Intracellular Environment Research Articles

Insights into the Mechanism of Tryptophan Fluorescence Quenching due to Synthetic Crowding Agents: A Combined Experimental and Computational Study.

Intrinsic tryptophan fluorescence spectroscopy is an important tool for examining the effects of molecular crowding and confinement on the structure, dynamics, and function of proteins. Synthetic crowders such as dextran, ficoll, polyethylene glycols, polyvinylpyrrolidone, and their respective monomers are used to mimic crowded intracellular environments. Interactions of these synthetic crowders with tryptophan and the subsequent impact on its fluorescence properties are therefore critically important for understanding the possible interference created by these crowders. In the present study, the effects of polymer and monomer crowders on tryptophan fluorescence were assessed by using experimental and computational approaches. The results of this study demonstrated that both polymer and monomer crowders have an impact on the tryptophan fluorescence intensity; however, the molecular mechanisms of quenching were different. Using Stern-Volmer plots and a temperature variation study, a physical basis for the quenching mechanism of commonly used synthetic crowders was established. The quenching of free tryptophan was found to involve static, dynamic, and sphere-of-action mechanisms. In parallel, computational studies employing Kohn-Sham density functional theory provided a deeper insight into the effects of intermolecular interactions and solvation, resulting in differing quenching modes for these crowders. Taken together, the study offers new physical insights into the quenching mechanisms of some commonly used monomer and polymer synthetic crowders.

A novel long-tailed myovirus represents a new T4-like cyanophage cluster.

Cyanophages affect the abundance, diversity, metabolism, and evolution of picocyanobacteria in marine ecosystems. Here we report an estuarine Synechococcus phage, S-CREM2, which represents a novel viral genus and leads to the establishment of a new T4-like cyanophage clade named cluster C. S-CREM2 possesses the longest tail (~418 nm) among isolated cyanomyoviruses and encodes six tail-related proteins that are exclusively homologous to those predicted in the cluster C cyanophages. Furthermore, S-CREM2 may carry three regulatory proteins in the virion, which may play a crucial role in optimizing the host intracellular environment for viral replication at the initial stage of infection. The cluster C cyanophages lack auxiliary metabolic genes (AMGs) that are commonly found in cyanophages of the T4-like clusters A and B and encode unique AMGs like an S-type phycobilin lyase gene. A variation in the composition of tRNA and cis-regulatory RNA genes was observed between the marine and freshwater phage strains in cluster C, reflecting their different modes of coping with hosts and habitats. The cluster C cyanophages are widespread in estuarine and coastal regions and exhibit equivalent or even higher relative abundance compared to those of clusters A and B cyanophages in certain estuarine regions. The isolation of cyanophage S-CREM2 provides new insights into the phage-host interactions mediated by both newly discovered AMGs and virion-associated proteins and emphasizes the ecological significance of cluster C cyanophages in estuarine environments.

Quality of approximating a mass-emitting object by a point source in a diffusion model

For the sake of computational efficiency and for theoretical purposes, in mathematical modelling, the Dirac Delta distributions are often utilized as a replacement for cells or vesicles, since the size of cells or vesicles is much smaller than the size of the surrounding tissues. Here, we consider the scenario that the cell or the vesicle releases the diffusive compounds to the immediate environment, which is modelled by the diffusion equation. Typically, one separates the intracellular and extracellular environment and uses homogeneous Neumann boundary condition for the cell boundary (so-called spatial exclusion model), while the point source model neglects the intracellular environment. We show that extra conditions are needed such that the solutions to the two models are consistent. We prove a necessary and sufficient condition for the consistency. Suggested by the numerical results, we conclude that an initial condition in the form of Gaussian kernel in the point source model compensates for a time-delay discrepancy between the solutions to the two models in the numerical solutions. Various approaches determining optimal amplitude and variance of the Gaussian kernel have been discussed.

Real-time monitoring of the reaction of KRAS G12C mutant specific covalent inhibitor by in vitro and in-cell NMR spectroscopy

KRAS mutations are major drivers of various cancers. Recently, allele-specific inhibitors of the KRAS G12C mutant were developed that covalently modify the thiol of Cys12, thereby trapping KRAS in an inactive GDP-bound state. To study the mechanism of action of the covalent inhibitors in both in vitro and intracellular environments, we used real-time NMR to simultaneously observe GTP hydrolysis and inhibitor binding. In vitro NMR experiments showed that the rate constant of ARS-853 modification is identical to that of GTP hydrolysis, indicating that GTP hydrolysis is the rate-limiting step for ARS-853 modification. In-cell NMR analysis revealed that the ARS-853 reaction proceeds significantly faster than that in vitro, reflecting acceleration of GTP hydrolysis by endogenous GTPase proteins. This study demonstrated that the KRAS covalent inhibitor is as effective in the cell as in vitro and that in-cell NMR is a valuable validation tool for assessing the pharmacological properties of the drug in the intracellular context.

How it feels in a cell.

Life emerges from thousands of biochemical processes occurring within a shared intracellular environment. We have gained deep insights from in vitro reconstitution of isolated biochemical reactions. However, the reaction medium in test tubes is typically simple and diluted. The cell interior is far more complex: macromolecules occupy more than a third of the space, and energy-consuming processes agitate the cell interior. Here, we review how this crowded, active environment impacts the motion and assembly of macromolecules, with an emphasis on mesoscale particles (10-1000 nm diameter). We describe methods to probe and analyze the biophysical properties of cells and highlight how changes in these properties can impact physiology and signaling, and potentially contribute to aging, and diseases, including cancer and neurodegeneration.

An Anaplasma phagocytophilum T4SS effector, AteA, is essential for tick infection.

Ticks are the number one vector of pathogens for livestock worldwide and for humans in the United States. The biology of tick transmission is an understudied area. Understanding this critical interaction could provide opportunities to affect the course of disease spread. In this study, we examined the zoonotic tick-borne agent Anaplasma phagocytophilum and identified a secreted protein, AteA, which is expressed in a tick-specific manner. These secreted proteins, termed effectors, are the first proteins to interact with the host environment. AteA is essential for survival in ticks and appears to interact with cortical actin. Most effector proteins are studied in the context of the mammalian host; however, understanding how this unique set of proteins affects tick transmission is critical to developing interventions.

Alphavirus-induced transcriptional and translational shutoffs play major roles in blocking the formation of stress granules.

Our study highlights the mechanisms behind the cell's resistance to stress granule (SG) formation after infection with Old World alphaviruses. Shortly after infection, the replication of these viruses hinders the cell's ability to form SGs, even when exposed to chemical inducers such as sodium arsenite. This resistance is primarily attributed to virus-induced transcriptional and translational shutoffs, rather than interactions between the viral nsP3 and the key components of SGs, G3BP1/2, or the ADP-ribosylhydrolase activity of nsP3 macro domain. While interactions between G3BPs and nsP3 are essential for the formation of viral replication complexes, their role in regulating SG development appears to be small, if any. Cells harboring replicating viruses or replicons with lower abilities to inhibit transcription and/or translation, but expressing wild-type nsP3, retain the ability for SG development. Understanding these mechanisms of regulation of SG formation contributes to our knowledge of viral replication and the intricate relationships between alphaviruses and host cells.

Spinal cord injury significantly alters the properties of reticulospinal neurons: delayed repolarization mediated by potassium channels.

After rostral spinal cord injury (SCI) of lampreys, the descending axons of injured (axotomized) reticulospinal (RS) neurons regenerate and locomotor function gradually recovers. Our previous studies indicated that relative to uninjured lamprey RS neurons, injured RS neurons display several dramatic changes in their biophysical properties, called the "injury phenotype." In the present study, at the onset of applied depolarizing current pulses for membrane potentials below as well as above threshold for action potentials (APs), injured RS neurons displayed a transient depolarization consisting of an initial depolarizing component followed by a delayed repolarizing component. In contrast, for uninjured neurons the transient depolarization was mostly only evident at suprathreshold voltages when APs were blocked. For injured RS neurons, the delayed repolarizing component resisted depolarization to threshold and made these neurons less excitable than uninjured RS neurons. After block of voltage-gated sodium and calcium channels for injured RS neurons, the transient depolarization was still present. After a further block of voltage-gated potassium channels, the delayed repolarizing component was abolished or significantly reduced, with little or no effect on the initial depolarizing component. Voltage-clamp experiments indicated that the delayed repolarizing component was due to a noninactivating outward-rectifying potassium channel whose conductance (gK) was significantly larger for injured RS neurons compared to that for uninjured neurons. Thus, SCI results in an increase in gK and other changes in the biophysical properties of injured lamprey RS neurons that lead to a reduction in excitability, which is proposed to create an intracellular environment that supports axonal regeneration.NEW & NOTEWORTHY After spinal cord injury (SCI), lamprey reticulospinal (RS) neurons responded to subthreshold depolarizing current pulses with a transient depolarization, which included an initial depolarization that was due to passive channels followed by a delayed repolarization that was mediated by voltage-gated potassium channels. The conductance of these channels (gK) was significantly increased for RS neurons after SCI and contributed to a reduction in excitability, which is expected to provide supportive conditions for subsequent axonal regeneration.

The secreted tyrosine phosphatase PtpA promotes Staphylococcus aureus survival in RAW 264.7 macrophages through decrease of the SUMOylation host response.

Staphylococcus aureus uses numerous strategies to survive and persist in the intracellular environment of professional phagocytes, including modulation of the SUMOylation process. This study aims to understand how S. aureus alters host SUMOylation to enhance its intracellular survival in professional phagocytes. Our results indicate that S. aureus strain Newman utilizes PtpA-driven phosphorylation to decrease the amount of SUMOylated proteins in murine macrophages to facilitate its survival in this immune cell type.

Response mechanisms to acid stress promote LF82 replication in macrophages.

Adherent-invasive E. coli (AIEC) LF82 is capable of adhering to and invading intestinal epithelial cells, as well as replicating within macrophages without inducing host cell death. We compared the transcriptomics of LF82 at pH=7.5 and pH=5.8 by RNA-sequencing, and qRT-PCR verified differentially expressed genes (DEGs). The deletion mutants of DEGs in the treatment group (pH=5.8) compared to the control group (pH=7.5) were constructed by λ recombinant. The replication differences between the mutants and WT infected Raw 264.7 at 24 h.p.i were analyzed by combining LB solid plate count and confocal observation. NH4Cl and chloroquine diphosphate (CQ) were used for acid neutralization to study the effect of pH on the replication of LF82 in macrophages. Na2NO3 was added to RPMI 1640 to study the effect of nitrate on the replication of LF82 in macrophages. 0.3% solid LB was used for flagellar motility assay and Hela was used to study flagellar gene deletion mutants and WT adhesion and invasion ability. In this study, we found that infection with LF82 results in acidification of macrophages. Subsequent experiments demonstrated that an intracellular acidic environment is necessary for LF82 replication. Transcriptome and phenotypic analysis showed that high expression of acid shock genes and acid fitness genes promotes LF82 replication in macrophages. Further, we found that the replication of LF82 in macrophages was increased under nitrate treatment, and nitrogen metabolism genes of LF82 were upregulated in acid treatment. The replication in macrophages of ΔnarK, ΔnarXL, ΔnarP, and Δhmp were decreased. In addition, we found that the expression of flagellar genes was downregulated in acidic pH and after LF82 invading macrophages. Motility assay shows that the movement of LF82 on an acidic semisolid agar plate was limited. Further results showed that ΔfliC and ΔfliD decreased in motility, adhesion ability, and invasion of host cells, but no significant effect on replication in macrophages was observed. In this study, we simulated the acidic environment in macrophages, combined with transcriptome technology, and explained from the genetic level that LF82 promotes replication by activating its acid shock and fitness system, enhancing nitrate utilization, and inhibiting flagellar function.

Understanding the Impacts of Molecular and Macromolecular Crowding Agents on Protein-Polymer Complex Coacervates.

Complex coacervation refers to the liquid-liquid phase separation (LLPS) process occurring between charged macromolecules. The study of complex coacervation is of great interest due to its implications in the formation of membraneless organelles (MLOs) in living cells. However, the impacts of the crowded intracellular environment on the behavior and interactions of biomolecules involved in MLO formation are not fully understood. To address this knowledge gap, we investigated the effects of crowding on a model protein-polymer complex coacervate system. Specifically, we examined the influence of sucrose as a molecular crowder and polyethylene glycol (PEG) as a macromolecular crowder. Our results reveal that the presence of crowders led to the formation of larger coacervate droplets that remained stable over a 25-day period. While sucrose had a minimal effect on the physical properties of the coacervates, PEG led to the formation of coacervates with distinct characteristics, including higher density, increased protein and polymer content, and a more compact internal structure. These differences in coacervate properties can be attributed to the effects of crowders on individual macromolecules, such as the conformation of model polymers, and nonspecific interactions among model protein molecules. Moreover, our results show that sucrose and PEG have different partition behaviors: sucrose was present in both the coacervate and dilute phases, while PEG was observed to be excluded from the coacervate phase. Collectively, our findings provide insights into the understanding of crowding effects on complex coacervation, shedding light on the formation and properties of coacervates in the context of MLOs.

ROS meditated paralytic shellfish toxins production changes of Alexandrium tamarense caused by microplastic particles

A variety of studies have investigated the toxic effects of microplastics (MPs) on microalgae, but few of them considered their influence on dinoflagellate toxins production, which could cause significant ecological safety concerns in coastal areas. This research investigated the impacts of 5 μg L−1 and 5 mg L−1 polystyrene (PS) MPs on the changes of paralytic shellfish toxins (PSTs) production and their relationship with cellular oxidative stress of Alexandrium tamarense, a common harmful algal blooms causative dinoflagellate. The results showed elevation of reactive oxygen species (ROS) levels, activation of antioxidant system and overproduction of PSTs were positively correlated under PS MPs exposure (especially under 5 mg L−1 PS MPs), and the PSTs changes were eliminated by the ROS inhibitor. Further transcriptomic analysis revealed that ROS could enhance biosynthesis of glutamate, providing raw materials for PSTs precursor arginine, accompanied with enhanced acetyl-CoA and ATP production, finally leading to the overproduction of PSTs. Moreover, the oxidative intracellular environments might block the reduction process from STX to C1&C2, leading to the increase of STX and decrease of C1&C2 proportions. This work brings the first evidence that ROS could mediate PSTs production and compositions of Alexandrium under MPs exposure, with important scientific and ecological significance.

Burkholderia pseudomallei biofilm resists Acanthamoeba sp. grazing and produces 8-O-4′-diferulic acid, a superoxide scavenging metabolite after passage through the amoeba

Burkholderia pseudomallei, an etiological agent of melioidosis is an environmental bacterium that can survive as an intracellular pathogen. The biofilm produced by B. pseudomallei is crucial for cellular pathogenesis of melioidosis. The purpose of this investigation is to explore the role of biofilm in survival of B. pseudomallei during encounters with Acanthamoeba sp. using B. pseudomallei H777 (a biofilm wild type), M10 (a biofilm defect mutant) and C17 (a biofilm-complemented strain). The results demonstrated similar adhesion to amoebae by both the biofilm wild type and biofilm mutant strains. There was higher initial internalisation, but the difference diminished after longer encounter with the amoeba. Interestingly, confocal laser scanning microscopy demonstrated that pre-formed biofilm of B. pseudomallei H777 and C17 were markedly more persistent in the face of Acanthamoeba sp. grazing than that of M10. Metabolomic analysis revealed a significant increased level of 8-O-4′-diferulic acid, a superoxide scavenger metabolite, in B. pseudomallei H777 serially passaged in Acanthamoeba sp. The interaction between B. pseudomallei with a free-living amoeba may indicate the evolutionary pathway that enables the bacterium to withstand superoxide radicals in intracellular environments. This study supports the hypothesis that B. pseudomallei biofilm persists under grazing by amoebae and suggests a strategy of metabolite production that turns this bacterium from saprophyte to intracellular pathogen.

Effect of In Vitro Gastrointestinal Digestion on Phytochemicals and Antioxidant Activities in Cherry Tomatoes (Solanum lycopersicum var. cerasiforme).

We investigated the impact of simulated in vitro gastrointestinal digestion on the levels of total polyphenols, total flavonoids, carotenoids, and antioxidant capacity in cherry tomatoes. The initial total polyphenol content of fresh tomatoes was 220.51 μg GAE/g, which decreased to 203.24 μg GAE/g after 120 min of stomach treatment and further decreased to 138.23 μg GAE/g after 120 min of small intestine treatment. Similarly, the initial total flavonoid content in fresh tomatoes was 43.28 μg QE/g, but after 120 min of small intestine digestion, it decreased by approximately 50.72% to 21.33 μg QE/g. Lycopene, lutein, and β-carotene also experienced a decrease of 69.71∼78.38% during the digestion process compared to fresh tomatoes. The antioxidant activity exhibited a reduction of 34.95∼37.67% compared to fresh tomatoes after digestion in the stomach and intestines. The bioactive compounds present in tomatoes undergo decomposition and conversion into other substances during digestion, and these degradation products are believed to inhibit the growth of SK-Hep1 human hepatoma cells while enhancing antioxidant activity within the intracellular environment.

Radiochemical Oxygen Depletion Depends on Chemical Environment and Dose-Rate: Implications for FLASH Effect

FLASH (dose rates > 40 Gy/s) radiotherapy (RT) protects normal tissues from radiation damage, compared with conventional RT (∼Gy/min). Radiation-chemical oxygen depletion (ROD) has been the leading and most-studied mechanism to explain FLASH. Previous studies have reported low ROD values of ∼0.35 µM/Gy, however, they do not accurately model (utilizing water or oxidized substrates such as albumin) the intracellular environment. It has been proposed that the intracellular environment may support higher ROD values due to its specific chemical makeup. As chemical reducing agents, which play a critical role in the cell, may promote ROD, we measured ROD for several well-known cellular reducing agents. Precision polarographic oxygen sensors were used to measure ROD over a ∼100-0 µM oxygen concentration range in solutions containing various types of intracellular reducing agents, either alone or combined with glycerol (1M; to simulate the intracellular hydroxyl-radical-scavenging capacity). A Cs irradiator and a research proton beamline were used for radiation beam generation over a 10,000-fold (0.0085 to 100 Gy/s) dose rate range. Reagents were grouped generally into primary free-radical scavengers (glycerol & lysozyme) and reducing agents (cysteamine, cysteine, glutathione, dextrin-conjugated linoleic acid, NAD(P)H, ascorbate and uric acid). Reducing agents at 1 mM significantly altered ROD values. ROD, at 0.1 Gy/s varied from greater than 1 µM/Gy for NADPH to less than 0.1 µM/Gy for uric acid. Even higher RODs were found at lower dose rates and/or higher reducing agent concentrations. Although most greatly increased ROD, ascorbate and uric acid decreased ROD and additionally imposed an oxygen dependence of ROD at low oxygen concentrations. Interestingly, all agents having high rates of ROD at low dose rates show a decrease at FLASH dose rates and this seems to be a proportional response (i.e., the greater the ROD at low dose rate, the greater the inhibition by FLASH). At FLASH dose rates our data for a simple mixture of glycerol and glutathione showed the same trend as recent studies that, in general, FLASH had ∼25% lower ROD rates than conventional demonstrating agreement between polarographic sensors and optical phosphorescence decay methods. ROD was greatly augmented by some intracellular reducing agents but others effectively reversed this effect. Ascorbate had its greatest impact at low oxygen concentrations. ROD decreased with increasing dose rate and in all cases was less than 1 µM/Gy at 100 Gy/s, potentially posing a fundamental limit of max ROD for FLASH.

Salmonella Gallinarum mgtC mutant shows a delayed fowl typhoid progression in chicken

Salmonella Gallinarum (SG) provokes fowl typhoid, an infectious disease of acute clinical course that affects gallinaceous of any age and leads to high mortality rates. During the typhoid-like systemic infection of S. Typhimurium (STM) in mice, the bacterium expresses the mgtC gene, which is encoded in the Salmonella Pathogenecity Island – 3 (SPI-3). In this serovar, the function is linked to bacterial replication within macrophages, and its absence attenuates the pathogen. We hypothesized that deleting mgtC from SG genome would alter the microorganism pathogenicity in susceptible commercial poultry in a similar manner. Thus, the present study sought to elucidate the importance of mgtC on SG pathogenicity. For this, a mgtC-mutant lacking S. Gallinarum mutant was constructed (SG ΔmgtC). Its ability to replicate in medium that mimicries the mgtC-related intracellular environment of macrophages as well as in primary macrophages from chicken was evaluated. Moreover, the infection of susceptible chickens was performed to elucidate its pathogenicity and the elicited immune responses by measuring key interleukins by qRT-PCR and the population of macrophages and lymphocytes T CD4+ and CD8+ by means of immunohistochemistry. It was observed that mgtC was required for S. Gallinarum replication in acidified low-Mg2+ media and survival within macrophages. However, unlike its requirement for initial phase of STM infection in mice, lower bacterial counts were only observed at the late stage of macrophage infection without affecting the citotoxicity. Experiments showed that knocking-out the mgtC gene neither altered bacterial uptake by macrophages nor affects bacterial counts in liver and spleen and total chicken mortality. However, plotting a survival curve and analyzing the clinical-pathologic conditions, it was observed a slower progression of the disease in chickens infected by SG ΔmgtC compared to those challenged by the wild-type strain. Furthermore, the mRNA expression of IFN-γ and LITAF were similar between the infected chickens, but higher than in the uninfected group. The same was observed in macrophages and lymphocytes T CD4+ populations. On the other hand, the presence of lymphocytes T CD8+ was increased in the initial phase of the disease provoked by the wild-type strain over the mutant strain. We concluded that the role of mgtC in Fowl Typhoid in susceptible chickens differs from the role in typhoid-like infections in mammals. Thus, the deletion of mgtC gene from S. Gallinarum genome does not affect the overall pathogenicity, but slightly alters the pathogenesis.

The post-translational regulation of transcription factor EB (TFEB) in health and disease.

Transcription factor EB (TFEB) is a basic helix-loop-helix leucine zipper transcription factor that acts as a master regulator of lysosomal biogenesis, lysosomal exocytosis, and macro-autophagy. TFEB contributes to a wide range of physiological functions, including mitochondrial biogenesis and innate and adaptive immunity. As such, TFEB is an essential component of cellular adaptation to stressors, ranging from nutrient deprivation to pathogenic invasion. The activity of TFEB depends on its subcellular localisation, turnover, and DNA-binding capacity, all of which are regulated at the post-translational level. Pathological states are characterised by a specific set of stressors, which elicit post-translational modifications that promote gain or loss of TFEB function in the affected tissue. In turn, the resulting increase or decrease in survival of the tissue in which TFEB is more or less active, respectively, may either benefit or harm the organism as a whole. In this way, the post-translational modifications of TFEB account for its otherwise paradoxical protective and deleterious effects on organismal fitness in diseases ranging from neurodegeneration to cancer. In this review, we describe how the intracellular environment characteristic of different diseases alters the post-translational modification profile of TFEB, enabling cellular adaptation to a particular pathological state.

Polysiloxane probes with main chain modifications regulating subcellular organelles localization for dynamic observation of lysosomal and mitochondrial interactions during cellular hypoxia

As an important pathological state, hypoxia is closely associated with the occurrence of numerous diseases. The degree of hypoxia can reflect the related pathological conditions. The expression of heme oxygenase-1 (HO-1) will lead to the increase of endogenous carbon monoxide (CO) during hypoxia. Abnormal concentrations of CO involved in regulating various physiological activities of cells may result in the damage of organelles from lysosomes to mitochondria and further disrupt the normal communication between organelles. However, normal intercellular communication and interactions have profound significance for the dynamic equilibrium of the intracellular environment. Hence, it is a challenging task to develop new tools to visualize the effect of CO on the interaction of different organelles during hypoxia. In this paper, we designed fluorescent probes that could switch between lysosomes and mitochondria by using polysiloxane platforms (P1-CORM-Lyso and P1-CORM-Mito) for the first time. P1-CORM-Lyso and P1-CORM-Mito successfully enabled monitor the CO generated during anoxia with excellent biocompatibility. We expect that this switchable targeting silicon-based fluorescent materials will open up new fields for the design of new fluorescent probes and get a deeper understanding of the cellular hypoxia process.

Exosomal miRNA analysis provides new insights into exposure to nanoplastics and okadaic acid

As an emerging environmental pollutant, nanoplastics (NPs) have attracted wide attention in terms of their impact on the ecological environment and human health. Currently, researches on the cytotoxicity of NPs mainly focus on oxidative stress, damage to the cell membrane and organelles, induction of immune response and genotoxicity. Okadaic acid (OA) is the main component of diarrheal shellfish toxin. Based on the previous combined toxicity exploration of polystyrene (PS) NPs and (OA) to human gastric adenocarcinoma (AGS) cells, cell-derived exosomes were extracted and exosomal miRNA profiles were analyzed for the first time in this study. The results showed that the composition of miRNAs varied after the exposure of NPs and OA. Specifically, the expression of miR-1-3p in both PS-Exo and PS-OA-Exo was significantly reduced. And the expression of miR-1248 was upregulated most significantly by comparing the DE miRNAs between PS-Exo and PS-OA-Exo. MiR-1-3p and miR-1248 may be the key genes for the combined toxicity of NPs and OA. After analysis, we found that both the decreased expression of miR-1-3p and the increased expression of miR-1248 can increase the expression of FN1 and affect DNA replication, which was surprisingly consistent with the results of our previous cytotoxicity studies. Since exosomal miRNAs are selectively encapsulated by donor cell, we speculate that the changes of exosomal miRNAs may due to the synchronous changes of intracellular environment and the downregulation of intracellular FN1 may be attributed to decreased expression of miR-1-3p and increased expression of miR-1248 in donor cells. Accordingly, we come to the conclusion that the changes of miRNAs in the exosomes derived from AGS cells after environmental stimulation could reflect the biological effects of donor cells.

Itaconic acid and dimethyl itaconate exert antibacterial activity in carbon-enriched environments through the TCA cycle

Itaconic acid (IA), a metabolite generated by the tricarboxylic acid (TCA) cycle in eukaryotic immune cells, and its derivative dimethyl itaconate (DI) exert antibacterial functions in intracellular environments. Previous studies suggested that IA and DI only inhibit bacterial growth in carbon-limited environments; however, whether IA and DI maintain antibacterial activity in carbon-enriched environments remains unknown. Here, IA and DI inhibited the bacteria with minimum inhibitory concentrations of 24.02 mM and 39.52 mM, respectively, in a carbon-enriched environment. The reduced bacterial pathogenicity was reflected in cell membrane integrity, motility, biofilm formation, AI-2/luxS, and virulence. Mechanistically, succinate dehydrogenase (SDH) activity and fumaric acid levels decreased in the IA and DI treatments, while isocitrate lyase (ICL) activity was upregulated. Inhibited TCA circulation was also observed through untargeted metabolomics. In addition, energy-related aspartate metabolism and lysine degradation were suppressed. In summary, these results indicated that IA and DI reduced bacterial pathogenicity while exerting antibacterial functions by inhibiting TCA circulation. This study enriches knowledge on the inhibition of bacteria by IA and DI in a carbon-mixed environment, suggesting an alternative method for treating bacterial infections by immune metabolites.