Detection of α-Thrombin with Platelet Glycoprotein Ibα (GP1bα) for the Development of a Coagulation Marker.

The detection of prothrombotic markers is crucial for understanding thromboembolism and assessing the effectiveness of anticoagulant drugs. α-Thrombin is a marker that plays a critical role in the coagulation cascade process. However, the detection of this enzymatic molecule was hindered by the absence of an efficient modality in the clinical environment. Previously, we reported that one α-thrombin interacts with two α-chains of glycoprotein Ib (GPIbα), i.e., multivalent protein binding (MPB), using bioresponsive hydrogel nanoparticles (nanogels) and optical microscopy. In this study, we demonstrated that GPIbα-mediated platforms led to the highly sensitive and quantitative detection of α-thrombin in various diagnostic systems. Initially, a bioresponsive nanogel-based surface plasmon resonance (nSPR) assay was developed that responds to the MPB of α-thrombin to GPIbα. The use of GPIbα for the detection of α-thrombin was further validated using the enzyme-linked immunosorbent assay, which is a gold-standard protein detection technique. Additionally, GPIbα-functionalized latex beads were developed to perform latex agglutination (LA) assays, which are widely used with hospital diagnostic instruments. Notably, the nSPR and LA assays exhibited a nearly 1000-fold improvement in sensitivity for α-thrombin detection compared to our previous optical microscopy method. The superiority of our GPIbα-mediated platforms lies in their stability for α-thrombin detection through protein-protein interactions. By contrast, assays relying on α-thrombin enzymatic activity using substrates face the challenge of a rapid decrease in postsample collection. These results suggested that the MPB of α-thrombin to GPIbα is an ideal mode for clinical α-thrombin detection, particularly in outpatient settings.

Method for the Rapid Detection of SARS-CoV-2-Neutralizing Antibodies Using a Nanogel-Based Surface Plasmon Resonance Biosensor.

The efficacy of coronavirus disease 2019 (COVID-19) vaccination is closely related to the serum levels of SARS-CoV-2-neutralizing antibodies (NAb) that bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Therefore, the rapid and quantitative measurement of SARS-CoV-2 NAb in the sera of vaccinated individuals is essential to develop an effective vaccine and further achieve population immunity, that is, herd immunity. The plaque reduction neutralization test, the gold standard for NAb effectiveness in serological tests, is accurate but requires biosafety level 3 facilities because of the use of the virus, which hampers its application in common laboratories and clinical practice. Here, we developed a bioresponsive nanogel-based surface plasmon resonance (nSPR) platform that detects SARS-CoV-2 NAb in clinical samples without complicated pretreatment. We found that multivalent protein binding (MPB) between the nanogel-conjugated RBD protein and SARS-CoV-2 NAb yields significantly enhanced SPR signals compared to the nonspecific interference from serum proteins in the nSPR assay. The excellence of our nanogel-based SARS-CoV-2 NAb test is due to its selectivity for NAb, with resistance to all other proteins, allowing the rapid detection and quantification of NAbs in each individual. Importantly, this nSPR assay provides a NAb detection platform for easier and safer COVID-19 vaccination strategies.

Tuning Surface Plasmon Resonance Responses through Size and Crosslinking Control of Multivalent Protein Binding-Capable Nanoscale Hydrogels.

Surface plasmon resonance (SPR) phenomena have been widely studied to detect biomolecules because of their high sensitivity and ability to determine biomolecular interactions with kinetic information. However, highly selective detection in specific concentration ranges relevant to target biomolecules is still a challenging task. Recently, we developed bioresponsive nanoscale hydrogels to selectively intensify SPR signals through multivalent protein binding (MPB) events with target biomolecules, including IL-2, where we were able to demonstrate exceptional selectivity for target biomolecules with minimal responses to nonspecific and monovalent binding events. In this work, we systematically explored the relationship between the physical properties of MPB-capable nanoscale hydrogels and their SPR response induced in the presence of the programmed cell death protein 1 antibody (PD-1Ab) as a model target biomolecule. First, we developed a synthetic protocol by controlling various reaction parameters to construct a library of nanoscale poly(N-isopropylacrylamide-co-acrylic acid) hydrogels (NHs) with different sizes (from 400 nm to 1 µm) and degrees of crosslinking (from 2 to 8%). Then, by incorporating MPB-capable PD-1 receptors onto the surface of NHs to form PD-1-responsive nanoscale hydrogels (PNHs), the hydrogel size and crosslinking dependency of their SPR responses were investigated. Our results reveal the appropriate hydrogel size regime and degree of crosslinking for effective PD-1Ab detection at specific concentrations range between a few nM and 1 µM. Overall, our study demonstrates that by tuning the physical properties of the nanoscale hydrogel matrix, the sensitivity and detection range of MPB-based SPR sensors can be modulated to potentially benefit clinical applications such as monitoring diverse therapeutic biomolecules.

New Tool for Rapid and Accurate Detection of Interleukin-2 and Soluble Interleukin-2 Receptor α in Cancer Diagnosis Using a Bioresponsive Microgel and Multivalent Protein Binding.

Interleukin-2 (IL-2) and its α receptor in soluble form (sIL-2Rα) are considered biomarkers for cancers and immune-related diseases. Enzyme-linked immunosorbent assay is the most common method used to evaluate biomarkers in clinical practice; it is precise but time-consuming and involves complicated procedures. Here, we have developed a rapid yet accurate modality for cancer diagnosis that enables on-site evaluation of cancer markers, that is, IL-2 and sIL-2Rα, without complicated pretreatment of cancer patient-derived blood samples. Surface plasmon resonance and bioresponsive microgels conjugated with IL-2 receptors, that is, IL-2Rß and IL-2Rγ, were utilized to measure IL-2 and sIL-2Rα levels via multivalent protein binding (MPB) between the ligands and their receptors. Our results showed that this novel method enables us to perform cancer diagnosis with a 1000-fold dilution of serum in 10 min. The advantage of MPB-based cancer diagnosis originates from its great selectivity for a target molecule and tolerance to a myriad of nonspecific substances in serum, which allows on-site clinical evaluation. Importantly, our finding implies that MPB-based cancer diagnosis provides a new paradigm not only for improving cancer treatment but also for evaluating a target molecule in unpurified and complex solutions such as blood.

Nitric oxide attenuated transforming growth factor-ß induced myofibroblast differentiation of human keratocytes.

Nitric oxide (NO) has the potential to modulate myofibroblast differentiation. In this study, we investigated the effect of exogenous NO on the myofibroblast differentiation of human keratocytes using sodium nitrite as a NO donor. Myofibroblasts were induced by exposing resting keratocytes to transforming growth factor (TGF)-ß1. N-cadherin and α-smooth muscle actin (αSMA) were used as myofibroblast markers. Both resting keratocytes and -stimulated keratocytes were exposed to various concentrations of sodium nitrite (1 µM to 1000 mM) for 24 to 72 h. Exposure to sodium nitrite did not alter keratocytes' viability up to a 10 mM concentration for 72 h. However, significant cytotoxicity was observed in higher concentrations of sodium nitrite (over 100 mM). The expression of αSMA and N-cadherin was significantly increased in keratocytes by TGF-ß1 stimulation after 72 h incubation. The addition of sodium nitrite (1 mM) to TGF-ß1-stimulated keratocytes significantly decreased αSMA and N cadherin expression. Smad3 phosphorylation decreased after sodium nitrite (1 mM) exposure in TGF-ß1-stimulated keratocytes. The effect of NO was reversed when NO scavenger, 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) was added in the culture medium. Application of sodium nitrite resulted in significant decrease of corneal opacity when measured at 2 weeks after the chemical burn in the mouse. These results verified the potential therapeutic effect of NO to decrease myofibroblast differentiation of human keratocytes and corneal opacity after injury.

Development and Evaluation of Olive Flounder cyp1a1-Luciferase Assay for Effective Detection of CYP1A-Inducing Contaminants in Coastal Sediments.

Flounders have been widely used as indicator species for monitoring the benthic environment of marine coastal regions owing to their habitat and feeding preferences in or on sandy sediments. Here, a single-step, sensitive, specific, and simple luciferase assay was developed, using the olive flounder cyp1a1 gene, for effective detection of CYP1A-inducing contaminants in coastal sediments. The developed cyp1a1-luciferase assay was highly sensitive to the widely used CYP1A inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo[a]pyrene (B[a]P), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). In the case of TCDD, significant dose-dependent increases in luciferase activity (0.3-300 ng/L) were detected. The assay was more sensitive to PCB 126 than to B[a]P. The assay also involved the highly sensitive expression of luciferase to extracted mixtures of PCBs and polycyclic aromatic hydrocarbons (PAHs) collected from coastal sediments. PCBs were more capable of cyp1a1 induction in the assay system at small doses than PAHs in environmental samples. Using the cyp1a1-luciferase assay along with water or sediment chemistry will certainly aid in diagnosing CYP1A-inducing contaminants in coastal environments.

Effect of Nitric Oxide on Acanthamoeba castellanii.

Purpose: Acanthamoeba keratitis is a well-known intractable corneal infectious disease. We investigated the anti-Acanthamoeba effect of exogenous nitric oxide (NO). Methods: Acanthamoeba castellanii was axenically cultured and exposed to various concentrations of NO donors, such as sodium nitrite, sodium nitroprusside (SNP), and NO-releasing silica nanoparticles (coated in branched polyethylene imine, size:100 nm), for 1 to 7 days (sodium nitrite and SNP: 0, 0.1, 1, 10, 100, and 1000 µM; silica nanoparticles: 0, 6.25, 12.5, 25, 50, and 100 µg/mL). Human corneal epithelial cells (HCECs) were cultured and exposed to sodium nitrite, SNP (0, 0.1, 1, 10, 100, and 1000 µM), and silica nanoparticles for 1, 2, and 3 days. Results: Sodium nitrite and SNP showed a dose-dependent inhibitory effect on A. castellanii viability. A more prominent inhibitory effect was observed with SNP (less than 10% of organisms survived at 7-day culture with 1000 µM) compared with sodium nitrite. However, more cytotoxicity on HCEC was observed with SNP. NO-releasing silica nanoparticles were successfully internalized into the amoebic cytoplasm and accumulated in large vacuoles. Although blank silica nanoparticles had no inhibitory effect on A. castellanii viability, NO-releasing silica nanoparticles showed a dose-dependent amoebicidal effect. Furthermore, no cystic transformation of A. castellanii was observed under a phase contrast microscope or transmission electron microscope after exogenous NO treatment. Conclusions: Our results demonstrated the anti-Acanthamoeba effect of exogenous NO. This finding suggests that NO-releasing drug platforms, including nano-carriers, can be a promising therapeutic strategy for Acanthamoeba keratitis.

Identification and molecular characterization of two Cu/Zn-SODs and Mn-SOD in the marine ciliate Euplotes crassus: Modulation of enzyme activity and transcripts in response to copper and cadmium.

The superoxide dismutase (SOD) family is a first line antioxidant enzyme group involved in transformation of the superoxide anion (O2-) into hydrogen peroxide (H2O2) and O2. SOD gene expression patterns and enzyme activities therefore have a role as molecular biomarkers in evaluating the oxidative stress status of aquatic organisms. However, antioxidant enzyme systems are yet to be fully explored in the marine ciliates. In this study, we identified and characterized two types of Cu/Zn SODs (Ec-Cu/ZnSOD1 and Ec-Cu/ZnSOD2) and Ec-Mn SOD in the marine ciliate Euplotes crassus. Subsequently, SOD activity and transcriptional modulation of the relevant genes were investigated after the exposure to Cd and Cu for 8 h. All Ec-SODs showed conserved domains and metal binding sites on their active sites. Total SOD activity was induced at 1 h after exposure to Cd (125 and 1000 µg/L), and showed a marginal increase at 1-h exposure to Cu (10 and 100 µg/L). However, SOD activity was maintained at a steady level under Cd and decreased under Cu exposure conditions at 3 h and 8 h. mRNA expression of both the Ec-Cu/Zn-SODs and Mn-SOD were remarkably elevated after the exposure to Cd (250-1000 µg/L, maximum 4-fold, p < 0.05) and, in particular, Cu (25-100 µg/L, maximum > 20-fold, p < 0.05), in a concentration - dependent manner. These findings suggest that Ec-SODs may be actively involved in cellular protection against metal - mediated oxidative stress. This study is therefore helpful in understanding the molecular responses for metal toxicity in the ciliates.

Identification and molecular characterization of cytochrome P450 (CYP450) family genes in the marine ciliate Euplotes crassus: The effect of benzo[a]pyrene and beta-naphthoflavone.

Marine ciliate Euplotes crassus, a single-cell eukaryote, and has been considered as a model organism for monitoring of environmental pollutions in sediments. Cytochrome P450 (CYP450) monooxygenase are phase I enzyme involved in detoxification of environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs). However, little information on CYP450 family genes in ciliate is available. In the present study, acute toxicity of PAH, benzo[a]pyrene (B[a]P) and PAH-like model compound, beta-naphthoflavone (ß-NF), was investigated; full-length cDNA sequences and genomic structure of five CYP450 genes (CYP5680A1, CYP5681A1, CYP5681B1, CYP5682A1, and CYP5683A1) were analyzed; and finally their activities and transcriptional changes were measured after exposure to PAHs for 48h. According to the results, B[a]P exposure showed a negative effect on E. crassus survival, whereas ß-NF exposure showed no significant effect. The 8h-LC50 value of B[a]P was determined to be 2.449µM (95%-C.L., 7.726-3.619µM). Five genes belonging to the CYP450 family had conserved domains and clustered with those of ciliate group, as revealed in phylogenetic analysis. CYP activity did not change after exposure to B[a]P, whereas it was slightly, but significantly, induced after exposure to ß-NF. The mRNA expression of five CYP450 genes was significantly modulated in a concentration- and time-dependent manner after exposure to both the chemicals. Our findings suggest that CYP450 genes in E. crassus may be involved in detoxification of B[a]P and ß-NF. This study would give a better understanding about the mode of action of B[a]P and ß-NF in marine ciliates at the molecular level.

Molecular characterization of ABC transporters in marine ciliate, Euplotes crassus: Identification and response to cadmium and benzo[a]pyrene.

ATP-binding cassette (ABC) transporters participate in transporting various substances, including xenobiotics, in or out of cells. However, their genetic information and function in ciliates remain still unclear. In this study, we sequenced and characterized two ABC transporter genes (EcABCB and EcABCC), and investigated the effect of cadmium (Cd) and benzo[a]pyrene (B[a]P) on their function and gene expression, using efflux assay and real-time reverse transcription-polymerase chain reaction (qRT-PCR), respectively, in the marine ciliate, Euplotes crassus. Sequencing analysis and efflux assay showed that EcABCB and EcABCC are typical ABC transporters, possessing conserved function. Exposure to Cd (≥5mg/L) and B[a]P (≥50.5µg/L) enhanced accumulation of a substrate. A significant increase in the expression of EcABCB and EcABC mRNA was observed at lower concentration in response to Cd and B[a]P. Our findings indicate that Cd and B[a]P could inhibit the efflux function of ABC transporters, leading to cellular toxicity in the ciliate.

The Effects of Nonporous Silica Nanoparticles on Cultured Human Keratocytes.

Purpose: Silica nanoparticles (SiNPs) are promising carriers for ophthalmic drug delivery. In this study, we investigated the effect of various sizes of nonporous SiNPs on cultured human keratocytes. Methods: Three different sizes of SiNPs (50, 100, and 150 nm) were manufactured. Primarily cultured human keratocytes were exposed to different concentrations (0, 25, 50, and 100 µg/mL) of three sizes of SiNPs for up to 72 hours. Intracellular reactive oxygen species (ROS) generation, cellular viability, lactate dehydrogenase (LDH) assay, autophagy, vimentin expression, and mammalian target of rapamycin (mTOR) pathway activation were evaluated. Intracellular distribution of SiNPs was evaluated with transmission electron microscopy. Results: Transmission electron microscopy revealed SiNPs were taken up by keratocytes inside cytoplasmic vacuoles. Neither nuclear entry of SiNPs nor mitochondrial structural damage was observed. Both intracellular ROS generation and LDH level remained unchanged with up to 100 µg/mL SiNP treatment. Cellular viability was not affected by SiNP treatment. Autophagy showed significant dose-dependent activation with 50- and 100-nm SiNPs. However, mTOR activation remained unchanged. Vimentin expression did not show any significant increase with SiNPs. Conclusions: Our findings suggested that 50-, 100-, and 150-nm SiNPs did not induce significant cytotoxicity in cultured human keratocytes at concentrations up to 100 µg/mL.