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Cerium dioxide nanoparticles (CeONPs) have been extensively applied in research for future energy development due to two common oxidation states on their surface. Considering that shape (aspect ratio) is a key determinant of NPs-induced toxicity, we compared the toxicity of hexagonal (H)- and rod-shaped (R)-CeONPs in mice. At 24h after pharyngeal aspiration, both types of CeONPs recruited surrounding immune cells (monocytes and neutrophils) into the lung, and R-CeONPs induced a more severe pulmonary inflammatory response compared with H-CeONPs. To identify an indicator to predict pulmonary inflammatory responses at the cellular level, we also investigated their responses in alveolar macrophage cells. At 24h after treatment, both types of CeONPs were mainly located within the vacuoles (partially, in the lysosome) in the cytoplasm. Mitochondrial damage, intracellular calcium accumulation, and increased NO production were observed in cells exposed to both types of CeONPs, ultimately resulting in a decrease in cell viability. More interestingly, both types of CeONPs formed multinucleated giant cells. Meanwhile, contrary to when suspended in deionized water, R-CeONPs were strongly aggregated with a negative charge in cell culture media, whereas H-CeONPs were relatively well-dispersed with a positive charge. R-CeONPs-induced lysosomal extension was also recovered by premix with negatively charged DNA, and even NPs suspended in cell culture media without cells were detected under the FACS system, suggesting interference by protein corona. Therefore, we suggest that shape (aspect ratio) is an important factor determining inhaled NPs-induced pathology and that the effect of the surface charge and protein corona should be carefully considered in interpreting results derived from in vitro tests. Furthermore, we propose that the relationship between the formation of multinucleated giant cells and the inflammatory response of inhaled CeONPs should be further studied.

MicroRNAs (miRNAs) are short non-coding RNAs that play an important role in regulating gene expression. Since miRNAs are abnormally expressed in various cancers, they are considered to be promising biomarkers for early cancer diagnosis. However, the short length and strong sequence similarity among miRNAs make their reliable quantification very challenging. We developed a highly selective amplification-free miRNA detection method based on Förster resonance energy transfer (FRET)-aided single-molecule counting. miRNAs were selectively labeled with FRET probes using splinted ligation. When imaged with a single-molecule FRET setup, the miRNA molecules were accurately identified by the probe's FRET. miRNA concentrations were estimated from the count of molecules. The high sensitivity of the method in finding sparse molecules enabled us to achieve a limit of detection of 31-56 amol for miR-125b, miR-100, and miR-99a. Single nucleotide mismatch could be discriminated with a very high target-to-mismatch ratio. The method accurately measured the high expression of miR-125b in gastric cancer cells, which agreed well with previous reports. The high sensitivity and accuracy of this technique demonstrated its clinical potential as a robust miRNA detection method.

To prevent the ongoing spread of the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), accurate and early detection based on a rapid, ultrasensitive, and highly reliable sensing method is crucially important. Here, we present a bumpy core-shell surface-enhanced Raman spectroscopy (SERS) nanoprobe-based sensing platform with single-nanoparticle (SNP)-based digital SERS analysis. The tailorable bumpy core-shell SERS nanoprobe with an internal self-assembled monolayer of 4-nitrobenzenethiol Raman reporters, synthesized using HEPES biological buffer, generates a strong, uniform, and reproducible SERS signal with an SNP-level sensitive and narrowly distributed enhancement factor (2.1 × 10

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.

The importance of multi-omic-based approaches to better understand diverse pathological mechanisms including neurodegenerative diseases has emerged. Spatial information can be of great help in understanding how biomolecules interact pathologically and in elucidating target biomarkers for developing therapeutics. While various analytical methods have been attempted for imaging-based biomolecule analysis, a multi-omic approach to imaging remains challenging due to the different characteristics of biomolecules. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a powerful tool due to its sensitivity, chemical specificity, and high spatial resolution in visualizing chemical information in cells and tissues. In this paper, we suggest a new strategy to simultaneously obtain the spatial information of various kinds of biomolecules that includes both labeled and label-free approaches using ToF-SIMS. The enzyme-assisted labeling strategy for the targets of interest enables the sensitive and specific imaging of large molecules such as peptides, proteins, and mRNA, a task that has been, to date, difficult for any MS analysis. Together with the strength of the analytical performance of ToF-SIMS in the label-free tissue imaging of small biomolecules, the proposed strategy allows one to simultaneously obtain integrated information of spatial distribution of metabolites, lipids, peptides, proteins, and mRNA at a high resolution in a single measurement. As part of the suggested strategy, we present a sample preparation method suitable for MS imaging. Because a comprehensive method to examine the spatial distribution of multiple biomolecules in tissues has remained elusive, our strategy can be a useful tool to support the understanding of the interactions of biomolecules in tissues as well as pathological mechanisms.

Biomolecule detection based on surface-enhanced Raman scattering (SERS) for application to biosensors and bio-imaging requires the fabrication of SERS nanoprobes that can generate strong Raman signals as well as surface modifications for analyte-specific recognition and binding. Such requirements lead to disadvantages in terms of reproducibility and practicality, and thus, it has been difficult to apply biomolecule detection utilizing the advantages of the SERS phenomenon to actual clinically relevant analysis. To achieve reproducible and practical SERS signal generation in a biomolecule-specific manner without requiring the synthesis of nanostructures and their related surface modification to introduce molecules for specific recognition, we developed a new type of SERS probe formed by enzyme reactions in the presence of Raman reporters. By forming unique plasmonic structures, our method achieves the detection of biomolecules on chips with uniform and stable signals over long periods. To test the proposed approach, we applied it to a SERS-based immunohistochemistry assay and found successful multiplexed protein detection in brain tissue from transgenic mice.

African swine fever virus (ASFV) causes a highly contagious and fatal disease affecting both domesticated and wild pigs. Substandard therapies and inadequate vaccinations cause severe economic damages from pig culling and removal of infected carcasses. Therefore, there is an urgent need to develop a rapid point-of-use approach that assists in avoiding the spread of ASFV and reducing economic loss. In this study, we developed a colorimetric sensing platform based on dual enzymatic amplification that combined the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system and the enzyme urease for accurate and sensitive detection of ASFV. The mechanism of the sensing platform involves a magnetic bead-anchored urease-conjugated single-stranded oligodeoxynucleotide (MB@urODN), which in the presence of ASFV dsDNA is cleaved by activated CRISPR/Cas12a. After magnetically separating the free urease, the presence of virus can be confirmed by measuring the colorimetric change in the solution. The advantage of this method is that it can detect the presence of virus without undergoing a complex target gene duplication process. The established method detected ASFV from three clinical specimens collected from porcine clinical tissue samples. The proposed platform is designed to provide an adequate, simple, robust, highly sensitive and selective analytical technique for rapid zoonotic disease diagnosis while eliminating the need for vast or specialized tools.

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.

For organ transplantation patients, the therapeutic drug monitoring (TDM) of immunosuppressive drugs is essential to prevent the toxicity or rejection of the organ. Currently, TDM is done by immunoassays or liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods; however, these methods lack specificity or are expensive, require high levels of skill, and offer limited sample throughput. Although matrix-assisted (MA) laser desorption ionization (LDI) mass spectrometry (MS) can provide enhanced throughput and cost-effectiveness, its application in TDM is limited due to the limitations of the matrixes such as a lack of sensitivity and reproducibility. Here, we present an alternative quantification method for the TDM of the immunosuppressive drugs in the blood of organ transplant patients by utilizing laser desorption ionization mass spectrometry (LDI-MS) based on a tungsten disulfide nanosheet, which is well-known for its excellent physicochemical properties such as a strong UV absorbance and high electron mobility. By adopting a microliquid inkjet printing system, a high-throughput analysis of the blood samples with enhanced sensitivity and reproducibility was achieved. Furthermore, up to 80 cases of patient samples were analyzed and the results were compared with those of LC-MS/MS by using Passing-Bablok regression and Bland-Altman analysis to demonstrate that our LDI-MS platform is suitable to replace current TDM techniques. Our approach will facilitate the rapid and accurate analysis of blood samples from a large number of patients for immunosuppressive drug prescriptions.

To make surface enhanced Raman scattering (SERS) sensors more practical, we propose nanoporous Ag disks as SERS-active plasmonic structures that can be readily inkjet-printed just before use to avoid degradation of SERS enhancement. Together with the aid of the enhanced plasmonic fields from the nanoporous Ag (confirmed by electromagnetic simulation), we utilize a coffee-ring effect to concentrate target analytes, which is demonstrated by confocal Raman measurements. By using the proposed SERS sensor, Raman signals of TiO2 nanoparticles with a concentration of ppm to sub-ppb have been successfully measured. TiO2 in commercial consumables has been also detected by distinguishing its crystalline phase.

Retinal pigment epithelium (RPE) damage is a major factor in age-related macular degeneration (AMD). The RPE in AMD shows mitochondrial dysfunction suggesting an association of AMD with mitochondrial function. Therefore, exogenous mitochondrial transplantation for restoring and replacing dysfunctional mitochondria may be an effective therapeutic strategy for AMD. Here, we investigated the effects of extrinsic mitochondrial transplantation on senescence-induced ARPE-19 cells. We demonstrated mitochondrial dysfunction in replicative senescence-induced ARPE-19 cells after repeated passage. Imbalanced mitophagy and mitochondrial dynamics resulted in increased mitochondrial numbers and elevated levels of mitochondrial and intracellular reactive oxygen species. Exogenous mitochondrial transplantation improved mitochondrial dysfunction and alleviated cellular senescence hallmarks, such as increased cell size, increased senescence-associated β-galactosidase activity, augmented NF-κB activity, increased inflammatory cytokines, and upregulated the cyclin-dependent kinase inhibitors p21 and p16. Further, cellular senescence properties were improved by exogenous mitochondrial transplantation in oxidative stress-induced senescent ARPE-19 cells. These results indicate that exogenous mitochondrial transplantation modulates cellular senescence and may be considered a novel therapeutic strategy for AMD.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the identification of peptide sample TOF-SIMS spectra by machine learning. More than 1000 time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra of six peptide model samples (one of them was a test sample) were collected using 27 TOF-SIMS instruments from 25 institutes of six countries, the U. S., the U. K., Germany, China, South Korea, and Japan. Because peptides have systematic and simple chemical structures, they were selected as model samples. The intensity of peaks in every TOF-SIMS spectrum was extracted using the same peak list and normalized to the total ion count. The spectra of the test peptide sample were predicted by Random Forest with 20 amino acid labels. The accuracy of the prediction for the test spectra was 0.88. Although the prediction of an unknown peptide was not perfect, it was shown that all of the amino acids in an unknown peptide can be determined by Random Forest prediction and the TOF-SIMS spectra. Moreover, the prediction of peptides, which are included in the training spectra, was almost perfect. Random Forest also suggests specific fragment ions from an amino acid residue Q, whose fragment ions detected by TOF-SIMS have not been reported, in the important features. This study indicated that the analysis using Random Forest, which enables translation of the mathematical relationships to chemical relationships, and the multi labels representing monomer chemical structures, is useful to predict the TOF-SIMS spectra of an unknown peptide.

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.

TiO2 is the most commonly used photocatalyst in water treatment. The particle size of TiO2 is an important factor that significantly influences its activity during photocatalytic degradation. In the presence of liquid, the properties of nanopowders composed of exactly the same product clearly differ according to their aggregation size. In this study, TiO2 nanoparticles with a controlled size were fabricated by focused ultrasound dispersion. The high energy generated by this system was used to control the size of TiO2 particles in the suspension. The constant high energy released by cavitation enabled the dispersion of the particles without a surfactant. The activities of the prepared TiO2 photocatalysts for methylene blue (MB) degradation were then compared. The dye degradation effect of the photocatalyst was as high as 61.7% after 10 min when the size of the powder was controlled in the solution, but it was only as high as 41.0% when the aggregation size was not controlled. Furthermore, when the TiO2 concentration exceeded a certain level, the photocatalytic activity of TiO2 decreased. Controlling the size of the aggregated photocatalyst particles is, therefore, essential in water-treatment technologies utilizing TiO2 photocatalytic properties, and adjusting the TiO2 concentration is an important economic factor in this photocatalytic technology. This study contributes to the development of processes for degrading dyes, such as MB, released from wastewater into aquatic environments.

This year, France banned the application of titanium dioxide nanoparticles as a food additive (hereafter, E171) based on the insufficient oral toxicity data. Here, we investigated the subchronic toxic responses of E171 (0, 10, 100, and 1,000 mg/kg) and tried to elucidate the possible toxic mechanism using AGS cells, a human stomach epithelial cell line. There were no dose-related changes in the Organisation for Economic Cooperation and Development test guideline-related endpoints. Meanwhile, E171 deeply penetrated cells lining the stomach tissues of rats, and the IgM and granulocyte-macrophage colony-stimulating factor levels were significantly lower in the blood from rats exposed to E171 compared with the control. The colonic antioxidant protein level decreased with increasing Ti accumulation. Additionally, after 24-h exposure, E171 located in the perinuclear region of AGS cells and affected expression of endoplasmic reticulum stress-related proteins. However, cell death was not observed up to the used maximum concentration. A gene profile analysis also showed that immune response-related microRNAs were most strongly affected by E171 exposure. Collectively, we concluded that the NOAEL of E171 for 90 days repeated oral administration is between 100 and 1,000 mg/kg for both male and female rats. Additionally, further study is needed to clarify the possible carcinogenesis following the chronic accumulation in the colon.

A potential method to tune reactivity In organic chemistry, reaction rates are often sensitive to substituents that either donate or withdraw electron density from a reactive carbon center. Heo et al. now report that when reactants are tethered to an electrode, the polarization effect of an applied potential can exert an analogous influence, essentially acting as a tunable functional group. The authors demonstrate rate modulations in ester hydrolysis and arene cross-coupling, as well as a two-step acid-to-amide conversion that benefits from inversion of the polarization midway through the reaction. Science , this issue p. 214

Nanomaterial toxicity tests using normal and cancer cells may yield markedly different results. Here, nanomaterial toxicity between cancer and primary human cells was compared to determine the basic cell line selection criteria for nanomaterial toxicity analyses. Specifically, we exposed two cancer (A549 and HepG2) and two normal cell lines (NHBE and HH) cell lines to SiO2 nanoparticles (NPs) and evaluated the cytotoxicity (MTS assay), cell death mode, and intracellular NP retention. MTS assay results revealed higher sensitivity of HH cells to SiO2 NPs than HepG2 cells, while no difference was observed between NHBE and A549 cells. In addition, SiO2 NPs primarily induced necrosis in all the cell lines. Moreover, we evaluated NP accumulation by treating the cell lines with fluorescein-isothiocyanate-labeled SiO2 NPs. After 48 h of treatment, less than 10% of A549 and HepG2 cells and more than 30% of NHBE and HH cells contained the labeled NPs. Collectively, our results suggest that cell viability, death mode, and intracellular compound accumulation could be assessed using cancer cells. However, the outcomes of certain investigations, such as intracellular NP retention, may differ between cancer and normal cells.

Chemical characterizations of biochemically functionalized single nanoparticles are necessary to optimize the nanoparticle surface functionality in recently advanced nanobiological applications but have not yet been fully explored because of technical difficulties. Exploiting the photoinduced force exerted on a light-illuminated nanoscale tip, nanoscale mid-infrared hyperspectral images with a 10 nm spatial resolution of a monolayer ligand-functionalized single gold nanoparticle under ambient and environmental conditions are presented. We extend our study to the diagnosis of nanoscale heterogeneous chemical contaminants which come from a particle functionalization process but are undetectable in conventional ensemble-averaged imaging technique. High sensitivity and high spatial resolution are achieved via the strongly localized tip-enhanced force at the junction between the gold-coated tip and the functionalized nanoparticle in photoinduced force microscopy, which far exceeds the capability of the conventional methods. The present study paves a new way to directly detect heterogeneous nanochemicals at the single-component level, which is necessary to evaluate nanomaterial safety in biomedical applications.

Reliable quantification of nanoparticle uptake in mammalian cells is essential to study the effects of nanoparticles in the fields of medicine and environmental science. Most conventional quantification methods, such as electron microscopy or confocal imaging, are laborious and semi-quantitative and therefore not readily applicable to routine analyses. Here, we developed assays to quantify fluorescently labelled nanoparticle uptake in mammalian cells using a flow cytometer. The first approach was to measure the percentage of nanoparticle-containing cells based on a cutoff fluorescence intensity as set from a histogram of control cells, which is a quick and easy way to relatively compare nanoparticle uptake in the same set of experiments. The second approach was to measure the calibrated fluorescence intensity of the nanoparticle-treated cells in molecules of equivalent soluble fluorophore (MESF) values using calibration beads, which allows for comparisons between different sets of experiments. We successfully applied the developed assays to more readily measure fluorescence-labelled silica nanoparticle uptake in A549 lung carcinoma cells in a quantitative rather than semi-quantitative manner. We further tested the assays with nine different types of mammalian cells and investigated the correlation between cell type/size and nanoparticle uptake.

Fulminant hepatitis is a serious inflammatory condition of the liver characterized by massive necrosis of liver parenchyma following excessive immune cell infiltration into the liver, and possibly causing sudden hepatic failure and medical emergency. However, the underlying mechanisms are not fully understood. Here, we investigated the role of cyclic AMP-responsive element-binding protein, hepatocyte specific (CREBH) in concanavalin A (ConA)-driven hepatitis-evoked liver injury. C57BL/6J (WT) and Crebh knockout (KO) mice injected with ConA (7.5 or 25 mg/kg) and bone marrow (BM) chimeric mice, generated by injection of BM cells into sub-lethally irradiated recipients followed by ConA injection (22.5 or 27.5 mg/kg) 8 weeks later, were used for in vivo study. Primary mouse hepatocytes and HEK293T cells were used for a comparative in vitro study. Crebh KO mice are highly susceptible to ConA-induced liver injury and prone to death due to increased neutrophil infiltration driven by enhanced hepatic expression of neutrophil-attracting chemokines. Notably, BM chimera experiment demonstrated that Crebh-deficient hepatocytes have an enhanced ability of recruiting neutrophils to the liver, thereby promoting hepatotoxicity by ConA. Intriguingly, in vitro assays showed that p65, a subunit of NF-κB and common transcription factor for various chemokines, dependent transactivation was inhibited by CREBH. Furthermore, p65 expression was inversely correlated with CREBH level in ConA-treated mice liver and TNFα-stimulated primary mouse hepatocytes. This is the first demonstration that CREBH deficiency aggravates inflammatory liver injury following chemokine-dependent neutrophil infiltration via NF-κB p65 upregulation. CREBH is suggested to be a novel therapeutic target for treatment of fulminant hepatitis.

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1–100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NMbearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is UltravioletVisible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5–100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization., peer-reviewed

This study aimed to investigate the long-term clinical efficacy of and satisfaction with integrative Korean medicine (KM) treatment in patients with shoulder osteoarthritis (SOA). We conducted a prospective observational study of patients with SOA. Patients aged 19 years and older who underwent inpatient treatment for more than 1 week were eligible for enrollment in the study. The primary evaluation index was the numeric rating scale for shoulder pain. Sub-evaluation indices included the Shoulder Pain and Disability Index for shoulder function, EuroQol-5-dimension score for overall quality of life, and Patient Global Impression of Change. Outcome measures were assessed at admission, discharge, and follow-up. For the follow-up questionnaire survey, the following information was collected: current status, surgery after discharge, reasons for finding integrative KM treatment satisfactory/unsatisfactory, and quality of life after discharge. In total, 186 patients were enrolled in the primary analysis, and 103 patients completed the follow-up survey. The mean number of days of follow-up was 1019 ± 439. Compared with the baseline, the mean differences in the numeric rating scale and Shoulder Pain and Disability Index were 3.05 ± 0.34 and 36.06 ± 5.53, respectively. Regarding the Patient Global Impression of Change, 89 out of 103 (86.4%) patients chose "minimally improved" or better. Furthermore, the EuroQol-5-dimension score also increased, showing an improvement of health-related quality of life after treatment. Integrative KM treatment is a potential option for reducing pain severity and improving function and health-related quality of life in patients with SOA. Prospective randomized studies would support this finding for the next step.

In our previous study, 20 nm-sized amorphous silica nanoparticles (20-SiNPs), but not 50 nm-sized amorphous silica nanoparticles (50-SiNPs), induced pulmonary inflammatory response in rats exposed repeatedly for 14 days (12.5, 25, and 50 μg/time, total six times). In this study, we tried to clarify the causes of different responses induced by both SiNPs using mice (12.5, 25, and 50 μg/lung) and mouse alveolar macrophage cells. When exposed to alveolar macrophage cells for 24 h, both SiNPs decreased cell viability and enhanced ROS generation compared to controls. The 20- and 50-SiNPs also formed giant and autophagosome-like vacuoles in the cytoplasm, respectively. Structural damage of organelles was more pronounced in 20-SiNPs-treated cells than in 50-SiNPs-treated cells, and an increased mitochondrial membrane potential and mitochondrial calcium accumulation were observed only in the 20-SiNPs-treated cells. Additionally, a single intratracheal instillation of both sizes of SiNPs to mice clearly elevated the relative proportion of neutrophils and inhibited differentiation of macrophages and expression of an adhesion molecule. Meanwhile, interestingly, the total number of pulmonary cells and the levels of pro-inflammatory mediators more notably increased in the lungs of mice exposed to 20-SiNPs compared to 50-SiNPs. Given that accumulation of giant vacuoles and dilation of the ER and mitochondria are key indicators of paraptosis, we suggest that 20-SiNPs-induced pulmonary inflammation may be associated with paraptosis of alveolar macrophages.

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.

The increasingly widespread use of engineered nanoparticles in medical, industrial, and food applications has raised concerns regarding their potential toxicity to humans and the environment. Silicon dioxide nanoparticles (SiO2 NPs), which have relatively low direct toxicity, have been increasingly applied in both consumer products and biomedical applications, leading to significantly higher exposure for humans and the environment. We carried out a toxicity assessment of SiO2 NPs using the common water flea D. magna by focusing on physiological and behavioral indicators such as heart rate, swimming performance, and growth. Exposure to SiO2 NPs did not produce acute or chronic toxicity at limited concentrations (

Appropriate surface designs and precise characterizations of nanoparticles (NPs) are important but as of yet unrealized in biomedical applications of nanomaterials. We report a facile synthetic approach to the preparation of iron oxide (Fe3O4) NPs with different surface charges through exchange with polymeric ligands. An accurate analysis method for characterization of the surface-modified Fe3O4 NPs using a combination of the time-of-flight secondary ion mass spectrometry (ToF-SIMS) and Fourier transform infrared spectroscopy (FT-IR) techniques is demonstrated. A comparison of the spatial distribution of the ligand and core particles with ToF-SIMS and FT-IR verified the successful modification and purification. In addition, the surface was shown to play an important role in interactions with live cells in terms of cytotoxicity, cellular uptake, and even gene transfection. Interestingly, precise control over the surface chemistry of NP enabled efficient transfection of siRNA into suspension type cell, the Jurkat human leukemic T cell line, achieving successful down regulation of NOTCH1 whose signaling pathway is known to be involved in acute leukemia. Our report presents an in-depth study of the interactions between the NPs and cells, as well as their synthesis, characterization, and surface-dependent performance properties.

We report polyethylene glycol (PEG)-grafting antifouling surfaces using a plasma copolymerized (PcP) technique to monitor protease activity in complex media. By varying the mixing ratio of the PEG and ethylenediamine (EDA) precursors, the PcP-PEG-EDA (PcP-PE) film was able to easily control surface amine density with good preservation of the internal PEG structure. We found that nonspecific protein adsorption was dramatically reduced in serum-containing media on the PcP-PE films, as opposed to that on plasma polymerized-EDA (PP-E) films without PEG. When SPR sensor chips coated with PcP-PE film were employed to detect protease activity, biotinylated luciferase probes (luciferase-peptide-biotin) on streptavidin-conjugated SPR chips enabled real-time and label-free measurement of matrix metalloproteinase activity in cell culture media. Owing to its excellent antifouling ability, this newly developed method boasts minimal nonspecific binding and can serve as a biochip platform to promote a wide range of applications in the biological field.

In this study, ten individual green-to-red stimulated Raman scattering (SRS) peaks are optimally generated for wavelength-dependent real-time optical-resolution photoacoustic microscopy (OR-PAM) with contrast agents. Exogenous contrast agents, such as dyes, semiconducting polymers, and metallic nanoparticles, are frequently used in OR-PAM to provide structural, functional, and molecular images of biological samples with a high spatial resolution. For an effective use of various exogenous contrast agents for functional or molecular OR-PAM, selectable visible and near-infrared wavelengths are needed to select the absorption wavelengths of the contrast agents. Therefore, we optimized an SRS source, which can selectively generate ten narrow-band SRS peaks with a bandwidth smaller than 11 nm and individually produce sufficient threshold pulse energies (116–756 nJ) at a high pulse repetition rate (PRR) of 300 kHz. The wavelength-dependent photoacoustic signal was easily obtained in a methylene blue phantom and in vivo mouse studies using the optimal generation conditions of the ten-wavelength green-to-red SRS source, from 545 to 695 nm. In particular, for the first time, wavelength-dependent real-time in vivo OR-PAM images at a PRR of hundreds of kilohertz were obtained to distinguish gold nanoparticles from distributed blood vessels of a mouse ear.

This study investigated the effects of the packing density and particle size distribution of TiO2 nanoparticles on the mechanical properties of TiO2–epoxy nanocomposites (NCs). The uniform dispersion and good interfacial bonding of TiO2 in the epoxy resin resulted in improved mechanical properties with the addition of nanoparticles. Reinforcement nano-TiO2 particles dispersed in deionized water produced by three different ultrasonic dispersion methods were used; the ultrasonication effects were then compared. The nano-TiO2 suspension was added at 0.5–5.0 wt.%, and the mechanical and thermal properties of TiO2–epoxy NCs were compared using a universal testing machine, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC). The tensile strength of the NCs was improved by the dispersion strengthening effect of the TiO2 nanoparticles, and focused sonication improved the tensile strength the most when nano-TiO2 suspensions with a particle size of 100 nm or smaller were used. Thus, the reinforcing effect of TiO2 nanoparticles on the epoxy resin was observed, and the nano-TiO2 suspension produced by focused sonication showed a more distinct reinforcing effect.

Nanomaterials are used in a variety of fields and toxicity assessment is paramount for their development and application. Although most toxicity assessments have been performed in 2D (2-Dimensional) cell culture, the inability to adequately replicate thein vivoenvironment and toxicity is a limitation. To overcome the limitation, a 3D (3-Dimensional) cell culture method has been developed to make an environment closer to anin vivosystem. In this study, 20 nm SiO2nanoparticles were dispersed in serum-containing (SC) and serum-free (SF) media to compare 2D cell culture and 3D cell culture toxicity. The cells were subjected to a 3D cell culture method in which HepG2, a human-derived liver cancer cell line, was mixed on a scaffold. We found that nanoparticles induced toxicity in 2D cell culture, but toxicity was not observed in 3D cell culture similar toin vivoenvironment. However, differences in toxicity were observed between the three types of scaffolds in the absence of serum as the number of cells decreased.

Carbon nanotubes have recently been rated as an effective biomaterial owing to their functionalization ability. However, the safety of multi-walled carbon nanotubes (MWCNTs) has yet to be clearly understood. To investigate how cells differentially react to minor geometric differences, we prepared well-dispersed and stable long and short MWCNTs showing an approximately 100-nm length difference in an in vitro system. Through an optimal combination of bovine serum albumin (BSA) and fetal bovine serum (FBS) biosurfactants and ultrasonication, we first confirmed that the MWCNTs were maintained without aggregation throughout the experiments. Internalized MWCNTs in human coronary artery smooth muscle cells were then quantified in a label-free manner using coherent anti-Stokes Raman scattering, followed by an analysis of their localization via two-photon excitation fluorescence. Intracellular MWCNTs were found to primarily localize in mitochondria with abnormal morphologies. Mitochondrial dysfunction, which was found to result from early stages of oxidative stress that consequently lead to cell death, was then proved via decreasing mitochondrial membrane potentials, with short MWCNTs showing significantly greater cytotoxicity than long MWCNTs. Our results suggest that even small length differences of MWCNTs may lead to differential responses in cells.

In this study, the soft-type and hard-type lead zirconate titanate (PZT) ceramics were compared in order to create an optimal system for ultrasonic dispersion of nanoparticles, and sound pressure energy for each PZT ceramic was analyzed and closely examined with ultrasonic energy. TiO2 was water-dispersed using the soft-type and hard-type PZT transducer, possessing different characteristics, and its suspension particle size and distribution, polydispersity index (PDI), zeta potential, and dispersion were evaluated for 180 days. Furthermore, it was confirmed that the particles dispersed using the hard-type PZT transducer were smaller than the particles dispersed using the soft-type PZT by 15 nm or more. Because the hard-type PZT transducer had a lower PDI, uniform particle size distribution was also confirmed. In addition, by measuring the zeta potential over time, it was found that the hard-type PZT transducer has higher dispersion safety. In addition, it was confirmed that the ultrasonically dispersed TiO2 suspension using a hard-type PZT transducer maintained constant particle size distribution for 180 days, whereas the suspension from the soft-type PZT aggregated 30 days later. Therefore, the hard-type PZT is more suitable for ultrasonic dispersion of nanoparticles.

Multispectral photoacoustic microscopy uses a wavelength-dependent absorption difference as a contrast mechanism to image the target molecule. In this paper, we present a novel multispectral pulsed fiber laser source, which selectively alternates the excitation wavelengths between green and red colors based on the stimulated Raman scattering (SRS) effect for imaging. This laser has a high pulse repetition rate (PRR) of 300 kHz and high pulse energy of more than 200 nJ meeting the real-time requirements of optical-resolution photoacoustic microscopy imaging. By switching the polarization state of the pump light and optical paths of the pump light, the operating wavelengths of the light source can be selectively alternated at the same fast PRR for any two SRS peak wavelengths between 545 and 655 nm. At 545 nm excitation wavelength, molecular photoacoustic signals from both blood vessels and gold nanorods were obtained simultaneously. However, at 655 nm, the photoacoustic signals of gold nanorods were dominant because the absorption of light by the blood vessels decreased drastically in the spectral region over 600 nm. Thus the multispectral photoacoustic system designed using the novel laser source implemented here could simultaneously monitor the time-dependent fast movement of two molecules independently, having different wavelength-dependent absorption properties at a high repetition rate of 0.49 frames per second (fps).

Background The International Agency for Research on Cancer classified as a Group B carcinogen inhaled titanium dioxide (TiO 2 ) nanoparticles, and France banned the application of TiO 2 nanoparticles as a food additive based on the insufficient oral toxicity data. However, there still remains controversial due to the insufficient evidence for its safety. Here, we explored the toxicity of food-grade TiO 2 nanoparticles (hereafter, E171) in vivo and in vitro . Methods We investigated the subchronic toxic responses of E171 (0, 10, 100, and 1,000 mg/kg) under the Good Laboratory Practice (GLP) system and tried to elucidate the possible toxic mechanism using AGS cells, a human stomach epithelial cell line. Results There were no dose-related changes in the Organization for Economic Co-operation and Development test guideline-related endpoints. Meanwhile, E171 deeply penetrated cells lining the stomach tissues of rats administered the maximum dose, and blood IgM (male and female) and GM-CSF (female) levels were significantly lower in the E171-treated rats than in the control rats. Colonic SOD-1 (male and female) and SOD-2 (female) protein levels decreased with increasing Ti accumulation. When exposed to AGS cells for 24 h, E171 (40 μg/mL) located in the perinuclear region. The E171 treatment affected expression of ER stress-related proteins but did not induce cell death up to the used maximum concentration (40 μg/mL). A gene profile analysis also showed that immune response-related microRNAs were most strongly affected by E171 exposure. Conclusion Considering the potential toxicity observed in vivo and in vitro test, we concluded that the NOAEL of E171 for 90-days repeated oral administrations is less than 1,000 mg/kg for both male and female rats. Additionally, E171 may attenuate host's defense function against foreign bodies by decreasing antioxidant capacity, thus we propose that chronic toxicity studies on E171 are required to warrant the safety for use in the food industry.

Background Nanotechnology is indispensable to many different applications. Although nanoparticles have been widely used in, for example, cosmetics, sunscreen, food packaging, and medications, they may pose human safety risks associated with nanotoxicity. Thus, toxicity testing of nanoparticles is essential to assess the relative health risks associated with consumer exposure. Methods In this study, we identified the NOAEL (no observed adverse effect level) of the agglomerated/aggregated TiO2 P25 (approximately 180 nm) administered at repeated doses to Sprague-Dawley (SD) rats for 28 and 90 days. Ten of the 15 animals were necropsied for toxicity evaluation after the repeated-dose 90-day study, and the remaining five animals were allowed to recover for 28 days. The agglomerated/aggregated TiO2 P25 dose levels used included 250 mg kg− 1 d− 1 (low), 500 mg kg− 1 d− 1 (medium), and 1000 mg kg− 1 d− 1 (high), and their effects were compared with those of the vehicle control. During the treatment period, the animals were observed for mortality, clinical signs (detailed daily and weekly clinical observations), functional observation battery, weekly body weight, and food and water consumption and were also subjected to ophthalmological examination and urinalysis. After termination of the repeated-dose 28-day, 90-day, and recovery studies, clinical pathology (hematology, blood coagulation time, and serum biochemistry), necropsy (organ weights and gross findings), and histopathological examinations were performed. Results No systemic toxicological effects were associated with the agglomerated/aggregated TiO2 P25 during the repeated-dose 28-day, 90-day, and recovery studies in SD rats. Therefore, the NOAEL of the agglomerated/aggregated TiO2 P25 was identified as 1000 mg kg− 1 d− 1, and the substance was not detected in the target organs. Conclusion Subacute and subchronic oral administration of the agglomerated/aggregated TiO2 P25 was unlikely to cause side effects or toxic reactions in rats.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra, leading to motor symptoms. Despite the remarkable improvements in the management of PD in recent decades, many patients remain significantly disabled. Metformin is a primary medication for the management of type 2 diabetes. We previously showed that co-treatment with metformin and 3,4-dihydroxyphenyl-l-alanine (l-DOPA) prevented the development of l-DOPA-induced dyskinesia in a 6-hydroxydopamine (6-OHDA)-lesioned animal model of PD. However, effects of metformin on PD- and aging-induced genes in reactive astrocytes remain unknown. In this study, we assessed the effect of metformin on motor function, neuroprotection, and reactive astrocytes in the 6-OHDA-induced PD animal model. In addition, the effects of metformin on the genes expressed by specific types of astrocytes were analyzed in PD model and aged mice. Here, we showed that metformin treatment effectively improves the motor symptoms in the 6-OHDA-induced PD mouse model, whereas metformin had no effect on tyrosine hydroxylase-positive neurons. The activation of AMPK and BDNF signaling pathways was induced by metformin treatment on the 6-OHDA-lesioned side of the striatum. Metformin treatment caused astrocytes to alter reactive genes in a PD animal model. Moreover, aging-induced genes in reactive astrocytes were effectively regulated or suppressed by metformin treatment. Taken together, these results suggest that metformin should be evaluated for the treatment of Parkinson's disease and related neurologic disorders characterized by astrocyte activation.

Although detailed pathophysiological mechanisms of fulminant hepatitis remain elusive, immune cell recruitment with excessive cytokine production is a well-recognized hallmark of the disease. We determined the function of orphan nuclear receptor small heterodimer partner (SHP) in concanavalin A (ConA)-induced hepatitis model. Male C57BL/6 J mice were injected intravenously with either a lethal dose (25 mg/kg) or a sub-lethal dose (15 mg/kg) of ConA. For the C-X-C motif chemokine ligand (CXCL) 2 neutralization study, mice were intravenously administered anti-mouse CXCL2 antibody (100 μg/mouse). Thirty-six hours following lethal dose of ConA administration, 47% wild type (WT) mice were alive, whereas >85% of Shp knockout (KO) were dead. Shp KO mice were highly susceptible to ConA-induced liver injury and exhibited increased liver necrosis upon sub-lethal dose of ConA administration. FACS analysis and immunohistochemical staining showed significantly higher neutrophil infiltration in Shp KO mice, as compared with WT mice. We found that also in the WT situation, Shp expression gradually decreased, while Cxcl2 expression increased until 6 h, and vice versa at 24 h upon ConA-treatment, indicating an inverse correlation between Shp and Cxcl2 expression during ConA-induced hepatitis. Furthermore, in vivo neutralization of CXCL2 with neutralizing antibody reduces ConA-induced plasma ALT and AST levels, hepatocyte death and neutrophil infiltration in Shp KO mice. Collectively, these results confirm that lacking of SHP results in CXCL2-dependent neutrophil infiltration in ConA-induced liver damage. SHP plays a protective, anti-inflammatory role in liver during acute liver inflammation.

In recent years, many methods have been investigated to improve imaging speed in photoacoustic microscopy (PAM). These methods mainly focused upon three critical factors contributing to fast PAM: laser pulse repetition rate, scanning speed, and computing power of the microprocessors. A high laser repetition rate is fundamentally the most crucial factor to increase the PAM speed. In this paper, we review methods adopted for fast PAM systems in detail, specifically with respect to light sources. To the best of our knowledge, ours is the first review article analyzing the fundamental requirements for developing high-speed PAM and their limitations from the perspective of light sources.