Dissemination of pathogenic bacteria is reinforced by a MARTX toxin effector duet.

Multiple bacterial genera take advantage of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin to invade host cells. Secretion of the MARTX toxin by Vibrio vulnificus, a deadly opportunistic pathogen that causes primary septicemia, the precursor of sepsis, is a major driver of infection; however, the molecular mechanism via which the toxin contributes to septicemia remains unclear. Here, we report the crystal and cryo-electron microscopy (EM) structures of a toxin effector duet comprising the domain of unknown function in the first position (DUF1)/Rho inactivation domain (RID) complexed with human targets. These structures reveal how the duet is used by bacteria as a potent weapon. The data show that DUF1 acts as a RID-dependent transforming NADase domain (RDTND) that disrupts NAD+ homeostasis by hijacking calmodulin. The cryo-EM structure of the RDTND-RID duet complexed with calmodulin and Rac1, together with immunological analyses in vitro and in mice, provide mechanistic insight into how V. vulnificus uses the duet to suppress ROS generation by depleting NAD(P)+ and modifying Rac1 in a mutually-reinforcing manner that ultimately paralyzes first line immune responses, promotes dissemination of invaders, and induces sepsis. These data may allow development of tools or strategies to combat MARTX toxin-related human diseases.

Angiotensin-converting enzyme inhibition prevents l-dopa-induced dyskinesia in a 6-ohda-induced mouse model of Parkinson's disease.

Parkinson's disease (PD) is characterised by severe movement defects and the degeneration of dopaminergic neurones in the midbrain. The symptoms of PD can be managed with dopamine replacement therapy using L-3, 4-dihydroxyphenylalanine (L-dopa), which is the gold standard therapy for PD. However, long-term treatment with L-dopa can lead to motor complications. The central renin-angiotensin system (RAS) is associated with the development of neurodegenerative diseases in the brain. However, the role of the RAS in dopamine replacement therapy for PD remains unclear. Here, we tested the co-treatment of the angiotensin-converting enzyme inhibitor (ACEI) with L-dopa altered L-dopa-induced dyskinesia (LID) in a 6-hydroxydopamine (6-OHDA)-lesioned mouse model of PD. Perindopril, captopril, and enalapril were used as ACEIs. The co-treatment of ACEI with L-dopa significantly decreased LID development in 6-OHDA-lesioned mice. In addition, the astrocyte and microglial transcripts involving Ccl2, C3, Cd44, and Iigp1 were reduced by co-treatment with ACEI and L-dopa in the 6-OHDA-lesioned striatum. In conclusion, co-treatment with ACEIs and L-dopa, such as perindopril, captopril, and enalapril, may mitigate the severity of L-DOPA-induced dyskinesia in a mouse model of PD.

Evaluation of the effect of dimethyl fumarate on human bone marrow-derived mesenchymal stem cells using bottom-up proteomics.

Mesenchymal stem cells (MSCs) have potential as a viable treatment option in the field of regenerative medicine, but MSC-based therapy needs to be more efficient. Preconditioning is a method to improve MSC-based therapy, and dimethyl fumarate (DMF) - an agent that can enhance the antioxidative capacity of cells - can be considered for preconditioning of MSCs. In this study, we treated bone marrow-derived MSCs with DMF and evaluated their proteome using bottom-up proteomics. The MSCs were exposed to 10 µM DMF for 24 h, followed by lysis with an SDS solution, digestion with trypsin using an s-trap column, and analysis using nanoLC-MS/MS, which identified 2262 proteins with confidence. Bioinformatic analysis of the identified proteins revealed 47 upregulated proteins and 81 downregulated proteins upon DMF treatment. Pathway enrichment analysis suggested a possible decrease in autophagy and a decrease in the activity of the TCA cycle, while indicating a potential increase in proliferation and antioxidant activity in DMF-treated MSCs compared to untreated MSCs. Our findings suggest that DMF can enhance the proliferation of MSCs and increase their stability, and that preconditioning could improve the therapeutic efficacy of MSCs for the treatment of regenerative diseases.

Hypoxia stabilizes SETDB1 to maintain genome stability.

Von Hippel-Lindau (VHL) is a tumor suppressor that functions as the substrate recognition subunit of the CRL2VHL E3 complex. While substrates of VHL have been identified, its tumor suppressive role remains to be fully understood. For further determination of VHL substrates, we analyzed the physical interactome of VHL and identified the histone H3K9 methyltransferase SETBD1 as a novel target. SETDB1 undergoes oxygen-dependent hydroxylation by prolyl hydroxylase domain proteins and the CRL2VHL complex recognizes hydroxylated SETDB1 for ubiquitin-mediated degradation. Under hypoxic conditions, SETDB1 accumulates by escaping CRL2VHL activity. Loss of SETDB1 in hypoxia compared with that in normoxia escalates the production of transposable element-derived double-stranded RNAs, thereby hyperactivating the immune-inflammatory response. In addition, strong derepression of TEs in hypoxic cells lacking SETDB1 triggers DNA damage-induced death. Our collective results support a molecular mechanism of oxygen-dependent SETDB1 degradation by the CRL2VHL E3 complex and reveal a role of SETDB1 in genome stability under hypoxia.

Secreted Akkermansia muciniphila threonyl-tRNA synthetase functions to monitor and modulate immune homeostasis.

Commensal bacteria are critically involved in the establishment of tolerance against inflammatory challenges, the molecular mechanisms of which are just being uncovered. All kingdoms of life produce aminoacyl-tRNA synthetases (ARSs). Thus far, the non-translational roles of ARSs have largely been reported in eukaryotes. Here, we report that the threonyl-tRNA synthetase (AmTARS) of the gut-associated bacterium Akkermansia muciniphila is secreted and functions to monitor and modulate immune homeostasis. Secreted AmTARS triggers M2 macrophage polarization and orchestrates the production of anti-inflammatory IL-10 via its unique, evolutionary-acquired regions, which mediates specific interactions with TLR2. This interaction activates the MAPK and PI3K/AKT signaling pathways, which converge on CREB, leading to an efficient production of IL-10 and suppression of the central inflammatory mediator NF-κB. AmTARS restores IL-10-positive macrophages, increases IL-10 levels in the serum, and attenuates the pathological effects in colitis mice. Thus, commensal tRNA synthetases can act as intrinsic mediators that maintain homeostasis.

Comparison study of mouse brain tissue by using ToF-SIMS within static limits and hybrid SIMS beyond static limits (dynamic mode).

In the study of degenerative brain diseases, changes in lipids, the main component of neurons, are particularly important because they are used as indicators of pathological changes. One method for the sensitive measurement of biomolecules, especially lipids, is time-of-flight secondary ion mass spectrometry (ToF-SIMS) using pulsed argon cluster ions. In this study, biomolecules including various lipids present in normal mouse brain tissue were measured using ToF-SIMS equipped with pulsed argon cluster primary ions. Based on the ToF-SIMS measurement results, hybrid SIMS (OrbiSIMS), which is a ToF-SIMS system with the addition of an orbitrap mass analyzer, was used to directly identify the biomolecules by the region in the real tissue samples. For this, the results of ToF-SIMS, which measured the tissue samples from a single mouse brain within static limits, were compared with those from OrbiSIMS measured beyond the static limits in terms of the differences in molecular profiling. From this analysis, two types of positive and negative ions were selected for identification, with the OrbiSIMS MS/MS results indicating that the positive ions were glycerophosphocholine and the negative ions were glycerophosphoinositol and sulfatide, a sphingolipid. Then, to confirm the identification of the molecular candidates, lipids were extracted from mirror image tissue samples, and LC-MS/MS also using an orbitrap mass analyzer was performed. As a result, the direct identification of molecular candidate groups distributed in particular regions of the tissue samples via OrbiSIMS was found to be consistent with the identification results by LC-MS/MS for extracted samples.

Phytochrome B photobodies are comprised of phytochrome B and its primary and secondary interacting proteins.

Phytochrome B (phyB) is a plant photoreceptor that forms a membraneless organelle called a photobody. However, its constituents are not fully known. Here, we isolated phyB photobodies from Arabidopsis leaves using fluorescence-activated particle sorting and analyzed their components. We found that a photobody comprises ~1,500 phyB dimers along with other proteins that could be classified into two groups: The first includes proteins that directly interact with phyB and localize to the photobody when expressed in protoplasts, while the second includes proteins that interact with the first group proteins and require co-expression of a first-group protein to localize to the photobody. As an example of the second group, TOPLESS interacts with PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) and localizes to the photobody when co-expressed with PCH1. Together, our results support that phyB photobodies include not only phyB and its primary interacting proteins but also its secondary interacting proteins.

Shear stress induces monocyte/macrophage-mediated inflammation by upregulating cell-surface expression of heat shock proteins.

The loss of endothelial cells is associated with the accumulation of monocytes/macrophages underneath the surface of the arteries, where cells are prone to mechanical stimulation, such as shear stress. However, the impact of mechanical stimuli on monocytic cells remains unclear. To assess whether mechanical stress affects monocytic cell function, we examined the expression of inflammatory molecules and surface proteins, whose levels changed following shear stress in human THP-1 cells. Shear stress increased the inflammatory chemokine CCL2, which enhanced the migration of monocytic cells and tumor necrosis factor (TNF)-α and interleukin (IL)- 1ß at transcriptional and protein levels. We identified that the surface levels of heat shock protein 70 (HSP70), HSP90, and HSP105 increased using mass spectrometry-based proteomics, which was confirmed by western blot analysis, flow cytometry, and immunofluorescence. Treatment with HSP70/HSP105 and HSP90 inhibitors suppressed the expression and secretion of CCL2 and monocytic cell migration, suggesting an association between HSPs and inflammatory responses. We also demonstrated the coexistence and colocalization of increased HSP90 immunoreactivity and CD68 positive cells in atherosclerotic plaques of ApoE deficient mice fed a high-fat diet and human femoral artery endarterectomy specimens. These results suggest that monocytes/macrophages affected by shear stress polarize to a pro-inflammatory phenotype and increase surface protein levels involved in inflammatory responses. The regulation of the abovementioned HSPs upregulated on the monocytes/macrophages surface may serve as a novel therapeutic target for inflammation due to shear stress.

Investigation of Phospholipid Differences in Valproic Acid-Induced Autistic Mouse Model Brain Using Mass Spectrometry Imaging.

Autism is a neurodevelopmental disorder for which the cause and treatment have yet not been determined. The polyunsaturated fatty acid (PUFA) levels change rapidly in the blood or cerebrospinal fluid of autistic children and PUFAs are closely related to autism spectrum disorder (ASD). This finding suggests that changes in lipid metabolism are associated with ASD and result in an altered distribution of phospholipids in cell membranes. To further understand ASD, it is necessary to analyze phospholipids in organs consisting of nerve cells, such as the brain. In this study, we investigated the phospholipid distribution in the brain tissue of valproic acid-induced autistic mice using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Phospholipids including phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were identified in each brain region and exhibited differences between the ASD and control groups. These phospholipids contain docosahexaenoic acid and arachidonic acid, which are important PUFAs for cell signaling and brain growth. We expect that the differences in phospholipids identified in the brain tissue of the ASD model with MALDI-MSI, in conjunction with conventional biological fluid analysis, will help to better understand changes in lipid metabolism in ASD.

Heating-mediated purification of active FGF21 and structure-based design of its variant with enhanced potency.

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 °C returned to that at 20 °C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between ß10 and ß12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to surface plasmon resonance (SPR) experiments, in vitro cell-based assays, and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.

The developmental competence of human metaphase I oocytes with delayed maturation in vitro.

OBJECTIVE: To evaluate whether metaphase I (MI) oocytes completing maturation in vitro to metaphase II ("MI-MII oocytes") have similar developmental competence as the sibling metaphase II (MII) oocytes that reached maturity in vivo. DESIGN: Retrospective cohort study. SETTING: Academic medical center. PATIENT(S): A total of 1,124 intracytoplasmic sperm injection (ICSI) cycles from 800 patients at a single academic center between April 2016 and December 2020 with at least 1 MII oocyte immediately after retrieval and at least 1 sibling "MI-MII oocyte" that was retrieved as MI and matured to MII in culture before ICSI were included in the study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): A total of 7,865 MII and 2,369 sibling MI-MII oocytes retrieved from the same individuals were compared for the fertilization and blastocyst formation rates. For patients who underwent single euploid blastocyst transfers (n = 406), the clinical pregnancy, spontaneous pregnancy loss, and live birth rates were compared between the 2 groups. RESULT(S): The fertilization rate was significantly higher in MII oocytes than in delayed matured MI-MII oocytes (75.9% vs. 56.1%). Similarly, the blastocyst formation rate was higher in embryos derived from MII oocytes than in those from MI-MII oocytes (53.8% vs. 23.9%). The percentage of euploid embryos derived from MII oocytes was significantly higher than that of those from MI-MII oocytes (49.2% vs. 34.7%). Paired comparison of sibling oocytes within the same cycle showed higher developmental competence of the MII oocytes than that of MI-MII oocytes. However, the pregnancy, spontaneous pregnancy loss, and live birth rates after a single euploid blastocyst transfer showed no statistically significant difference between the 2 groups (MII vs. MI-MII group, 65.7% vs. 74.1%, 6.4% vs. 5.0%, and 61.5% vs. 70.0%, respectively). CONCLUSION(S): Compared with oocytes that matured in vivo and were retrieved as MII, the oocytes that were retrieved as MI and matured to MII in vitro before ICSI showed lower developmental competence, including lower fertilization, blastocyst formation, and euploidy rates. However, euploid blastocysts from either cohort resulted in similar live birth rates, indicating that the MI oocytes with delayed maturation can still be useful even though the overall developmental competence was lower than that of their in vivo matured counterparts.

Endothelial PTP4A1 mitigates vascular inflammation via USF1/A20 axis-mediated NF-κB inactivation.

AIMS: The nuclear factor-κB (NF-κB) signalling pathway plays a critical role in the pathogenesis of multiple vascular diseases. However, in endothelial cells (ECs), the molecular mechanisms responsible for the negative regulation of the NF-κB pathway are poorly understood. In this study, we investigated a novel role for protein tyrosine phosphatase type IVA1 (PTP4A1) in NF-κB signalling in ECs. METHODS AND RESULTS: In human tissues, human umbilical artery ECs, and mouse models for loss of function and gain of function of PTP4A1, we conducted histological analysis, immunostaining, laser-captured microdissection assay, lentiviral infection, small interfering RNA transfection, quantitative real-time PCR and reverse transcription-PCR, as well as luciferase reporter gene and chromatin immunoprecipitation assays. Short hairpin RNA-mediated knockdown of PTP4A1 and overexpression of PTP4A1 in ECs indicated that PTP4A1 is critical for inhibiting the expression of cell adhesion molecules (CAMs). PTP4A1 increased the transcriptional activity of upstream stimulatory factor 1 (USF1) by dephosphorylating its S309 residue and subsequently inducing the transcription of tumour necrosis factor-alpha-induced protein 3 (TNFAIP3/A20) and the inhibition of NF-κB activity. Studies on Ptp4a1 knockout or transgenic mice demonstrated that PTP4A1 potently regulates the interleukin 1ß-induced expression of CAMs in vivo. In addition, we verified that PTP4A1 deficiency in apolipoprotein E knockout mice exacerbated high-fat high-cholesterol diet-induced atherogenesis with upregulated expression of CAMs. CONCLUSION: Our data indicate that PTP4A1 is a novel negative regulator of vascular inflammation by inducing USF1/A20 axis-mediated NF-κB inactivation. Therefore, the expression and/or activation of PTP4A1 in ECs might be useful for the treatment of vascular inflammatory diseases.

Discovery of pan-IAP degraders via a CRBN recruiting mechanism.

Inhibitors of apoptosis proteins (IAPs), defined by the presence of baculovirus IAP repeat (BIR) protein domain, are critical regulators of cell survival and cell death processes. Cellular IAP 1/2 (cIAP1/2) and X-linked IAPs (XIAPs) regulate the innate immune signaling pathway through their E3 ubiquitin ligase activity. Peptidomimetics or small-molecule IAP antagonists have been developed to treat various diseases, such as cancer, infection, and inflammation. In this study, we synthesized and characterized IAP-cereblon (CRBN) heterodimerizing proteolysis-targeting chimera (PROTAC), which induces the degradation of cIAP1/2 and XIAP but not CRBN. We demonstrated that this PROTAC inhibits tumor necrosis factor alpha (TNFα)-induced innate immune response and cancer cell migration and invasion, leading to apoptotic cell death. Our study is the first to demonstrate that both cIAPs and XIAP are degradable when applied to the PROTAC strategy.

Glutamyl-prolyl-tRNA synthetase 1 coordinates early endosomal anti-inflammatory AKT signaling.

The AKT signaling pathway plays critical roles in the resolution of inflammation. However, the underlying mechanisms of anti-inflammatory regulation and signal coordination remain unclear. Here, we report that anti-inflammatory AKT signaling is coordinated by glutamyl-prolyl-tRNA synthetase 1 (EPRS1). Upon inflammatory activation, AKT specifically phosphorylates Ser999 of EPRS1 in the cytoplasmic multi-tRNA synthetase complex, inducing release of EPRS1. EPRS1 compartmentalizes AKT to early endosomes via selective binding to the endosomal membrane lipid phosphatidylinositol 3-phosphate and assembles an AKT signaling complex specific for anti-inflammatory activity. These events promote AKT activation-mediated GSK3ß phosphorylation, which increase anti-inflammatory cytokine production. EPRS1-deficient macrophages do not assemble the early endosomal complex and consequently exacerbate inflammation, decreasing the survival of EPRS1-deficient mice undergoing septic shock and ulcerative colitis. Collectively, our findings show that the housekeeping protein EPRS1 acts as a mediator of inflammatory homeostasis by coordinating compartment-specific AKT signaling.

Policosanol suppresses tumor progression in a gastric cancer xenograft model.

Gastric cancer (GC) is the most common cancer worldwide and the third leading cause of cancer death, with the fifth highest incidence. The development of effective chemotherapeutic agents is needed to decrease GC mortality. Policosanol (PC) extracted from Cuban sugar cane wax is a healthy functional food ingredient that helps improve blood cholesterol levels and blood pressure. Its various physiological activities, such as antioxidant, anti-inflammatory, and anticancer activities, have been reported recently. Nevertheless, the therapeutic efficacy of PC in gastric xenograft models is unclear. We aimed to investigate the anticancer effect of PC on human GC SNU-16 cells and a xenograft mouse model. PC significantly inhibited GC cell viability and delayed tumor growth without toxicity in the SNU-16-derived xenograft model. Therefore, we investigated protein expression levels in tumor tissues; the expression levels of Ki-67, a proliferation marker, and cdc2 were decreased. In addition, we performed proteomic analysis and found thirteen differentially expressed proteins. Our results suggested that PC inhibited GC progression via cdc2 suppression and extracellular matrix protein regulation. Notably, our findings might contribute to the development of novel and effective therapeutic strategies for GC.

Chrysin-Induced G Protein-Coupled Estrogen Receptor Activation Suppresses Pancreatic Cancer.

Pancreatic cancer (PC) has a high mortality rate due to its poor prognosis and the possibility of surgical resection in patients with the disease. Importantly, adjuvant chemotherapy is necessary to improve PC prognosis. Chrysin, a natural product with anti-inflammatory, antioxidant, and anticancer properties, has been studied for several years. Our previous study demonstrated that chrysin induced G protein-coupled estrogen receptor (GPER) expression and regulated its activity in breast cancer. Herein, we investigated whether chrysin-induced GPER activation suppresses PC progression in MIA PaCa-2 cells and a xenograft model. To determine its mechanism of action, cytotoxicity and clonogenic assays, a FACS analysis, and Western blotting were performed. Furthermore, the delay in tumor growth was evaluated in the MIA PaCa-2-derived xenograft model. Tumor tissues were investigated by Western blotting, immunohistochemistry, and a proteomic analysis. Chrysin caused cell cycle arrest and significantly decreased cell viability. Following co-treatment with chrysin and 17ß-estradiol, the inhibitory effect of chrysin on cell proliferation was enhanced. In the xenograft model, chrysin and G1 (a GPER agonist) significantly delayed tumor growth and reduced both Ki-67 (a proliferation marker) and c-Myc expressions in tumor tissues. The proteomic analysis of tumor tissues identified that rho-associated coiled-coil containing protein kinase 1 (ROCK1), transgelin 2 (TAGLN2), and FCH and Mu domain containing endocytic adaptor 2 (FCHO2) levels were significantly reduced in chrysin-treated tumor tissues. High ROCK1, TAGLN2, and FCHO2 expressions were indicative of low overall PC survival as found using the Kaplan-Meier plotter. In conclusion, our results suggest that chrysin suppresses PC progression through the activation of GPER and reductions in ROCK1, TAGLN2, and FCHO2 expressions.

Structure-activity relationship analysis of novel GSPT1 degraders based on benzotriazinone scaffold and its antitumor effect on xenograft mouse model.

Molecular glue degraders, such as lenalidomide and pomalidomide, bind to cereblon (CRBN) E3 ligase and subsequently recruit neosubstrate proteins, Ikaros (IKZF1) and Aiolos (IKZF3), for the ubiquitination-proteasomal degradation process. In this study, we explored structure-activity relationship analysis for novel GSPT1 degraders utilizing a benzotriazinone scaffold previously discovered as a novel CRBN binder. In particular, we focused on the position of the ureido group on the benzotriazinone scaffold, substituent effect on the phenylureido group, and methyl substitution on the benzylic position of benzotriazinone. As a result, we identified 34f (TD-522), which exhibits strong anti-proliferative effects in both KG-1 (EC50 = 0.5 nM) and TMD-8 (EC50 = 5.2 nM) cell lines. Compound 34f effectively induced GSPT1 degradation with a DC50 of 0.269 nM and Dmax of >95 % at 10 nM concentration in KG-1 cells. An in vivo xenograft study showed that compound 34f effectively suppressed TMD8-driven tumor growth, suggesting a potential role in the development of novel GSPT1 degraders.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging of Phospholipid Changes in a Drosophila Model of Early Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a degenerative disease caused by motor neuron damage in the central nervous system, and it is difficult to diagnose early. Drosophila melanogaster is widely used to investigate disease mechanisms and discover biomarkers because it is easy to induce disease in Drosophila through genetic engineering. We performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to investigate changes in phospholipid distribution in the brain tissue of an ALS-induced Drosophila model. Fly brain tissues of several hundred micrometers or less were sampled using a fly collar to obtain reproducible tissue sections of similar sizes. MSI of brain tissues of Drosophila cultured for 1 or 10 days showed that the distribution of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylinositol (PI), was significantly different between the control group and the ALS group. In addition, the lipid profile according to phospholipids differed as the culture time increased from 1 to 10 days. These results suggest that disease indicators based on lipid metabolites can be discovered by performing MALDI-MSI on very small brain tissue samples from the Drosophila disease model to ultimately assess the phospholipid changes that occur in early-stage ALS.

Global Proteomics to Study Silica Nanoparticle-Induced Cytotoxicity and Its Mechanisms in HepG2 Cells.

Silica nanoparticles (SiO2 NPs) are commonly used in medical and pharmaceutical fields. Research into the cytotoxicity and overall proteomic changes occurring during initial exposure to SiO2 NPs is limited. We investigated the mechanism of toxicity in human liver cells according to exposure time [0, 4, 10, and 16 h (h)] to SiO2 NPs through proteomic analysis using mass spectrometry. SiO2 NP-induced cytotoxicity through various pathways in HepG2 cells. Interestingly, when cells were exposed to SiO2 NPs for 4 h, the morphology of the cells remained intact, while the expression of proteins involved in mRNA splicing, cell cycle, and mitochondrial function was significantly downregulated. These results show that the toxicity of the nanoparticles affects protein expression even if there is no change in cell morphology at the beginning of exposure to SiO2 NPs. The levels of reactive oxygen species changed significantly after 10 h of exposure to SiO2 NPs, and the expression of proteins associated with oxidative phosphorylation, as well as the immune system, was upregulated. Eventually, these changes in protein expression induced HepG2 cell death. This study provides insights into cytotoxicity evaluation at early stages of exposure to SiO2 NPs through in vitro experiments.

Comparative study on formation of protein coronas under three different serum origins.

Nanomaterials form a complex called "protein corona" by contacting with protein-containing biological fluids such as serum when they are exposed to physiological environments. The characteristics of these proteins, which are one of the substantial factors in cellular response, are affected by the interactions between the nanomaterials and the biological systems. Many studies have investigated the biological behaviors of nanomaterials by conducting experiments in vitro and in vivo; however, the origin of the biological materials used is rather inconsistent. This is due to the fact that the composition of the protein coronas may differ depending on the animal origin, not on the composition or size of the nanoparticles. The resulting differences in the composition of the protein coronas can lead to different conclusions. To identify the differences in protein corona formation among sera of different species, we investigated protein coronas of gold and silica nanoparticles in serum obtained from various species. Using comparative proteomic analysis, common proteins adsorbed onto each nanoparticle among the three different sera were identified as highly abundant proteins in the serum. These findings indicate that protein corona formation is dependent on the serum population rather than the size or type of the nanoparticles. Additionally, in the physiological classification of protein coronas, human serum (HS) was found to be rich in apolipoproteins. In conclusion, our data indicate that HS components are different from those of bovine or mouse, indicating that the serum species origin should be carefully considered when selecting a biological fluid.