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Abstract Acetaminophen (APAP)-induced liver injury (APAP-ILI), which occurs during APAP overdose, has been extensively studied. The production of N-acetyl-p-benzoquinone imine (NAPQI), the reactive metabolite of APAP, primarily contributes to liver injury. However, the mechanism underlying APAP-ILI has not been fully characterized. For further clarification, it is important to consider drug localization and endogenous substances in the injured liver. Herein, we show the localization of NAPQI metabolites and the injury site–specific changes in endogenous substances in the rat liver following APAP overdose using a mass microscope. Our results of on-tissue derivatization matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) showed that the glutathione metabolite of APAP, a detoxified metabolite of NAPQI, localized in the damaged central vein region in the rat liver following APAP administration. Moreover, in the conventional MALDI-MSI, the intensities of some phospholipids, phosphocreatine, and ceramides decreased or increased in the damaged regions compared with those in non-damaged regions. Phosphocreatine was localized in the damaged cells, whereas its related mitochondrial creatine kinase was localized in the non-damaged cells. These results are expected to contribute to further elucidation of the mechanisms underlying APAP-ILI. Our findings illustrate the localization of NAPQI-related metabolites and endogenous molecules associated with APAP-ILI, which may be related to apoptosis or metabolic adaptation ultimately protecting the cells. As MALDI-MSI can analyze and differentiate regions with tissue damage, it is a valuable tool for analyzing the mechanism underlying drug-induced liver injury to identify novel biomarkers.

Understanding the relationship between the concentration of a drug and its therapeutic efficacy or side effects is crucial in drug development, especially to understand therapeutic efficacy in central nervous system drug, quantifying drug-induced site-specific changes in the levels of endogenous metabolites, such as neurotransmitters. In recent times, evaluation of quantitative distribution of drugs and endogenous metabolites using matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) has attracted much attention in drug discovery research. However, MALDI-MSI quantification (quantitative mass spectrometry imaging, QMSI) is an emerging technique, and needs to be further developed for practicable and convenient use in drug discovery research. In this study, we developed a reliable QMSI method for quantification of clozapine (antipsychotic drug) and dopamine and its metabolites in the rat brain using MALDI-MSI. An improved mimetic tissue model using powdered frozen tissue for QMSI was established as an alternative method, enabling the accurate quantification of clozapine levels in the rat brain. Furthermore, we used the improved method to evaluate drug-induced fluctuations in the concentrations of dopamine and its metabolites. This method can quantitatively evaluate drug localization in the brain and drug-induced changes in the concentration of endogenous metabolites, demonstrating the usefulness of QMSI.

Periodontitis is caused by a pathogenic shift in subgingival biofilm ecosystems, which is accompanied by alterations in microbiome composition and function, including changes in the metabolic activity of the biofilm, which comprises multiple commensals and pathogens. While Fusobacterium nucleatum is a common constituent of the supra- and subgingival biofilms, its metabolic integration within polymicrobial communities and the impact on periodontal pathogenesis are poorly understood. Fusobacterium nucleatum is a common constituent of the oral microbiota in both periodontal health and disease. Previously, we discovered ornithine cross-feeding between F. nucleatum and Streptococcus gordonii , where S. gordonii secretes ornithine via an arginine-ornithine antiporter (ArcD), which in turn supports the growth and biofilm development of F. nucleatum ; however, broader metabolic aspects of F. nucleatum within polymicrobial communities and their impact on periodontal pathogenesis have not been addressed. Here, we show that when cocultured with S. gordonii , F. nucleatum increased amino acid availability to enhance the production of butyrate and putrescine, a polyamine produced by ornithine decarboxylation. Coculture with Veillonella parvula , another common inhabitant of the oral microbiota, also increased lysine availability, promoting cadaverine production by F. nucleatum . We confirmed that ArcD-dependent S. gordonii -excreted ornithine induces synergistic putrescine production, and mass spectrometry imaging revealed that this metabolic capability creates a putrescine-rich microenvironment on the surface of F. nucleatum biofilms. We further demonstrated that polyamines caused significant changes in the biofilm phenotype of a periodontal pathogen, Porphyromonas gingivalis , with putrescine accelerating the biofilm life cycle of maturation and dispersal. This phenomenon was also observed with putrescine derived from S. gordonii - F. nucleatum coculture. Lastly, analysis of plaque samples revealed cooccurrence of P. gingivalis with genetic modules for putrescine production by S. gordonii and F. nucleatum . Overall, our results highlight the ability of F. nucleatum to induce synergistic polyamine production within multispecies consortia and provide insight into how the trophic web in oral biofilm ecosystems can eventually shape disease-associated communities. IMPORTANCE Periodontitis is caused by a pathogenic shift in subgingival biofilm ecosystems, which is accompanied by alterations in microbiome composition and function, including changes in the metabolic activity of the biofilm, which comprises multiple commensals and pathogens. While Fusobacterium nucleatum is a common constituent of the supra- and subgingival biofilms, its metabolic integration within polymicrobial communities and the impact on periodontal pathogenesis are poorly understood. Here, we report that amino acids supplied by other commensal bacteria induce polyamine production by F. nucleatum , creating polyamine-rich microenvironments. Polyamines reportedly have diverse functions in bacterial physiology and possible involvement in periodontal pathogenesis. We show that the F. nucleatum -integrated trophic network yielding putrescine from arginine through ornithine accelerates the biofilm life cycle of Porphyromonas gingivalis , a periodontal pathogen, from the planktonic state through biofilm formation to dispersal. This work provides insight into how cooperative metabolism within oral biofilms can tip the balance toward periodontitis.

Drosophila melanogaster is a useful and highly tractable model organism for understanding the molecular mechanisms of human diseases. We previously characterized a new dUbqn knockdown model that induces learning-memory and locomotive deficits mediated by impaired proteostasis. Although proteinopathies are the main causes of neurodegenerative diseases, limited information is currently available on the relationship between proteostasis and neurodegenerative-related behavioral perturbations, such as locomotion, wakefulness, and sexual activities. Thus, the present study aimed to elucidate the mechanisms by which dUbqn depletion which is known to cause proteinopathies, affects neurodegenerative-related behavioral perturbations. Pan-neuronal dUbqn -depleted flies showed significantly reduced evening activity along with altered pre- and postsynaptic structural NMJ’s proteins by attenuating signals of Bruchpilot puncta and GluRIIA clustering. In addition, the neurochemical profiles of GABA, glutamate, dopamine, and serotonin were disturbed and these changes also affected courtship behaviors in dUbqn -depleted flies. Collectively, these results extend our understanding on how dUbqn depletion affects neurochemical regulation to drive behavioral disturbances that are generally found in the early stage of neurodegenerative diseases. Moreover, the present study may contribute a novel finding to the design of new agents that prevent disease progression or even treat diseases related to neurodegeneration.

γ-Aminobutyric acid (GABA) accumulates in plants in response to environmental stresses. The activity levels of glutamate decarboxylase (GAD), an enzyme involved in GABA biosynthesis, are reported to increase during germination under salinity stress. However, it is not clear which tissues of the plant seeds are affected by GAD activity in response to salinity stress. In this study, the effects of salinity stress on the distribution of barley seeds GAD activity during germination were investigated. The mass spectrometry imaging (MSI) method was optimized, and the distribution of GAD activity in germinated seeds exposed to salinity stress at different germination stages from 12 to 48 h after imbibition was investigated. In this study, MSI was successfully applied to enzyme histochemistry to visualize the relative GAD activity in germinating barley seeds for the first time. The salinity stress increased the GAD activity, mostly due to the increase in relative GAD activity in the embryo. Higher GAD activity was detected in seeds exposed to salinity stress in the scutellum or aleurone layer, which are difficult to separate for extraction. This method can be used to clarify the role of GABA shunts, including GAD enzyme responses, in barley seeds under stress.

Testosterone is one of the androgens synthesized from cholesterol as a precursor in the Leydig cells of testes. Since the ionization efficiency of testosterone in matrix-assisted laser desorption ionization (MALDI) is quite low, visualization of testosterone by using MALDI-imaging mass spectrometry (MALDI-IMS) has been considered difficult. To overcome this problem, we used two types of on-tissue derivatization techniques, which were achieved by pyridine sulfur trioxide and Girard’s T (GT) reagent, to introduce a polar group into testosterone molecule with the aim to increase the sensitivity. Derivatization by use of GT reagent provided excellent results, superior to those obtained with pyridine sulfur trioxide, in terms of ionization efficiency, molecular specificity, and tissue damage. In GT derivatized testis tissues of mice treated with human chorionic gonadotropin (hCG), testosterone was broadly observed both inside and outside the seminiferous tubules by using an iMScope. To evaluate our imaging results, we performed quantification experiments of underivatized testosterone extracted from hCG-treated testes and control testes using LC-MS/MS. We confirmed the 256-fold concentration change between hCG-treated tissues and control tissues. We also confirmed the 228-fold change in detected peak intensities between hCG-treated tissue sections and control tissue sections in imaging results. We consider our tissue preparation methods for IMS provide high sensitivity with high precision. In addition, high-spatial definition IMS was also available, and we confirmed testosterone had mainly accumulated on the surface of the Leydig cells. Graphical abstract Girard’s T-testosterone (GT-Ts) provides the fragment ion at m/z 343.24. Clear GT-Ts signal was detected in hCG treated mouse testis not only as spectra but also as a mass image

Quorum sensing (QS) is generally used to describe the process involving the release and recognition of signaling molecules, such as N-acyl-homoserine lactones, by bacteria to coordinate their response to population density and biofilm development. However, detailed information on the heterogeneity of QS metabolites in biofilms remains largely unknown. Here, we describe the utilization of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to follow the production of specific metabolites, including QS metabolites, during Pseudomonas putida biofilm development. To do so, a method to grow an agar-based biofilm was first established, and MALDI-MSI was used to detect and visualize the distribution of QS metabolites in biofilms at different cultivation times. This study demonstrated that N-acyl-homoserine lactones are homogeneously produced in the early stages of P. putida biofilm formation. In contrast, the spatial distribution of quinolones and pyochelin correlated with the swarming motility of P. putida in mature biofilms. These two metabolites are involved in the production of extracellular polymeric substances and iron chelators. Our study thus contributes to establishing the specific temporal regulation and spatial distribution of N-acyl-homoserine lactone-related metabolites and quinolone and pyochelin in P. putida biofilms.

Gangliosides are particularly abundant in the central nervous system (CNS) and thought to play important roles in memory formation, neuritogenesis, synaptic transmission, and other neural functions. Although several molecular species of gangliosides have been characterized and their individual functions elucidated, their differential distribution in the CNS are not well understood. In particular, whether the different molecular species show different distribution patterns in the brain remains unclear. We report the distinct and characteristic distributions of ganglioside molecular species, as revealed by imaging mass spectrometry (IMS). This technique can discriminate the molecular species, raised from both oligosaccharide and ceramide structure by determining the difference of the mass-to-charge ratio, and structural analysis by tandem mass spectrometry. Gangliosides in the CNS are characterized by the structure of the long-chain base (LCB) in the ceramide moiety. The LCB of the main ganglioside species has either 18 or 20 carbons (i.e., C18- or C20-sphingosine); we found that these 2 types of gangliosides are differentially distributed in the mouse brain. While the C18-species was widely distributed throughout the frontal brain, the C20-species selectively localized along the entorhinal-hippocampus projections, especially in the molecular layer (ML) of the dentate gyrus (DG). We revealed development- and aging-related accumulation of the C-20 species in the ML-DG. Thus it is possible to consider that this brain-region specific regulation of LCB chain length is particularly important for the distinct function in cells of CNS.

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of α- and β-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular \"traffic sign\" for motor proteins in neuronal cells. To investigate whether polyglutamylated α-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of α-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated α-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated α-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of α-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission.

Direct tissue analysis using a novel tandem time-of-flight (TOF-TOF) mass spectrometer is described. This system consists of a matrix-assisted laser desorption/ionization ion source, a spiral ion trajectory TOF mass spectrometer \"SpiralTOF (STOF)\", a collision cell, and an offset parabolic reflectron (RTOF). The features of this system are high precursor ion selectivity due to a 17-m flight path length in STOF and elimination of post-source decay (PSD) ions. The acceleration energy is 20 keV, so that high-energy collision-induced dissociation (HE-CID) is possible. Elimination of PSD ions allows observation of the product ions inherent to the HE-CID process. By using this tandem TOF instrument, the product ion spectrum of lipids provided detailed structural information of fatty acid residues.