and inflammatory regions. CONCLUSIONS: Site-specific posttranslational regulation of collagen structure by proline hydroxylation may be involved in reactive stroma associated with PCa progression. Translational and posttranslational regulation of collagen protein structure has potential for new markers to understand PCa progression and outcomes.

Mapping Extracellular Matrix Proteins in Formalin-Fixed, Paraffin-Embedded Tissues by MALDI Imaging Mass Spectrometry.

Collagens and elastin form the fundamental framework of all tissues and organs, and their expression and post-translational processing are tightly regulated in disease and health. Because of their unique structural composition and properties, it is a recognized challenge to access these protein structures within the complex tissue microenvironment to understand how localized changes modulate tissue health. We describe a new workflow using a combination of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) with matrix metalloproteinase (MMP) enzymes to access and report on spatial localization of collagen and elastin sequences in formalin-fixed, paraffin-embedded (FFPE) tissues. The developed technology provides new access to collagens and elastin sequences localized to tissue features that were previously unattainable. This high-throughput technological advance should be applicable to any tissue regardless of disease type, tissue origin, or disease status and is thus relevant to all research: basic, translational, or clinical.

Membrane-lipid homeostasis in a prodromal rat model of Alzheimer's disease: Characteristic profiles in ganglioside distributions during aging detected using MALDI imaging mass spectrometry.

BACKGROUND: Accumulation of simple gangliosides GM2 and GM3, and gangliosides with longer long-chain bases (d20:1) have been linked to toxicity and the pathogenesis of Alzheimer's disease (AD). Conversely, complex gangliosides, such as GM1, have been shown to be neuroprotective. Recent evidence using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) has demonstrated that a-series gangliosides are differentially altered during normal aging, yet it remains unclear how simple species are shifting relative to complex gangliosides in the prodromal stages of AD. METHODS: Ganglioside profiles in wild-type (Wt) and transgenic APP21 Fischer rats were detected and quantified using MALDI-IMS at P0 (birth), 3, 12, and 20 months of age and each species quantified to allow for individual species comparisons. RESULTS: Tg APP21 rats were found to have a decreased level of complex gangliosides in a number of brain regions as compared to Wt rats and showed higher levels of simple gangliosides. A unique pattern of expression was observed in the white matter as compared to gray matter regions, with an age-dependent decrease in GD1 d18:1 species observed and significantly elevated levels of GM3 in Tg APP21 rats. CONCLUSIONS: These results are indicative of a pathological shift in ganglioside homeostasis during aging that is exacerbated in Tg APP21 rats. GENERAL SIGNIFICANCE: Ganglioside dysregulation may occur in the prodromal stages of neurodegenerative diseases like AD.

MALDI Mass Spectrometry Imaging of N-Linked Glycans in Tissues.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used for two decades to profile the glycan constituents of biological samples. An adaptation of the method to tissues, MALDI mass spectrometry imaging (MALDI-MSI), allows high-throughput spatial profiling of hundreds to thousands of molecules within a single thin tissue section. The ability to profile N-glycans within tissues using MALDI-MSI is a recently developed method that allows identification and localization of 40 or more N-glycans. The key component is to apply a molecular coating of peptide-N-glycosidase to tissues, an enzyme that releases N-glycans from their protein carrier. In this chapter, the methods and approaches to robustly and reproducibly generate two-dimensional N-glycan tissue maps by MALDI-MSI workflows are summarized. Current strengths and limitations of the approach are discussed, as well as potential future applications of the method.

Glucose Restriction Combined with Autophagy Inhibition and Chemotherapy in HCT 116 Spheroids Decreases Cell Clonogenicity and Viability Regulated by Tumor Suppressor Genes.

Drug resistance is a prevalent phenomenon that decreases the efficacy of cancer treatments and contributes to cancer progression and metastasis. Weakening drug-resistant cancer cells prior to chemotherapy is a potential strategy to combat chemoresistance. One approach to damage resistant cancer cells is modulation of nutritional intake. The combination of nutrient restriction with targeted compound treatment results in pronounced molecular changes. This study provides valuable information about augmenting existing chemotherapeutic regimes with simultaneous glucose restriction and autophagy inhibition in colorectal cancer cells. In this study, we explore the chemical pathways that drive the cellular response to nutrient restriction, autophagy inhibition, and the chemotherapy irinotecan using global quantitative proteomics and imaging mass spectrometry. We determined that significant pathways were altered including autophagy and metabolism via glycolysis, gluconeogenesis, and sucrose degradation. We also found that period circadian clock 2 (PER2), a tumor suppressor protein, was significantly up-regulated only when glucose was restricted with autophagy inhibition and chemotherapy. The upstream regulators of these differentially regulated pathways were determined to have implications in cancer, showing an increase in tumor suppressor proteins and a decrease in nuclear protein 1 (NUPR1) an important protein in chemoresistance. We also evaluated the phenotypic response of these cells and discovered autophagy inhibition and chemotherapy treatment increased apoptosis and decreased cell clonogenicity and viability. When glucose restriction was combined with autophagy inhibition and chemotherapy, all of the phenotypic results were intensified. In sum, our results indicate that glucose metabolism is of great importance in the ability of cancer cells to survive chemotherapy. By weakening cancer cells with glucose restriction and autophagy inhibition prior to chemotherapy, cancer cells become more sensitive to therapy.

Drug penetration and metabolism in 3D cell cultures treated in a 3D printed fluidic device: assessment of irinotecan via MALDI imaging mass spectrometry.

Realistic in vitro models are critical in the drug development process. In this study, a novel in vitro platform is employed to assess drug penetration and metabolism. This platform, which utilizes a 3D printed fluidic device, allows for dynamic dosing of three dimensional cell cultures, also known as spheroids. The penetration of the chemotherapeutic irinotecan into HCT 116 colon cancer spheroids was examined with MALDI imaging mass spectrometry (IMS). The active metabolite of irinotecan, SN-38, was also detected. After twenty-four hours of treatment, SN-38 was concentrated to the outside of the spheroid, a region of actively dividing cells. The irinotecan prodrug localization contrasted with SN-38 and was concentrated to the necrotic core of the spheroids, a region containing mostly dead and dying cells. These results demonstrate that this unique in vitro platform is an effective means to assess drug penetration and metabolism in 3D cell cultures. This innovative system can have a transformative impact on the preclinical evaluation of drug candidates due to its cost effectiveness and high throughput.

In situ detection of histone variants and modifications in mouse brain using imaging mass spectrometry.

Histone posttranslational modifications and histone variants control the epigenetic regulation of gene expression and affect a wide variety of biological processes. A complex pattern of such modifications and variants defines the identity of cells within complex organ systems and can therefore be used to characterize cells at a molecular level. However, their detection and identification in situ has been limited so far due to lack of specificity, selectivity, and availability of antihistone antibodies. Here, we describe a novel MALDI imaging MS based workflow, which enables us to detect and characterize histones by their intact mass and their correlation with cytological properties of the tissue using novel statistical and image analysis tools. The workflow allows us to characterize the in situ distribution of the major histone variants and their modification in the mouse brain. This new analysis tool is particularly useful for the investigation of expression patterns of the linker histone H1 variants for which suitable antibodies are so far not available.

Probing amyloid-ß pathology in transgenic Alzheimer's disease (tgArcSwe) mice using MALDI imaging mass spectrometry.

The pathological mechanisms underlying Alzheimer's disease (AD) are still not understood. The disease pathology is characterized by the accumulation and aggregation of amyloid-ß (Aß) peptides into extracellular plaques, however the factors that promote neurotoxic Aß aggregation remain elusive. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides and proteins in biological tissues. In the present study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)-based imaging was used to study Aß deposition in transgenic mouse brain tissue and to elucidate the plaque-associated chemical microenvironment. The imaging experiments were performed in brain sections of transgenic Alzheimer's disease mice carrying the Arctic and Swedish mutation of amyloid-beta precursor protein (tgArcSwe). Multivariate image analysis was used to interrogate the IMS data for identifying pathologically relevant, anatomical features based on their chemical identity. This include cortical and hippocampal Aß deposits, whose amyloid peptide content was further verified using immunohistochemistry and laser microdissection followed by MALDI MS analysis. Subsequent statistical analysis on spectral data of regions of interest revealed brain region-specific differences in Aß peptide aggregation. Moreover, other plaque-associated protein species were identified including macrophage migration inhibitory factor suggesting neuroinflammatory processes and glial cell reactivity to be involved in AD pathology. The presented data further highlight the potential of IMS as a powerful approach in neuropathology. Hanrieder et al. described an imaging mass spectrometry based study on comprehensive spatial profiling of C-terminally truncated Aß species within individual plaques in tgArcSwe mice. Here, brain region-dependent differences in Aß truncation and other plaque-associated proteins, such as macrophage migration inhibitory factor, were observed. The data shed further light on plaque-associated molecular mechanisms implicated in Alzheimer's pathogenesis. Cover image for this issue: doi: 10.1111/jnc.13328.

Tumoral heterogeneity of hepatic cholangiocarcinomas revealed by MALDI imaging mass spectrometry.

Cholangiocarcinoma (CC) is the second most common primary malignancy of the liver. Although all CC derive from biliary epithelial cells, two main subtypes, hilar (H), and peripheral (P) CC are described. The objective of the study was to compare, using MALDI imaging mass spectrometry (MALDI IMS), in situ proteomic profiles of H- and P-CC in order to assess whether these subtypes may express different markers and to describe their respective localizations. Twenty-seven CC (16 P-CC and 11 H-CC) were subjected to MALDI IMS. Proteomic data were submitted to a dedicated cross-classification comparative design, enabling comparison of the entire generated spectra. Immunohistochemistry was performed for validation. Comparative analysis yielded a list of 19 differential protein peaks for the two subtypes, 14 of which were overexpressed in H-CC and five in P-CC. Among H-CC protein markers, most discriminant were human neutrophil peptides 1-3 that were expressed mainly by tumor cells and S100 proteins (A6 and A11) that were restricted to the stromal area. In P-CC, thymosin ß4 was diffusely overexpressed. These results highlight the potential of MALDI IMS to discover new relevant biomarkers of CC and to characterize the heterogeneity of the two different subtypes.

Visualization and quantification of cerebral metabolic fluxes of glucose in awake mice.

Biotransformation of glucose in organs includes multiple pathways, while quantitative evaluation of percentages of its utilization for individual pathways and their spatial heterogeneity in vivo remain unknown. Imaging MS (IMS) and metabolomics combined with a focused microwave irradiation for rapidly fixing tissue metabolism allowed us to quantify and visualize metabolic fluxes of glucose-derived metabolites in the mouse brain in vivo. At 15 min after the intraperitoneal injection of (13) C6 -labeled glucose, the mouse brain was exposed to focused microwave irradiation, which can stop brain metabolism within 1 s. Quantification of metabolic intermediates containing (13) C atoms revealed that a majority of the (13) C6 -glucose was diverted into syntheses of glutamate, lactate, and uridine diphosphate (UDP)-glucose. IMS showed that regions rich in glutaminergic neurons exhibited a large signal of (13) C2 -labeled glutamate. On the other hand, the midbrain region was enriched with an intensive (13) C6 -labeled UDP-glucose signal, suggesting an active glycogen synthesis. Collectively, application of the current method makes it possible to examine the fluxes of glucose metabolism in a region-specific manner.

Multicenter matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) identifies proteomic differences in breast-cancer-associated stroma.

MALDI mass spectrometry imaging (MSI) has rapidly established itself as a powerful biomarker discovery tool. To date, no formal investigation has assessed the center-to-center comparability of MALDI MSI experiments, an essential step for it to develop into a new diagnostic method. To test such capabilities, we have performed a multicenter study focused on biomarkers of stromal activation in breast cancer. MALDI MSI experiments were performed in two centers using independent tissue banks, infrastructure, methods, and practitioners. One of the data sets was used for discovery and the other for validation. Areas of intra- and extratumoral stroma were selected, and their protein signals were compared. Four protein signals were found to be significantly associated with tumor-associated stroma in the discovery data set measured in Munich. Three of these peaks were also detected in the independent validation data set measured in Leiden, all of which were also significantly associated with intratumoral stroma. Hierarchical clustering displayed 100% accuracy in the Munich MSI data set and 80.9% accuracy in the Leiden MSI data set. The association of one of the identified mass signals (PA28) with stromal activation was confirmed with immunohistochemistry performed on 20 breast tumors. Independent and international MALDI MSI investigations could identify validated biomarkers of stromal activation.

Clinical response to chemotherapy in oesophageal adenocarcinoma patients is linked to defects in mitochondria.

Chemotherapeutic drugs kill cancer cells, but it is unclear why this happens in responding patients but not in non-responders. Proteomic profiles of patients with oesophageal adenocarcinoma may be helpful in predicting response and selecting more effective treatment strategies. In this study, pretherapeutic oesophageal adenocarcinoma biopsies were analysed for proteomic changes associated with response to chemotherapy by MALDI imaging mass spectrometry. Resulting candidate proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and investigated for functional relevance in vitro. Clinical impact was validated in pretherapeutic biopsies from an independent patient cohort. Studies on the incidence of these defects in other solid tumours were included. We discovered that clinical response to cisplatin correlated with pre-existing defects in the mitochondrial respiratory chain complexes of cancer cells, caused by loss of specific cytochrome c oxidase (COX) subunits. Knockdown of a COX protein altered chemosensitivity in vitro, increasing the propensity of cancer cells to undergo cell death following cisplatin treatment. In an independent validation, patients with reduced COX protein expression prior to treatment exhibited favourable clinical outcomes to chemotherapy, whereas tumours with unchanged COX expression were chemoresistant. In conclusion, previously undiscovered pre-existing defects in mitochondrial respiratory complexes cause cancer cells to become chemosensitive: mitochondrial defects lower the cells' threshold for undergoing cell death in response to cisplatin. By contrast, cancer cells with intact mitochondrial respiratory complexes are chemoresistant and have a high threshold for cisplatin-induced cell death. This connection between mitochondrial respiration and chemosensitivity is relevant to anticancer therapeutics that target the mitochondrial electron transport chain.

MALDI mass spectrometry-based identification of antifungal molecules from endophytic Bacillus strains with biocontrol potential of Lasiodiplodia theobromae, a grapevine trunk pathogen in Peru.

Lasiodiplodia theobromae, a grapevine trunk pathogen, is becoming a significant threat to vineyards worldwide. In Peru, it is responsible for Botryosphaeria dieback in many grapevine-growing areas and it has spread rapidly due to its high transmissibility; hence, control measures are urgent. It is known that some endophytic bacteria are strong inhibitors of phytopathogens because they produce a wide range of antimicrobial molecules. However, studies of antimicrobial features from endophytic bacteria are limited to traditional confrontation methods. In this study, a MALDI mass spectrometry-based approach was performed to identify and characterize the antifungal molecules from Bacillus velezensis M1 and Bacillus amyloliquefaciens M2 grapevine endophytic strains. Solid medium antagonism assays were performed confronting B. velezensis M1 - L. theobromae and B. amyloliquefaciens M2 - L. theobromae for antifungal lipopeptides identification. By a MALDI TOF MS it was possible identify mass spectra for fengycin, iturin and surfactin protoned isoforms. Masses spectrums for mycobacillin and mycosubtilin were also identified. Using MALDI Imaging MS we were able to visualize and relate lipopeptides mass spectra of fengycin (1463.9 m/z) and mycobacillin (1529.6 m/z) in the interaction zone during confrontations. The presence of lipopeptides-synthesis genes was confirmed by PCR. Liquid medium antagonism assays were performed for a proteomic analysis during the confrontation of B. velezensis M1 - L. theobromae. Different peptide sequences corresponding to many antifungal proteins and enzymes were identified by MALDI TOF MS/MS. Oxalate decarboxylase bacisubin and flagellin, reported as antifungal proteins, were identified at 99 % identity through peptide mapping. MALDI mass spectrometry-based identification of antifungal molecules would allow the early selection of endophytic bacteria with antifungal features. This omics tool could lead to measures for prevention of grapevine diseases and other economically important crops in Peru.

Optimization of Zebrafish Larvae Sectioning for Mass Spectrometry Imaging.

The utility of zebrafish is becoming more frequent due to lower costs and high similarities to humans. Zebrafish larvae are attractive subjects for drug screening and drug metabolism research. However, obtaining good quality zebrafish larvae sections for batch samples at designated planes, angles, and locations for comparison purposes is a challenging task. We report here the optimization of fresh frozen zebrafish larvae sectioning for mass spectrometry imaging. We utilized the gelatin solutions that were created at two different temperatures (50 and 85 °C) as embedding media. Gelatin-50 (gelatin created under 50 °C, solid gel under room temperature) was used to make a larvae-shaped mold and gelatin-85 (gelatin created under 85 °C, liquid under room temperature) was used to embed the larvae. H&E staining of sections shows well-preserved morphology and minimal histological interference. More importantly, the position of the larvae was well controlled resulting in more consistent sectioning of the larvae.

DDA-imaging with structural identification of lipid molecules on an Orbitrap Velos Pro mass spectrometer.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a useful technique for visualizing the spatial distribution of lipid molecules in tissues. Nevertheless, the use of MSI to investigate local lipid metabolic hallmarks has until recently been hampered by a lack of adequate technology that supports confident lipid identification. This limitation was recently mitigated by the development of DDA-imaging technology where high-resolution MSI is combined with parallel acquisition of lipid tandem MS2 spectra on a hybrid ion trap-Orbitrap Elite mass spectrometer featuring a resolving power of 240,000 and a scan time of 1 s. Here, we report the key tenets related to successful transfer of the DDA-imaging technology onto an Orbitrap Velos Pro instrument featuring a resolving power of 120,000 and a scan time of 2 s. Through meticulous performance assessments and method optimization, we tuned the DDA-imaging method to be able to confidently identify 73 molecular lipid species in mouse brain sections and demonstrate that the performance of the technology is comparable with DDA-imaging on the Orbitrap Elite. Altogether, our work shows that DDA-imaging on the Orbitrap Velos Pro instrument can serve as a robust workhorse for lipid imaging in routine applications.