Metallomics

Ferredoxins are central to cellular metabolism by mediating electron flow in energy conversion reactions. The focus of this study was to systematically examine twelve ferredoxin and ferredoxin-like proteins from Synechocystis sp. PCC 6803 to identify their properties, activities, and functions in electron transfer. Using electron paramagnetic resonance spectroscopy, we detected cluster types consistent with major ferredoxin families including plant-type [2Fe-2S], adrenodoxin, thioredoxin, and bacterial-type [4Fe- 4S] ferredoxins. In addition, we found that the ssr3184 ferredoxin-like protein exchanged between a [3Fe-4S] or a [4Fe-4S] cluster, pointing to a possible functional change in response to changes in oxygen or cellular redox poise. Electrochemical measurements demonstrated that these ferredoxins constitute a broad potential window, from -243 mV to -520 mV vs SHE. Investigations on their capacity to support electron-transfer focused on reactions with two major redox hubs: Photosystem I and pyruvate:ferredoxin oxidoreductase and included testing of binding interactions with nitrite reductase. Expression profiling under multiple environmental conditions was also used to predict function and revealed distinct regulatory patterns. Collectively, these findings identified a group of core ferredoxins that directly support photosynthetic electron transfer, and more specialized ones that may serve other functions. In summary, Synechocystis utilizes a suite of ferredoxins to maintain cellular redox homeostasis under dynamic environmental conditions.

Zinc is an essential transition metal that participates in many biological processes. In C. elegans, excess zinc is stored in lysosomes in intestinal cells; this process involves increasing the expression of the zinc transporter CDF-2 and remodeling of lysosomes characterized by an increase in the volume of the expansion compartment. To determine if this is a more general property, we investigated other metals. Here we report that lysosomes are remodeled in response to excess copper, manganese, and cadmium, with each metal causing an increase in the volume of the expansion compartment. Mutants with a reduced number of lysosomes were hypersensitive to growth retardation caused by excess copper and manganese, suggesting metal toxicity is prevented by metal sequestration in lysosomes. Using a novel method to analyze isolated lysosomes by X-ray Fluorescence Microscopy we demonstrated that zinc, copper and manganese are detectable in the lumen of lysosomes. To further analyze copper, we examined localization of CUA-1.1, a copper transporter that moves copper into the lumen of lysosomes. Like the zinc transporter CDF-2, CUA-1.1 localizes to both the acidified and expansion compartments in excess copper. These results indicate that the same intestinal lysosomes store zinc, copper and manganese. Lysosome remodeling characterized by an increase in volume of the expansion compartment is not specific to zinc but is a more general phenomenon during metal storage in lysosomes.

Oxidative stress results from excessive accumulation of reactive oxygen species (ROS) and plays a central role in numerous physiological and pathological processes. Accurate quantification of antioxidant enzyme activities is therefore essential in redox biology research. However, data analysis for commonly used assays, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), is frequently performed using spreadsheets or manual calculations, which are time-consuming and prone to error. Here, we present Redoxyme, a free, open-source, Python-based graphical user interface designed to standardize and automate the calculation of antioxidant enzyme activities. The software integrates protein normalization, enzyme-specific calculation routines, data visualization, and Excel export within an intuitive interface that does not require programming expertise. Redoxyme was validated using experimental data obtained from animal tissues (rats and mice), demonstrating excellent agreement with manual calculations and established analytical methods. Redoxyme provides a practical solution for improving reproducibility and efficiency in antioxidant enzyme activity analysis. The software is currently distributed as a standalone executable for Windows (locally installed), and an interactive web-based calculator implemented in Streamlit, enabling direct use without local installation. The source code and version-controlled development history are openly accessible via GitHub, promoting transparency, reproducibility, community-driven improvements, and can, in principle, be adapted for other operating systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/703993v2_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@120cc68org.highwire.dtl.DTLVardef@4be246org.highwire.dtl.DTLVardef@1f47134org.highwire.dtl.DTLVardef@1341100_HPS_FORMAT_FIGEXP M_FIG C_FIG

1O_LIZinc (Zn) deficiency limits rice productivity and poses a risk to human health, particularly in populations reliant on rice-based diets. Although rice germplasm exhibits wide variation in Zn-deficiency tolerance, the underlying physiological mechanisms remain poorly resolved. Evidence across the literature for Zn-deficiency-induced secretion of 2'-deoxymugineic acid (DMA) is inconsistent. This study clarifies the role of DMA secretion as a Zn-deficiency stress response. C_LIO_LIWe developed and validated a sensitive LC-ESI-Q-TOF-MS method for selective detection of DMA in rice root exudates. Five rice genotypes with contrasting Zn-deficiency tolerance were grown hydroponically and DMA secretion measured. C_LIO_LIZn-deficiency increased DMA exudation across all genotypes, with sensitive genotypes also showing higher secretion compared with control, supporting DMAs role as a general response to Zn stress rather than being restricted to efficient genotypes. C_LIO_LIFold-change responses exceeded previous studies, likely due to more severe stress exposure. Our results confirm that DMA secretion is induced under Zn-deficiency in rice as part of the micronutrient stress response. However, the lack of increased Zn uptake indicates that additional tolerance mechanisms are involved. These findings reconcile inconsistencies in the literature and position DMA secretion as an important, but not exclusive, component of Zn-deficiency adaptation in rice. C_LI

Understanding the toxicity of hazardous metals in microalgae is critical for environmental risk assessment and sustainable phycoremediation. Metal-tolerant organisms provide powerful models for dissecting the mechanisms that mitigate metal toxicity. Here, we investigated the cellular and molecular responses to uranium (U) chemotoxicity in the metal-tolerant microalga Coelastrella sp. PCV. We used an integrated multi-omics and high-resolution imaging approach, combined with physiological analyses, to elucidate the mechanisms underlying U tolerance in Coelastrella. Using TEM-EDX, U was localized to the cell wall, polyphosphate bodies within acidocalcisomes, and vacuoles. Three-dimensional cell reconstruction and morphometric analysis using FIB-SEM showed that U-challenged cells displayed increased vacuolization, reflecting sequestration of uranyl ions and autophagy-mediated detoxification. Transcriptome responses were rapid and extensive, characterized by repression of cell division and photosynthesis, and pronounced imbalance in protein turnover and trafficking. Uranium also disrupted the homeostasis of essential elements, with marked rewiring of gene networks governing molybdenum, manganese, phosphate, iron and calcium homeostasis, notably affecting transporters and metal-binding proteins. Coelastrella sp. PCV efficiently sequestered U in acidocalcisomes and vacuoles, while rapidly excluding U from the cell. These coordinated detoxification responses are likely mediated by calcium, iron, ABC, and MATE transporters among the strongly deregulated genes under U stress.

Promyelocytic leukemia (PML) proteins are known to form phase-separated nuclear punctate structures called PML-nuclear bodies (PML-NBs). The integrity disruption of PML-NBs is linked with the pathogenesis of acute promyelocytic leukemia (APL), and trivalent arsenic (As3+) has been used for the clinical treatment of APL to restore normal PML-NBs. As3+ is considered to bind to cysteine residues and enhances modification of PML with small-ubiquitin-like protein (SUMO). We exposed U-2OS and CHO-K1 cells stably overexpressing PML-VI to As3+ and found that the solubility of PML decreased and SUMOylation of PML increased after 2 h-exposure to 3 M As3+. Contrary to As3+-induced remarkable biochemical changes including the solubility change and SUMOylation of PML, microscopic observation of PML-NBs was not changed clearly after a short-term exposure to As3+. The number of PML-NBs decreased and extranuclear PML bodies (EnPBs), which are remniscences of PML-NBs after nuclear membrane breakdown at mitosis, increased after exposure to As3+ for 24 - 72 h. The amount of SUMOylated PML decreased after prolonged exposure to As3+ while the solubility of PML was kept low, suggesting that As3+ stabilized EnPB without SUMOylation. The effects of As3+ on EnPBs were clearly observed at as low as 0.3 M As3+ which corresponds to inorganic arsenic level in drinking water worldwide.

The invasive fungal pathogen Pseudogymnoascus destructans is responsible for the collapse of several North American bat species through an infectious fungal skin disease known as White-Nose Syndrome (WNS). Recent transcriptomic studies have suggested that trace copper ion acquisition is essential for P. destructans propagation on its animal hosts. However, little is known about the mechanistic details of P. destructans adaptation occurring at the protein level. In this study, we report the global proteomic adaptation of P. destructans under chronic Cu-stress growth conditions employing chemically defined media. We identify 4340 P. destructans proteins, or approximately 47.8% of the predicted proteome, spanning a dynamic intensity range of six orders of magnitude. Chronic Cu-withholding stress leads to substantial alterations in the proteome, with 1398 differentially abundant proteins (DAPs) exhibiting statistically significant (p < 0.05) changes in protein levels compared to control growth conditions. We find that Cu-withholding stress induces increased levels of proteins associated with high-affinity Cu-acquisition, changes in intracellular superoxide dismutase (SOD) levels, and alterations in mitochondrial proteins related to aerobic respiration. In contrast, chronic Cu-overload stress leads to 390 DAPs (p < 0.05), which are more widely distributed across the proteome, with several DAPs associated with genomic stability and basic metabolism. Additionally, in this report, we present assessment of antisera products against intracellular and cell-surface protein targets of P. destructans that are effective for indicating Cu-withholding stress by western blotting.

Oxidative DNA damage caused by endogenous reactive oxygen species (ROS) is a key driver of mutagenesis, cellular dysfunction, and aging, contributing to diseases like cancer, neurodegeneration, rheumatoid arthritis, cardiovascular disorders, and diabetes. Although more than 20 oxidative base lesions have been identified, ROS-induced DNA-protein crosslinks (DPCs) are poorly characterized. ROS-DPCs are unusually bulky and highly toxic lesions that accumulate in metabolically active tissues with age, but their identities, biological consequences, and repair in living cells have remained elusive. In the present work, we characterized ROS-DPCs in human fibrosarcoma (HT1080) cells treated with hydrogen peroxide (H2O2) and elucidated the mechanisms of their removal. Mass spectrometry-based proteomics has identified over 100 cellular proteins that participated in DPC formation, most of which are involved in DNA metabolism. Our data further reveal that DNA replication and transcription facilitate DPC detection and identify a critical role of the ubiquitin-proteasomal system (UPS), replication-coupled activity of SPRTN metalloprotease, and nucleotide excision repair (NER) in removing ROS-induced DPCs. ROS-DPC formation was blocked by pretreatment with metabolically stable and cell-permeable glutathione (GSH) analog ({Psi}-GSH), suggesting a possible therapeutic strategy for preventing diseases associated with increased ROS levels. KEY POINTSMass spectrometry-based proteomics identified over 100 proteins participating in DNA-protein cross-links in human cells treated with ROS Our work reveals the mechanisms through which living cells recognize and remove ROS-DPCs Our study demonstrates the potential of a glutathione analog to prevent ROS-DPC formation GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/704426v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@15d9c33org.highwire.dtl.DTLVardef@ba0307org.highwire.dtl.DTLVardef@1cd46dorg.highwire.dtl.DTLVardef@be80ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Prokaryotes, particularly those in extreme environments, are capable of diverse metabolic states resulting in altered cell envelope structure and function. However, these changes are difficult to assess as standard fluorescent probes are often incompatible with extreme conditions and/or extremophile cell physiology. Halophilic archaea present the challenge of near-saturated intra-/extra-cellular salts, high membrane potential, and extended survival in altered metabolic states including entrapped within salt crystal fluid inclusions. We evaluated the compatibility of six fluorescent markers of cell envelope stability and activity with two model species, Halobacterium salinarum and Haloferax volcanii. Redox activity markers alamarBlue and pure resazurin solutions, membrane potential probes MitoTracker Orange-CMTMRos and Rhodamine 123, and SYTO 9 and propidium iodide (LIVE/DEAD kit) to assess cell membrane integrity were evaluated for use in bulk (microplate reader) and cell-specific (microscopy) applications. Limitations of each probe were identified, clarifying the utilization of each based on cell physiology, growth phase, medium composition, and probe exposure time including extended timescales needed to simulate the environmental conditions of haloarchaea. Of particular note, propidium iodide behavior was unreliable leading to double-labeling of cells and false interpretation of cells as dead. These data provide important insights into the study of prokaryotes in non-standard conditions.

The global industrialization and rapid urbanization elevated the risk of toxic pollutant exposure, which affects human health specially during pregnancy. Pregnant mothers are daily exposed to bisphenol-A (BPA), which is a common plastic leachate and a prominent toxic pollutant present in our environment. BPA act as an endocrine disrupting chemical (EDCs) by altering feto-placental homeostasis. This persistent and potent exposure of BPA during gestation can trigger placental damage affecting trophoblast cell function and survival. BPA even disrupts specific signalling cascades by altering post translational protein phosphorylation. However, this BPA mediated dysregulation of signalling nodes in early trimester placenta is still unexplored. Therefore, this study investigates the global proteome changes in post-BPA exposed extravillous trophoblast (EVTs) cells, which revealed a BPA mediated dynamic regulation of phosphoproteome-signatures and their associated kinases. Further inspection showed that the altered phosphorylation of c-JUN (S63) and GSK3 (Y279) is associated with BPA toxicity in EVTs and placenta. This altered phosphorylation affects the cellular signalling downstream, imparting damage upon the growing feto-placental unit. This highlights an altered phosphorylation mediated mechanism of BPA toxicity in placenta which can cause an onset of adverse pregnancy outcome. Data are available via ProteomeXchange with the identifiers PXD074780.

The opportunistic pathogen Pseudomonas aeruginosa is highly adaptable to different environmental conditions due to its versatile sensing and metabolic capabilities. Both external temperature and metal availability have a strong influence on the virulence and pathogenicity of P. aeruginosa, but the coupling between these two factors is not well understood. While iron is recognized as major player in nutritional immunity, the role of cobalt and the cobalt-containing vitamin B12 (cobalamin) during host infection remains unclear. Here, we investigate the environmental isolate P. aeruginosa PA254 using high-resolution global proteomics and cellular cobalamin measurements over a temperature gradient spanning environmental, host-associated, and heat-stress conditions (22-42 {degrees}C). PA254 occupies a continuum between an ambient-temperature virulent state characterized by versatile secreted factors, exopolysaccharide-rich biofilms, and planktonic swimmers and surface swarmers; and a host-associated virulent state characterized by potent secretion effectors, alginate-dominated biofilms, and a strong proportion of surface twitching motility. Pathway analyses indicate a shift toward carbon sparing, energy conservation, redox control, and metabolic maintenance during a host-adapted lifestyle, along with the strong overexpression of alternative iron acquisition strategies relying on heme and siderophores. Proteins of the cobalamin biosynthetic pathway declined significantly above ambient temperatures, despite constant intracellular B12 concentrations across all conditions. This decoupling of biosynthesis from cellular pools implies prioritization and recycling within B12-dependent processes, while the lack of B12 production at human body temperatures creates avenues for therapeutics interfering with B12 supply. Altogether, this work highlights a gradual rather than stepwise reprogramming of the P. aeruginosa proteome in response to environmental cues, and highlights proteomics as a tool to investigate system level mechanisms of challenging pathogens.

Copper is widely used as a fast-acting antimicrobial, yet the strategies that allow bacteria to survive copper stress remain incompletely understood. Here, we characterize the transcriptional responses of Escherichia coli MG1655 to excess ionic copper using RNA sequencing and a genome-wide GFP-based promoter library. We applied 2 mM copper, which slows growth, and 8 mM copper, a near-lethal concentration. RNA-seq revealed extensive transcriptome remodeling, with 487 genes upregulated at 2 mM and 364 at 8 mM. Both concentrations strongly induced canonical copper-responsive systems, oxidative stress defenses, histidine biosynthesis, and multiple iron acquisition pathways - including enterobactin biosynthesis and transport - despite external iron failing to reduce copper toxicity. At 2 mM copper, additional pathways were activated, including heat-shock and protein-folding functions as well as lipid A, methionine and arginine biosynthesis. Copper exposure also repressed large gene sets: 486 genes at 2 mM, enriched for biofilm formation and pH elevation, and 217 genes at 8 mM, enriched for anaerobic metabolism. In contrast to the robust RNA seq results, we investigated the Horizon Discovery E. coli genome-wide GFP based promoter library as an alternative screening tool. However, in our experiments it showed low signal to noise ratios, limiting its suitability for large scale gene expression screening.

Staphylococcus aureus encounters massive oxidative stress during infection. To counter this, the bacterium developed robust antioxidative defense mechanism. Glutathione peroxidases (Gpx) are well characterized antioxidative enzymes in eukaryotes; however, their bacterial counterparts remain poorly explored. S. aureus possesses two putative Gpx genes but lacks GSH biosynthetic machinery and glutathione reductase required for canonical Gpx function, suggesting alternate electron donor system(s) may be involved. This study aimed to elucidate structure-based biochemical characterization of one of the S. aureus glutathione peroxidases homologs (SaGpx, Uniprot Id: Q2FYZ0) and identify its plausible electron donor system. Herein, we cloned, purified and determined the high-resolution crystal structure of SaGpx (1.5 [A] resolution) using X-ray diffraction crystallography. In vitro biochemical characterization of the highly conserved active site amino acid point mutants, as well as their structural disposition suggests their precise roles in the enzymes catalysis. The crystal structure of SaGpx revealed that the enzyme adopts a canonical glutathione peroxidase fold with conserved catalytic tetrad composed of C36, Q70, W124 and N125. Also, SaGpx shows similarity with mammalian Gpx4, which was previously shown to exert phospholipid hydroperoxide peroxidase activity. Furthermore, biochemical assays suggest that SaGpx utilizes Staphylococcal thioredoxin1 as its cognate electron donor. The catalytic mechanism follows an atypical 2-cysteine peroxiredoxin-like pathway involving the formation of a sulfenic acid intermediate, followed by an intramolecular disulfide bond subsequently resolved by thioredoxin. This work provides the first structure-based biochemical characterization of a bacterial glutathione peroxidase homolog, establishing the novel structural insights of SaGpx as a noncanonical thioredoxin-dependent glutathione peroxidase.

Phosphate (Pi) and phosphite (Phi), a non-metabolizable analogue of Pi, are taken up by plant roots through the same transport system. Whereas Pi is an essential nutrient for plants, Phi might function as a biostimulant and in protection against pathogens. However, how Phi mechanistically exerts beneficial effects on plants remains unsolved. We examined the impact of Phi and Pi on Arabidopsis thaliana and rice growth and upon pathogen infection. Phi inhibited the in vitro growth of Plectosphaerella cucumerina and Fusarium fujikuroi in a dose-dependent manner, whereas Magnaporthe oryzae growth was largely unaffected. Phis effect on plant growth was dependent on the plant species, the basal Pi level in the plant, and the ratio Pi to Phi. In Arabidopsis, Phi enhanced resistance to P. cucumerina by triggering a hypersensitive response-like cell death. Notably, Phi reversed Pi-induced susceptibility to blast (M. oryzae) and bakanae (F. fujikuroi) diseases in rice. Transcriptomic analysis revealed that Phi triggered extensive reprogramming in rice under high Pi, including the activation of signaling pathways enriched in phosphorylation-dependent processes, while attenuating induction of carbon metabolism. Phi acts as a multifaceted agent, promotes balanced metabolic state, improved plant performance, and reduced Pi-induced disease susceptibility when applied under appropriate Pi conditions. HighlightPhosphite application confers protection against fungal pathogens in Arabidopsis and rice plants by regulating signaling pathways depending on phosphorylation processes.

Glutathione peroxidases (GPXs) are widely recognized as key antioxidants that mitigate oxidative stress by detoxifying reactive oxygen species (ROS). However, GPXs are largely uncharacterized in citrus. Here, we demonstrated that Citrus sinensis contains four GPX proteins (CsGPX1-4). Unexpectedly, overexpression of CsGPX4, a homolog of AtGPX8 in Arabidopsis, in citrus resulted in typical oxidative stress phenotypes including severe growth inhibition, chlorosis, and elevated intracellular ROS accumulation. Transmission electron microscopy (TEM) analysis further revealed stress responses at cellular level. Whole genome shot gun sequencing analysis showed that T-DNA insertion occurs in the UTR of SWEET2 gene, which is unlikely to be responsible for the oxidative stress phenotypes. Immunoblotting revealed that CsGPX4 accumulates as a truncated protein in citrus, in contrast to the full-length version expressed in Nicotiana benthamiana. MALDI-TOF assays further confirmed the truncation of CsGPX4 in the transgenic line with the predicted cleavage site between L115-K117. This truncation was associated with altered subcellular localization, shifting from cytoplasmic and nuclear distribution in N. benthamiana to membrane association in citrus. Proteomic profiling further indicated extensive reprogramming of pathways involved in detoxification, cytoskeletal stability, hormone signaling, and cell wall modification. Our data suggests that de facto overexpression of truncated CsGPX4 may have dominant-negative effects on proteins interacting with CsGPX4, thus interfering with their normal functions. In conclusion, our study demonstrates CsGPX4 as a critical regulator of redox homeostasis and ROS homeostasis in citrus and reveals selective truncation of CsGPX4 as a unique proteolytic or regulatory strategies in such processes.

Bisphenol A (BPA) and Bisphenol S (BPS) exposure disrupt ovarian function and granulosa cell (GC) steroidogenesis. Extracellular vesicles (EVs) and their miRNA cargo, as mediators of cellular response to environmental stimuli, might be involved in fertility and folliculogenesis. This study explored modulation of microRNA expression after 48h BPA or BPS exposure (10 {micro}M) in ovine primary GC and EVs from corresponding conditioned medium (CM EVs). Small RNA sequencing of control (0h) and 48h treated GC, CM EVs as well as follicular fluid EVs allowed identification of 533 ovine miRNAs, including 129 new sequences. BPA did not alter miRNA expression in GC, while BPS decreased cellular oar-24b miR. In contrast, BPA modified expression of 4 miRNAs in CM-EVs, including 3 new sequences, and two miRNAs were modified by BPS. Both compounds reduced expression of sequence homologous to miR-1306. Further studies are required to decipher their roles in bisphenol toxicity in GC.

Cadmium is a very toxic heavy metal. We studied Cd-treated barley plants with especial focus on rare atypical plants with signs of chlorosis. Cd treatment decreased the maximal photochemical activities of both photosystems while the activity of photosystem I decreased more than activity of photosystem II. In photosystem II, Cd treatment inhibited non-photochemical quenching that increased portion of unquenched "closed" complexes of photosystem II. The latter effect increased balance of limitations between the acceptor side of photosystem II (qC) and the donor side of photosystem I (Y(ND)) and raised the ratio qC/Y(ND). All these effects were enhanced in the atypical more damaged plants. Cd treatment reduced K content in the first leaves; in atypical plants, K content decreased even more. Cd treatment changed a pattern of stomatal conductance possibly by means of reducing K content in leaves. The untreated barley plants kept different stomatal conductance at adaxial and abaxial sides of leaves and fulfilled a complicated diurnal dynamics with large ups and downs of stomatal conductance. The typical Cd-treated plants were less flexible and demonstrated medium values. Stomatal conductance in the untreated plants were higher or lower than in the typical Cd-treated plants depending on a particular time; average daytime stomatal conductance was equal in both variants. At 10.00, stomatal conductance in the atypical Cd-treated plants was smaller than in the typical ones. Levels of 13 chloroplast mRNAs remained unchanged, while psbD decreased in both types of Cd-treated plants. HighlightsO_LISeveral Cd effects were enhanced in more damaged (atypical) chlorotic plants C_LIO_LICd treatment decreased activity of photosystem I and non-photochemical quenching C_LIO_LIRatio of limitations between photosystems II and I [qC/Y(ND)] was rather constant C_LIO_LICd treatment reduced K content in the first leaves C_LIO_LICd treatment changed pattern of stomatal conductance C_LI

Electrolytic hydrogen water (EHW) plays a critical role in modulating cellular metabolism; yet, the underlying molecular mechanisms remain unclear. This study utilized next-generation sequencing (NGS) to assess mRNA and miRNA expression in EHW-treated Caco-2 cells. Bioinformatics analysis identified differentially expressed genes (DEGs) and pathways influenced by EHW and highlighted its involvement in the oxidative stress response and tight junction formation. Protein-protein interaction (PPI) network analysis of the DEGs identified first-neighbor genes, supporting the role of EHW in suppressing oxidative stress-related genes while also enhancing the expression of the TCEB2-CUL5-COMMD8 (ECS complex) genes, both of which converged on the HIF-1 signaling pathway. We also constructed an mRNA-miRNA competing endogenous RNA (ceRNA) network, which revealed four hub genes, two non-coding RNAs (miR-429 and miR-200c-3p) and two protein-coding RNAs (CUL5 and GOLGA7). These genes co-target the transcription factor KLF4 in Caco-2 cells, forming a TF-miRNA-gene network (TMGN). EHW treatment significantly decreased the levels of miR-429 and miR-200c-3p and stabilized CUL5 and GOLGA7 transcripts post-transcriptionally as compared to ACW. Concurrently, reduced miRNA expression weakened their pre-transcriptional competition with mRNAs for KLF4 binding, further enhancing CUL5 and GOLGA7 expression. Phenotypic assays confirmed that continuous EHW treatment promotes Caco-2 cell differentiation. This study underscores the regulatory role of EHW in intestinal cells via feed-forward loops (FFLs), offering novel insights into the molecular mechanisms and functions of EHW. HighlightsO_LIIdentification of Novel Key Regulatory Genes Modulated by Electrolytic Hydrogen Water (EHW) Treatment: PPI network analysis demonstrated that EHW downregulates mitochondrial oxidative metabolism-related genes while upregulating TCEB2-CUL5-COMMD8 (ECS complex) expression within the HIF-1 axis. C_LIO_LIConstruction of a ceRNA Network: By integrating transcriptome and miRNA sequencing data from EHW-treated samples, we assembled an associated network and identified four hub genes in intestinal cells within the mRNA-miRNA ceRNA network: miR-429, miR-200c-3p, CUL5, and GOLGA7. C_LIO_LINovel Mechanistic Insights of Post- and Pre-Transcriptional Regulation by EHW: We identified KLF4 as a key transcription factor regulating EHW hub genes and constructed a TF-miRNA-gene (TMGN) feed-forward loop (FFL) network, offering new insights into EHW biomarkers. Our analysis revealed that EHW reduces miR-429 and miR-200c-3p levels, thereby enhancing CUL5 and GOLGA7 expression through both pre-transcriptional and post-transcriptional regulation. C_LIO_LIPhenotypic Confirmation: Continuous EHW treatment shortened the time required for Caco-2 cell differentiation. C_LI

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme whose substrates are various phosphorylated extracellular molecules including pyridoxal phosphate (vitamin B6) and adenine nucleotides. Dysfunctions of TNAP result in hypophosphatasia, a rare disease characterized by defective bone mineralization and impaired brain functions. In the brain, TNAP expression peaks during development and is associated with various steps of neurogenesis. However, the influence of TNAP activity on neurogenesis remains poorly understood in its cellular and molecular aspects. Here we used the SK-N-SH D human neuroblastoma cell line as a cell culture model to further investigate the involvement of TNAP in neuronal precursor proliferation and neuronal differentiation. We also used 1H-NMR-based metabolomics to investigate the molecular correlates of TNAP action on SK-N-SH D cell proliferation and differentiation. We first observed an increase in alkaline phosphatase (AP) activity when the cells were placed in differentiation medium. We next found that inhibiting TNAP with a specific inhibitor (MLS-0038949) impeded neuroblastoma cell proliferation. TNAP inhibition also hindered neuronal differentiation, as evidenced by a decrease in the number of neurite-bearing cells. In contrast, neurite length was not affected by TNAP inhibition, suggesting that TNAP controls neurite sprouting, but not neurite outgrowth per se. The metabolomic results indicate that proliferation and differentiation are associated with a decrease in the amounts of proteinogenic amino acids as well as that of compounds potentially involved in lipid production. This analysis also revealed that proliferation and differentiation are associated with increased glutathione levels and decreased amounts of hypotaurine and taurine, supporting proposals that organosulfur compounds play an important role in these processes. Since pyridoxine was present in the culture media, these results suggest that TNAP is involved in neurogenesis through mechanisms in addition to its role in vitamin B6 metabolism and may instead involve the ectonucleotidase activity (or an unidentified activity) of TNAP.

Diverse bacterial pathogens have evolved complex regulatory mechanisms to adapt to various environmental stresses during infection. The uncertainty in mRNA-protein levels in response to environmental stressors complicates our understanding of bacterial physiology and their adaptation to stressful environments. To examine this issue, we have integrated transcriptomics and proteomics data on three human bacterial pathogens Salmonella enterica Typhimurium, Yersinia pseudotuberculosis, and Staphylococcus aureus under ten infection-relevant stress conditions. We observed positive correlations between mRNA and protein levels, which were decreased under different stress conditions. Essential genes exhibited higher expression levels with lower variation across the conditions and stronger mRNA-protein correlations compared to non-essential genes, highlighting their critical role in bacterial adaptability and survival. Moreover, we identified a substantial number of genes with stress-induced non-correlating mRNA-protein levels, particularly under conditions triggering strong stress responses. Particularly this level was dramatically lowered for osmotic stress specific genes affected by impaired translational activity under osmotic stress. Our findings highlight the prevalence of non-correlating mRNA-protein levels and the potential role of post-translational modifications in modulating protein levels in response to environmental stressors during infection. This study provides a comprehensive framework for integrating transcriptomics and proteomics data and identifies potential gene products that might significantly impact the ability of diverse bacterial pathogens to adapt to hostile infection environments.