Integrated spatial metabolomics and transcriptomics decipher the hepatoprotection mechanisms of wedelolactone and demethylwedelolactone on non-alcoholic fatty liver disease.

Eclipta prostrata L. has been used in traditional medicine and known for its liver-protective properties for centuries. Wedelolactone (WEL) and demethylwedelolactone (DWEL) are the major coumarins found in E. prostrata L. However, the comprehensive characterization of these two compounds on non-alcoholic fatty liver disease (NAFLD) still remains to be explored. Utilizing a well-established zebrafish model of thioacetamide (TAA)-induced liver injury, the present study sought to investigate the impacts and mechanisms of WEL and DWEL on NAFLD through integrative spatial metabolomics with liver-specific transcriptomics analysis. Our results showed that WEL and DWEL significantly improved liver function and reduced the accumulation of fat in the liver. The biodistributions and metabolism of these two compounds in whole-body zebrafish were successfully mapped, and the discriminatory endogenous metabolites reversely regulated by WEL and DWEL treatments were also characterized. Based on spatial metabolomics and transcriptomics, we identified that steroid biosynthesis and fatty acid metabolism are mainly involved in the hepatoprotective effects of WEL instead of DWEL. Our study unveils the distinct mechanism of WEL and DWEL in ameliorating NAFLD, and presents a "multi-omics" platform of spatial metabolomics and liver-specific transcriptomics to develop highly effective compounds for further improved therapy.

Efficient axonal transport of endolysosomes relies on the balanced ratio of microtubule tyrosination and detyrosination.

In neurons, the microtubule (MT) cytoskeleton forms the basis for long-distance protein transport from the cell body into and out of dendrites and axons. To maintain neuronal polarity, the axon initial segment (AIS) serves as a physical barrier, separating the axon from the somatodendritic compartment and acting as a filter for axonal cargo. Selective trafficking is further instructed by axonal enrichment of MT post-translational modifications, which affect MT dynamics and the activity of motor proteins. Here, we compared two knockout mouse lines lacking the respective enzymes for MT tyrosination and detyrosination, and found that both knockouts led to a shortening of the AIS. Neurons from both lines also showed an increased immobile fraction of endolysosomes present in the axon, whereas mobile organelles displayed shortened run distances in the retrograde direction. Overall, our results highlight the importance of maintaining the balance of tyrosinated and detyrosinated MTs for proper AIS length and axonal transport processes.

Mass spectrometry imaging-based metabolomics highlights spatial metabolic alterations in three types of liver injuries.

Liver's distinctive function renders it highly susceptible to diverse damage sources. Characterizing the metabolic profiles and spatial signatures in different liver injuries is imperative for early diagnosis and etiology-oriented treatment. In this comparative study, we conducted whole-body spatial metabolomics on zebrafish with liver injury induced by ethanol (EtOH), acetaminophen (APAP), and thioacetamide (TAA). The two specific levels, the whole-body and liver-specific metabolic profiles, as well as their regional distributions, were systematically mapped in situ by mass spectrometry imaging, which is distinct from conventional LC-MS and GC-MS methods. We found that liver injury regions exhibited more pronounced metabolic reprogramming than the entire organism, leading to significant alterations in eight fatty acids, three phospholipids, and four low-molecular-weight metabolites. More importantly, fatty acids as well as small molecule metabolites including glutamine, glutamate, taurine and malic acid displayed contrasting changes between alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). In addition, phospholipids, including Lyso PC (16:0) and Lyso PE (18:0), demonstrated notable down-regulation in all damaged liver, whereas PC (34:1) underwent upregulation. This study not only deepens insights into distinct potential biomarkers for liver injuries, but also underscores spatial metabolomics as a powerful tool to elucidate possible pathogenic mechanisms in other metabolic diseases.

Construction of rod-like cobalt-pyridinedicarboxylic acid/MXene nanosheets composites for hydrogen evolution reaction and supercapacitor.

Metal-organic frameworks (MOFs) have attracted considerable attention in the field of energy storage and conversion due to their large specific surface area, regulatable pore structure and composition. However, the poor electrical conductivity and few active sites of MOFs impede their application. Herein, highly conductive MXene nanosheets are introduced to modulate the electronic conductivity and structure of rod-like Co-pyridinedicarboxylic acid (Co-PDC), and thus enhancing the electrochemical performance of MOFs. The heterostructural Co-PDC/MXene (CPM) was facily synthesized at room temperature. The as-prepared CPM-30 with 30 % MXene only requires the overpotential of 75.1 mV to achieve a current density of 10 mA cm-2 for hydrogen evolution reaction (HER), and the assembled electrolytic cell with CPM-30 and RuO2 as cathode and anode electrodes can achieve a current density of 10 mA cm-2 at a voltage of 1.65 V. In addition, CPM-10 exhibits a high specific capacitance of 583.1 F g-1 at 0.5 A g-1 and an excellent rate performance of 41.6 % at 50 A g-1. Furthermore, the assembled asymmetric supercapacitor CPM-10//AC exhibited an energy density of 15.55 Wh kg-1 at a power density of 750 W kg-1 and excellent stability with a capacitance retention rate of 95 % after 10,000 cycles. The excellent electrochemical properties of Co-PDC/MXene are attributed to the unique structure and synergistic effect of Co-PDC and MXene.

Isotope-Coded On-Tissue Derivatization for Quantitative Mass Spectrometry Imaging of Short-Chain Fatty Acids in Biological Tissues.

Short-chain fatty acids (SCFAs), as the main metabolites of gut microbiota, are recognized as crucial players in the host's inflammatory response and metabolic disease. Imaging the spatial distributions and calculating the accurate contents of SCFAs in the heterogeneous intestinal tissue are critical to reveal their biological functions. Here, we develop an isotope-coded on-tissue derivatization method combined with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to map the spatial expressions of SCFAs in the colon tissue based on pair-labeled N,N,N-trimethyl-2-(piperazin-1-yl)ethan-1-aminium iodide (TMPA) and D3-TMPA. A noticeable increase in the MALDI-MSI sensitivity of SCFAs was achieved after on-tissue derivatization, which enables the visualization of acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, hydroxy acetic acid, and hydroxy propionic acid in the colon tissue. Moreover, the introduction of D3-TMPA-tagged SCFAs as internal standards can significantly reduce quantitation deviation from the matrix effects, ensuring the quantitative MALDI-MSI of SCFAs. We further used this method to characterize the spatial alterations of SCFAs in the colon tissues of mice with enterocolitis. The development of this strategy provides a reliable approach to image the spatial expressions of SCFAs in tissues and paves an insight way to study the roles of SCFAs in the gut microbiota and disease.

Spatially Resolved Metabolomics Combined with the 3D Tumor-Immune Cell Coculture Spheroid Highlights Metabolic Alterations during Antitumor Immune Response.

The metabolic cross-talk between tumor and immune cells plays key roles in immune cell function and immune checkpoint blockade therapy. However, the characterization of tumor immunometabolism and its spatiotemporal alterations during immune response in a complex tumor microenvironment is challenging. Here, a 3D tumor-immune cell coculture spheroid model was developed to mimic tumor-immune interactions, combined with mass spectrometry imaging-based spatially resolved metabolomics to visualize tumor immunometabolic alterations during immune response. The inhibition of T cells was simulated by coculturing breast tumor spheroids with Jurkat T cells, and the reactivation of T cells can be monitored through diminishing cancer PD-L1 expressions by berberine. This system enables simultaneously screening and imaging discriminatory metabolites that are altered during T cell-mediated antitumor immune response and characterizing the distributions of berberine and its metabolites in tumor spheroids. We discovered that the transport and catabolism of glutamine were significantly reprogrammed during the antitumor immune response at both metabolite and enzyme levels, corresponding to its indispensable roles in energy metabolism and building new biomass. The combination of spatially resolved metabolomics with the 3D tumor-immune cell coculture spheroid visually reveals metabolic interactions between tumor and immune cells and possibly helps decipher the role of immunometabolic alterations in tumor immunotherapy.

In-depth profiling of carbohydrate isomers in biological tissues by chemical derivatization-assisted mass spectrometry imaging.

Carbohydrates play crucial regulatory roles in various physiological and pathological processes. However, the low ionization efficiency and the presence of linkage pattern, monosaccharide composition and anomeric configuration isomers make their in-depth analysis very challenging, especially for heterogeneous biological tissues. In this study, we propose a high-sensitive and isomer-specific imaging approach to visualize the spatial distributions of monosaccharide and disaccharide isomers by integrating chemical derivatization and matrix-assisted laser desorption/ionization tandem mass spectrometry imaging (MALDI-MS2I). 2-Pyridinecarbohydrazide (PYD) is developed as a novel derivatization reagent which can not only improves the MS sensitivity of carbohydrates, but also enables the identification and visualization of ketose and aldose monosaccharide isomers, as well as linkage pattern, monosaccharide composition and anomeric configuration disaccharide isomers by mass spectrometry imaging of isomer-specific MS/MS fragment ions. Moreover, we build quantitative MALDI-MS2 and MALDI-MS2I methods for disaccharide isomers based on the diagnostic fragment ions, and good linear relationships could be achieved both in solution and on glass slides. We expect that this study should provide new ideas for in-depth profiling of the spatial signatures of carbohydrates in biological tissues and lay the foundation for a deeper understanding of carbohydrates' structure.

Imaging the metabolic reprograming of fatty acid synthesis pathway enables new diagnostic and therapeutic opportunity for breast cancer.

BACKGROUND: Reprogrammed metabolic network is a key hallmark of cancer. Profiling cancer metabolic alterations with spatial signatures not only provides clues for understanding cancer biochemical heterogeneity, but also helps to decipher the possible roles of metabolic reprogramming in cancer development. METHODS: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique was used to characterize the expressions of fatty acids in breast cancer tissues. Specific immunofluorescence staining was further carried out to investigate the expressions of fatty acid synthesis-related enzymes. RESULTS: The distributions of 23 fatty acids in breast cancer tissues have been mapped, and the levels of most fatty acids in cancer tissues are significantly higher than those in adjacent normal tissues. Two metabolic enzymes, fatty acid synthase (FASN) and acetyl CoA carboxylase (ACC), which being involved in the de novo synthesis of fatty acid were found to be up-regulated in breast cancer. Targeting the up-regulation of FASN and ACC is an effective approach to limiting the growth, proliferation, and metastasis of breast cancer cells. CONCLUSIONS: These spatially resolved findings enhance our understanding of cancer metabolic reprogramming and give an insight into the exploration of metabolic vulnerabilities for better cancer treatment.

Application of food exchange portion method in home-based nutritional intervention for elderly patients with chronic heart failure.

BACKGROUND: The home treatment of elderly patients with chronic heart failure (CHF) is often accompanied by malnutrition, which increases the risk of re-hospitalisation and affects the prognosis. Therefore, how to effectively improve the nutritional self-management of patients is a current focus of medical research. This study aims to test the effect of home-based nutritional intervention method on improving the nutritional status of elderly patients with CHF. METHODS: A total of 90 hospitalised elderly patients with CHF were randomly divided into the experimental group (n = 45) and the control group (n = 45). The patients in both groups were given standardised drug therapy and their nutritional status was evaluated using a body composition analyser prior to discharge (protein, body fat percentage, visceral fat area, skeletal muscle, upper arm muscle circumference, left lower limb and right lower limb muscle mass), with the cardiopulmonary function evaluated using a six-minute walk test and the metabolic equivalents method. The control group was given general nutrition education and routine dietary guidance from cardiac rehabilitation nurses, while the experimental group was given an individualised nutrition prescription by dietitians based on the evaluation results, according to which one-to-one food exchange dietary intervention training was given until the patients mastered the process. RESULTS: The nutritional indexes at the end of the study were significantly higher in the experimental group than in the control group and were higher than those before the intervention (P < 0.05). The muscle circumference of the upper arm, the muscle mass of the left lower limb and the right lower limb had no statistical significance following the intervention compared to the control group and before the intervention (P > 0.05). The cardiopulmonary function indexes were significantly better in the experimental group at the end of the study than before the intervention and were better than those in the control group, with statistically significant differences (P < 0.05), while no significant changes were observed in the control group before and after the intervention (P > 0.05). CONCLUSION: The home-based nutritional intervention method of food exchange portions can effectively improve the nutritional status of elderly patients with CHF, with the distribution of visceral fat more reasonable and the cardiopulmonary function and exercise endurance improved.

Matrix-assisted laser desorption/ionization mass spectrometry imaging reveals "Spatial-Temporal-Content" changes of parishins in Gastrodiae Rhizoma during the steaming process.

Gastrodiae Rhizoma is a commonly used plant material for both medicine and food in China for thousands of years. Steaming as the main pre-processing method of Gastrodiae Rhizoma is the key to its quality formation. In this study, we established a high-coverage matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) method to visualize the spatial distribution of phenols in Gastrodiae Rhizoma. By optimizing 1,5-diaminonaphthalene as MALDI matrix in negative mode, the "spatial-temporal-content" changes of 13 phenols including 11 parishins in Gastrodiae Rhizoma during the steaming process were imaged. We also characterized the activity of two hydrolases related to parishins degradation during the steaming. Moreover, the degradation pathways of parishins in Gastrodiae Rhizoma were summarized. The integration of spatially-resolved parishins and corresponding hydrolases information revealed the scientific connotation of steaming as the processing method of Gastrodiae Rhizoma is to inactivate hydrolases and protect parishins, which provided theoretical support for the quality improvement and the standardized processing of Gastrodiae Rhizoma.

On-tissue derivatization for isomer-specific mass spectrometry imaging and relative quantification of monosaccharides in biological tissues.

Spatially-resolved profiling of tissue monosaccharides not only gives an insight into the spatial heterogeneity of monosaccharides, but also helps to decipher the possible roles of monosaccharides in biological processes. Here, we develop an on-tissue derivatization method, coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to image and quantify the aldose and ketose isomers of monosaccharide in biological tissues. A new derivatization reagent, 1-naphthaleneacethydrazide (NAH) was synthesized for the on-tissue derivatization of monosaccharides, and it significantly enhanced the imaging sensitivity of monosaccharides. Moreover, the NAH-derivatized aldose and ketose can generate isomer-specific diagnostic ions during MALDI-MS/MS analysis, and thus paves way for the isomer-specific MS imaging of aldose and ketose monosaccharides. On this basis, we further constructed a quantitative MALDI-MS imaging model based on isomer-specific diagnostic ions, and calculated the expression contents of aldose and ketose monosaccharide isomers in different tissue regions of carrot section. We expect that the development of this method should provide more precise view on the spatial distributions and contents of different monosaccharides in heterogeneous biological tissues.

Grass-specific ABERRANT MICROSPORE DEVELOPMENT 1 is required for maintaining pollen fertility in rice.

Pollen development includes a series of biological events that require precise gene regulation. Although several transcription factors (TFs) have been shown to play roles in maintaining pollen fertility, the major regulatory networks underlying tapetum development and pollen wall formation are largely unknown. Herein, we report that ABERRANT MICROSPORE DEVELOPMENT1 (AMD1), a protein annotated previously as unknown protein, is required for tapetum development and pollen exine patterning in rice (Oryza sativa L.). AMD1 encodes a grass-specific protein exhibiting transactivation activity in the nucleus and is spatiotemporally expressed in the tapetum and microspores during pollen development. Further biochemical assays indicate that AMD1 directly activates the transcription of DEFECTIVE POLLEN WALL (DPW) and POLYKETIDE SYNTHASE2 (OsPKS2), which are both implicated in sporopollenin biosynthesis during exine formation. Additionally, AMD1 directly interacts with TAPETUM DEGENERATION RETARDATION (TDR), a key TF involved in the regulation of tapetum degradation and exine formation. Taken together, we demonstrate that AMD1 is an important regulatory component involved in the TDR-mediated regulatory pathway to regulate sporopollenin biosynthesis, tapetum degradation, and exine formation for pollen development. Our work provides insights into the regulatory network of rice sexual reproduction and a useful target for genetic engineering of new male-sterile lines for hybrid rice breeding.

SWOLLEN TAPETUM AND STERILITY 1 is required for tapetum degeneration and pollen wall formation in rice.

The pollen wall is important for protecting the male gametophyte and for fertilization. The lipid components of the pollen wall are mainly synthesized and transported from the sporophytic tapetum. Although several factors related to lipid biosynthesis have been characterized, the molecular mechanisms underlying lipid biosynthesis during pollen development in rice (Oryza sativa L.) remain elusive. Here, we showed that mutation in the SWOLLEN TAPETUM AND STERILITY 1 (STS1) gene causes delayed tapetum degradation and aborted pollen wall formation in rice. STS1 encodes an endoplasmic reticulum (ER)-localized protein that contains domain of unknown function (DUF) 726 and exhibits lipase activity. Lipidomic and transcriptomic analyses showed that STS1 is involved in anther lipid homeostasis. Moreover, STS1 interacts with Polyketide Synthase 2 (OsPKS2) and Acyl-CoA Synthetase 12 (OsACOS12), two enzymes crucial in lipidic sporopollenin biosynthesis in pollen wall formation, suggesting a potentially lipidic metabolon for sporopollenin biosynthesis in rice. Collectively, our results indicate that STS1 is an important factor for lipid biosynthesis in reproduction, providing a target for the artificial control of male fertility in hybrid rice breeding and insight into the function of DUF726-containing protein in plants.

DEAP1 encodes a fasciclin-like arabinogalactan protein required for male fertility in rice.

Arabinogalactan proteins (AGPs) are widely distributed in plant cells. Fasciclin-like AGPs (FLAs) belong to a subclass of AGPs that play important roles in plant growth and development. However, little is known about the biological functions of rice FLA. Herein, we report the identification of a male-sterile mutant of DEFECTIVE EXINE AND APERTURE PATTERNING1 (DEAP1) in rice. The deap1 mutant anthers produced aberrant pollen grains with defective exine formation and a flattened aperture annulus and exhibited slightly delayed tapetum degradation. DEAP1 encodes a plasma membrane-associated member of group III plant FLAs and is specifically and temporally expressed in reproductive cells and the tapetum layer during male development. Gene expression studies revealed reduced transcript accumulation of genes related to exine formation, aperture patterning, and tapetum development in deap1 mutants. Moreover, DEAP1 may interact with two rice D6 PROTEIN KINASE-LIKE3s (OsD6PKL3s), homologs of a known Arabidopsis aperture protein, to affect rice pollen aperture development. Our findings suggested that DEAP1 is involved in male reproductive development and may affect exine formation and aperture patterning, thereby providing new insights into the molecular functions of plant FLAs in male fertility.

Multi-wavelength visible-light induced [2+2] cycloaddition for identification of lipid isomers in biological samples.

Ultraviolet (UV) light-catalyzed Paternò-Büchi (PB) reaction has been developed as an efficient lipid C=C double bond (DB) derivatization strategy, which can accurately assign the position of C=C bond in unsaturated lipids when coupled with tandem mass spectrometry (MS/MS). Inspired by this, here we proposed a novel visible-light induced [2+2] cycloaddition reaction combined with ESI-MS/MS and MALDI-MS/MS to identify lipid C=C position isomers. Benz[g]isoquinoline-5,10-dione (BIQD) and 6,9-difluorobenzo[g]isoquinoline-5,10-dione (DF-BIQD) were developed as a new type of [2+2] cycloaddition reagent, which can not only react with C=C bond under 254 nm UV light irradiation, but also quickly combine with lipid C=C bond under the irradiation of 405 nm visible-light and > 400 nm compact fluorescent lamp visible-light. High cycloaddition reaction conversion efficiency can be achieved by irradiating under compact fluorescent lamp light for 2 min. Moreover, we discovered that 437 nm, 489 nm, 545 nm, 581 nm, and 613 nm monochromatic light appearing in compact fluorescent lamp can individually induce the [2 + 2] cycloaddition reaction between DF-BIQD and unsaturated lipids. Using this method, we found that the expressions of lipid DB-positional isomers in rat heart, brain, lung, spleen, thymus, kidney, liver and plasma vary greatly. The relative content of FA-18:1 (Δ9) in rat heart is only 1.49 times that of FA-18:1 (Δ11), while the relative content of FA-18:1 (Δ9) in rat plasma is 5.20 times that of FA-18:1 (Δ11). The above results offer new insight into the development of photocatalytic reagent for visible-light induced [2+2] cycloaddition and structural lipidomic studies.

Caldendrin and myosin V regulate synaptic spine apparatus localization via ER stabilization in dendritic spines.

Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in structural and molecular diversity of dendritic spines. Active spines with large calcium ion (Ca2+ ) transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines via the actin-based motor myosin V. An increase in synaptic strength correlates with stable anchoring of the ER, followed by the formation of an organelle referred to as the spine apparatus. Here, we show that myosin V binds the Ca2+ sensor caldendrin, a brain-specific homolog of the well-known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. In mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V-dependent pathway. We propose that caldendrin transforms myosin into a stationary F-actin tether that enables the localization of ER tubules and formation of the spine apparatus in dendritic spines.

Autism-associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses.

Members of the SH3- and ankyrin repeat (SHANK) protein family are considered as master scaffolds of the postsynaptic density of glutamatergic synapses. Several missense mutations within the canonical SHANK3 isoform have been proposed as causative for the development of autism spectrum disorders (ASDs). However, there is a surprising paucity of data linking missense mutation-induced changes in protein structure and dynamics to the occurrence of ASD-related synaptic phenotypes. In this proof-of-principle study, we focus on two ASD-associated point mutations, both located within the same domain of SHANK3 and demonstrate that both mutant proteins indeed show distinct changes in secondary and tertiary structure as well as higher conformational fluctuations. Local and distal structural disturbances result in altered synaptic targeting and changes of protein turnover at synaptic sites in rat primary hippocampal neurons.

Sodium arsenite induces spatial learning and memory impairment associated with oxidative stress and activates the Nrf2/PPARγ pathway against oxidative injury in mice hippocampus.

Arsenic (As) is a ubiquitous environmental and industrial toxin with known correlates of oxidative stress and cognitive deficits in the brain. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that represents a central cellular antioxidant defense mechanism and transcribes many antioxidant genes. Peroxisome proliferator-activated receptor-gamma (PPARγ) is a well-known nuclear receptor to regulate lipid metabolism in many tissues, and it has been also associated with the control of oxidative stress, neuronal death, neurogenesis and differentiation. The role of Nrf2 and PPARγ in As-induced neurotoxicity is still debated. The present study was designed to investigate the neurobehavioral toxic effect of sub-chronic and middle-dose sodium arsenite exposure in mice hippocampus, as well as the response of Nrf2/PPARγ expression and influence on protein expression levels of their downstream antioxidant genes. Our results showed that mice treated with intraperitoneal injection of sodium arsenite (50 mg/kg body wt.) twice a week for 7 weeks resulted in increased generation of reactive oxygen species and impairment of spatial cognitive function. The present study also found a positive association between Nrf2/PPARγ expression in hippocampus of mice, and activation of antioxidant defenses by the evidently upregulated expression of their downstream genes, including superoxide dismutase, heme oxygenase-1 and glutathione peroxidase-3. Therefore, our findings were helpful for further understanding the role of Nrf2/PPARγ feedback loop in As-induced neurobehavioral toxicity.