Revealing Redox Behavior of Molybdenum Disulfide and Its Application as Rechargeable Antioxidant Reservoir.

Molybdenum disulfide (MoS2) is a representative two-dimensional transition metal dichalcogenide and has a unique electronic structure and associated physicochemical properties. The redox property of MoS2 has recently attracted significant attention from various fields, such as biomedical applications. Intriguingly, MoS2 functions as an antioxidant in certain applications and as a pro-oxidant in others. We use the mediated electrochemical probing method to understand the redox behavior of MoS2. This method reveals that MoS2 (i) has a reversible and fast redox activity at a mild potential (between -0.20 and +0.25 V vs Ag/AgCl), (ii) functions as an antioxidant for molecules that have different redox mechanisms (electron or hydrogen atom transfer), and (iii) is electrochemically or molecularly rechargeable. Finally, we show that MoS2 reduces oxidized molecules more efficiently than the potent natural antioxidant, curcumin. This study enhances our understanding of MoS2 and shows its potential as an advanced antioxidant reservoir.

Recent Advances in Photoacoustic Agents for Theranostic Applications.

Photoacoustic agents are widely used in various theranostic applications. By evaluating the biodistribution obtained from photoacoustic images, the effectiveness of theranostic agents in terms of their delivery efficiency and treatment responses can be analyzed. Through this study, we evaluate and summarize the recent advances in photoacoustic-guided phototherapy, particularly in photothermal and photodynamic therapy. This overview can guide the future directions for theranostic development. Because of the recent applications of photoacoustic imaging in clinical trials, theranostic agents with photoacoustic monitoring have the potential to be translated into the clinical world.

Comparison of vaccination efficacy using live or ultraviolet-inactivated influenza viruses introduced by different routes in a mouse model.

Influenza is a major cause of highly contagious respiratory illness resulting in high mortality and morbidity worldwide. Annual vaccination is an effective way to prevent infection and complication from constantly mutating influenza strains. Vaccination utilizes preemptive inoculation with live virus, live attenuated virus, inactivated virus, or virus segments for optimal immune activation. The route of administration also affects the efficacy of the vaccination. Here, we evaluated the effects of inoculation with ultraviolet (UV)-inactivated or live influenza A virus strains and compared their effectiveness and cross protection when intraperitoneal and intramuscular routes of administration were used in mice. Intramuscular or intraperitoneal inoculation with UV-inactivated Influenza A/WSN/1933 provided some protection against intranasal challenge with a lethal dose of live Influenza A/WSN/1933 but only when a high dose of the virus was used in the inoculation. By contrast, inoculation with a low dose of live virus via either route provided complete protection against the same intranasal challenge. Intraperitoneal inoculation with live or UV-inactivated Influenza A/Philippines/2/1982 and intramuscular inoculation with UV-inactivated Influenza A/Philippines/2/1982 failed to produce cross-reactive antibodies against Influenza A/WSN/1933. Intramuscular inoculation with live Influenza A/Philippines/2/1982 induced small amounts of cross-reactive antibodies but could not suppress the cytokine storm produced upon intranasal challenge with Influenza A/WSN/1993. None of the tested inoculation conditions provided observable cross protection against intranasal challenge with a different influenza strain. Taken together, vaccination efficacy was affected by the state and dose of the vaccine virus and the route of administration. These results provide practical data for the development of effective vaccines against influenza virus.

Enzyme Nanosheet-Based Electrochemical Aspartate Biosensor for Fish Point-of-Care Applications.

Bacterial infections in marine fishes are linked to mass mortality issues; hence, rapid detection of an infection can contribute to achieving a faster diagnosis using point-of-care testing. There has been substantial interest in identifying diagnostic biomarkers that can be detected in major organs to predict bacterial infections. Aspartate was identified as an important biomarker for bacterial infection diagnosis in olive flounder (Paralichthys olivaceus) fish. To determine aspartate levels, an amperometric biosensor was designed based on bi-enzymes, namely, glutamate oxidase (GluOx) and aspartate transaminase (AST), which were physisorbed on copolymer reduced graphene oxide (P-rGO), referred to as enzyme nanosheets (GluOx-ASTENs). The GluOx-ASTENs were drop casted onto a Prussian blue electrodeposited screen-printed carbon electrode (PB/SPCE). The proposed biosensor was optimized by operating variables including the enzyme loading amount, coreactant (α-ketoglutarate) concentration, and pH. Under optimal conditions, the biosensor displayed the maximum current responses within 10 s at the low applied potential of -0.10 V vs. the internal Ag/AgCl reference. The biosensor exhibited a linear response from 1.0 to 2.0 mM of aspartate concentrations with a sensitivity of 0.8 µA mM-1 cm-2 and a lower detection limit of approximately 500 µM. Moreover, the biosensor possessed high reproducibility, good selectivity, and efficient storage stability.

Differential effect of SARS-CoV-2 infection on stress granule formation in Vero and Calu-3 cells.

Stress granule formation is induced by numerous environmental stressors, including sodium arsenite treatment and viral infection. Accordingly, stress granules can inhibit viral propagation and function as part of the antiviral host response to numerous viral infections. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antagonizes stress granule formation, in part, via interaction between SARS-CoV-2 nucleocapsid (N) protein and Ras-GTPase-activating SH3-domain-binding protein 1 (G3BP1). However, it is unclear whether there are differential effects in different cell types. In this study, we assessed interaction between the N protein of SARS-CoV-2 S clade and G3BP1/2 in Vero and Calu-3 cells and investigated the effect of various SARS-CoV-2 strains on sodium arsenite-induced stress granule formation. Our data show that SARS-CoV-2 S clade N protein interacts with both G3BP1 and G3BP2 more strongly in Calu-3 vs. Vero cells. Consistent with this observation, infection with SARS-CoV-2 S clade induces stress granule formation in Vero but not in Calu-3 cells. However, infection with SARS-CoV-2 S clade, as well as other SARS-CoV-2 variants, inhibits sodium arsenite-induced stress granule formation in both cell lines. Taken together, our results show differential effects of SARS-CoV-2 infection on stress granule formation that is dependent on host cell type, rather than virus strain type.

Abiraterone Acetate Attenuates SARS-CoV-2 Replication by Interfering with the Structural Nucleocapsid Protein.

The drug repurposing strategy has been applied to the development of emergency COVID-19 therapeutic medicines. Current drug repurposing approaches have been directed against RNA polymerases and viral proteases. Recently, we found that the inhibition of the interaction between the SARS-CoV-2 structural nucleocapsid (N) and spike (S) proteins decreased viral replication. In this study, drug repurposing candidates were screened by in silico molecular docking simulation with the SARS-CoV-2 structural N protein. In the ChEMBL database, 1994 FDA-approved drugs were selected for the in silico virtual screening against the N terminal domain (NTD) of the SARS-CoV-2 N protein. The tyrosine 109 residue in the NTD of the N protein was used as the center of the ligand binding grid for the docking simulation. In plaque forming assays performed with SARS-CoV-2 infected Vero E6 cells, atovaquone, abiraterone acetate, and digoxin exhibited a tendency to reduce the size of the viral plagues without affecting the plaque numbers. Abiraterone acetate significantly decreased the accumulation of viral particles in the cell culture supernatants in a concentration-dependent manner. In addition, abiraterone acetate significantly decreased the production of N protein and S protein in the SARS-CoV-2-infected Vero E6 cells. In conclusion, abiraterone acetate has therapeutic potential to inhibit the viral replication of SARS-CoV-2.

Targeting the Interaction Between Spike Protein and Nucleocapsid Protein for Suppression and Detection of Human Coronavirus OC43.

Human coronavirus OC43 (HCoV-OC43) is the coronavirus most associated with "common colds", infections of the upper respiratory tract. Previously, we reported that direct interactions of nucleocapsid (N) protein and C-terminal domain of Spike protein (Spike CD) are essential for replication of SARS-CoV-2 and MERS-CoV. Thus, we developed a novel ELISA-based strategy targeting these specific interactions to detect SARS-CoV-2 and MERS-CoV. Here, we investigated whether the same principles apply to HCoV-OC43. We discovered that the S protein of HCoV-OC43 interacts with N protein and that cell penetrating Spike CD peptide inhibits virus protein expression and replication of HCoV-OC43. The interaction between HCoV-OC43 S and N proteins were recapitulated with a recombinant HCoV-OC43 Spike CD fusion protein and a recombinant HCoV-OC43 N fusion protein in vitro. By producing an anti-HCoV-OC43 N protein-specific monoclonal antibody, we established a virus detection system based on the interaction between recombinant Spike CD and N protein of HCoV-OC43. We suggest that the interaction between Spike CD and N protein is conserved in coronaviruses and therefore could be a target for therapeutics against both novel coronavirus and its variants.

Graphene for Nanobiosensors and Nanobiochips.

Graphene family nanomaterials have interesting electronic structures which determine their electrical, optical, and mechanical properties. Especially, their unique chemical properties enable interactions with biological substances and chemical reagents, and the interactions have further an influence on the observable properties of the graphene family nanomaterials. Such aspects render graphene family nanomaterials versatile for various types of biosensing as a target recognition unit and a recognition-to-signal transduction unit. In this chapter, we look over the recent progress on the graphene-based biosensors, which is categorized in terms of (1) the role of graphene family nanomaterials (target recognition, signal transduction), (2) the sensing mechanisms and modes (electrochemical, electrical, fluorescent, Raman scattering), and (3) the formats of sensing devices (paper, lab-on-a-chip, wearable devices).

Wearable Sensing Systems for Monitoring Mental Health.

Wearable systems for monitoring biological signals have opened the door to personalized healthcare and have advanced a great deal over the past decade with the development of flexible electronics, efficient energy storage, wireless data transmission, and information processing technologies. As there are cumulative understanding of mechanisms underlying the mental processes and increasing desire for lifetime mental wellbeing, various wearable sensors have been devised to monitor the mental status from physiological activities, physical movements, and biochemical profiles in body fluids. This review summarizes the recent progress in wearable healthcare monitoring systems that can be utilized in mental healthcare, especially focusing on the biochemical sensors (i.e., biomarkers associated with mental status, sensing modalities, and device materials) and discussing their promises and challenges.

Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein.

SARS-CoV-2 infections continue to spread quickly by human-to-human transmission around the world. Therefore, developing methods to rapidly detect SARS-CoV-2 with high sensitivity are still urgently needed. We produced a monoclonal antibody that specifically detects the N protein of SARS-CoV-2 and recognizes N protein in cell lysates of SARS-CoV-2-infected Vero cells but not in cell lysates of MERS-CoV- or HCoV-OC43-infected Vero cells. This antibody recognized N protein in SARS-CoV-2 clades S, GR, and GH and recognized N protein in all the SARS-CoV-2 clades to ∼300 pfu. Previously, we reported that the coronavirus N protein interacts with the C-terminal domain of the spike protein (Spike CD). In this study, we developed an ELISA-based "bait and prey" system to confirm the interaction between SARS-CoV-2 Spike CD and N protein using recombinant fusion proteins. Furthermore, this system can be modified to quantitatively detect SARS-CoV-2 in culture media of infected cells by monitoring the interaction between the recombinant Spike CD fusion protein and the viral N protein, which is captured by the N protein-specific antibody. Therefore, we conclude that our N protein-specific monoclonal antibody and our ELISA-based bait and prey system could be used to diagnose SARS-CoV-2 infections.

Apoptosis Enhances the Replication of Human Coronavirus OC43.

Human coronavirus OC43 (HCoV-OC43) is one of the coronaviruses causing a mild common cold, but few studies have been made on this strain. Here, we identified the molecular mechanisms involved in HCoV-OC43-induced apoptosis and its implications for viral reproduction in Vero cells and MRC-5 cells. HCoV-OC43 infection induced apoptosis that was accompanied by cleavage of caspase-3 and PARP, degradation of cyclin D1, and cell cycle arrest at S and G2M phases. Dephosphorylation of STAT1 and STAT3, induced by HCoV-OC43 infection, was also associated with HCoV-OC43-mediated apoptosis. The pan-caspase inhibitor effectively prevented HCoV-OC43-induced apoptosis and reduced viral replication, suggesting that apoptosis contributes to viral replication. Collectively our results indicate that HCoV-OC43 induces caspase-dependent apoptosis to promote viral replication in Vero cells and MRC-5 cells.

ChemSkin Reference Chemical Database for the Development of an In Vitro Skin Irritation Test.

Since the animal test ban on cosmetics in the EU in 2013, alternative in vitro safety tests have been actively researched to replace in vivo animal tests. For the development and evaluation of a new test method, reference chemicals with quality in vivo data are essential to assess the predictive capacity and applicability domain. Here, we compiled a reference chemical database (ChemSkin DB) for the development and evaluation of new in vitro skin irritation tests. The first candidates were selected from 317 chemicals (source data n = 1567) searched from the literature from the last 20 years, including previous validation study reports, ECETOC, and published papers. Chemicals showing inconsistent classification or those that were commercially unavailable, difficult or dangerous to handle, prohibitively expensive, or without quality in vivo or in vitro data were removed, leaving a total of 100 chemicals. Supporting references, in vivo Draize scores, UN GHS/EU CLP classifications and commercial sources were compiled. Test results produced by the approved methods of OECD Test No. 439 were included and compared using the classification table, scatter plot, and Pearson correlation analysis to identify the false predictions and differences between in vitro skin irritation tests. These results may provide an insight into the future development of new in vitro skin irritation tests.

Me-too validation study for in vitro skin irritation test with a reconstructed human epidermis model, KeraSkin™ for OECD test guideline 439.

We conducted a me-too validation study to confirm the reproducibility, reliability, and predictive capacity of KeraSkin™ skin irritation test (SIT) as a me-too method of OECD TG 439. With 20 reference chemicals, within-laboratory reproducibility (WLR) of KeraSkin™ SIT in the decision of irritant or non-irritant was 100%, 100%, and 95% while between-laboratory reproducibility (BLR) was 100%, which met the criteria of performance standard (PS, WLR≥90%, BLR≥80%). WLR and BLR were further confirmed with intra-class correlation (ICC, coefficients >0.950). WLR and BLR in raw data (viability) were also shown with a scatter plot and Bland-Altman plot. Comparison with existing VRMs with Bland-Altman plot, ICC and kappa statistics confirmed the compatibility of KeraSkin™ SIT with OECD TG 439. The predictive capacity of KeraSkin™ SIT was estimated with 20 reference chemicals (the sensitivity of 98.9%, the specificity of 70%, and the accuracy of 84.4%) and additional 46 chemicals (for 66 chemicals [20 + 46 chemicals, the sensitivity, specificity and accuracy: 95.2%, 82.2% and 86.4%]). The receiver operating characteristic (ROC) analysis suggested a potential improvement of the predictive capacity, especially sensitivity, when changing cut-off (50% → 60-75%). Collectively, the me-too validation study demonstrated that KeraSkin™ SIT can be a new me-too method for OECD TG 439.

Tethered molecular redox capacitors for nanoconfinement-assisted electrochemical signal amplification.

Nanostructured materials offer the potential to drive future developments and applications of electrochemical devices, but are underutilized because their nanoscale cavities can impose mass transfer limitations that constrain electrochemical signal generation. Here, we report a new signal-generating mechanism that employs a molecular redox capacitor to enable nanostructured electrodes to amplify electrochemical signals even without an enhanced reactant mass transfer. The surface-tethered molecular redox capacitor engages diffusible reactants and products in redox-cycling reactions with the electrode. Such redox-cycling reactions are facilitated by the nanostructure that increases the probabilities of both reactant-electrode and product-redox-capacitor encounters (i.e., the nanoconfinement effect), resulting in substantial signal amplification. Using redox-capacitor-tethered Au nanopillar electrodes, we demonstrate improved sensitivity for measuring pyocyanin (bacterial metabolite). This study paves a new way of using nanostructured materials in electrochemical applications by engineering the reaction pathway within the nanoscale cavities of the materials.

Prediction of the skin sensitization potential of didecyldimethylammonium chloride and 3,7-dimethyl-2,6-octadienal and mixtures of these compounds with the excipient ethylene glycol through the human Cell Line Activation Test and the Direct Peptide Reactivity Assay.

In commercial products such as household deodorants or biocides, didecyldimethylammonium chloride (DDAC) often serves as an antimicrobial agent, citral serves as a fragrance agent, and the excipient ethylene glycol (EG) is used to dissolve the active ingredients. The skin sensitization (SS) potentials of each of these substances are still being debated. Moreover, mixtures of DDAC or citral with EG have not been evaluated for SS potency. The in vitro alternative assay called human Cell Line Activation Test (h-CLAT) and Direct Peptide Reactivity Assay (DPRA) served to address these issues. On three independent runs of h-CLAT, DDAC and citral were predicted to be sensitizers while EG was predicted to be a non-sensitizer and also by the DPRA. Mixtures of DDAC or citral with EG at ratios of 7:3 and 1:4 w/v were all positive by the h-CLAT in terms of SS potential but SS potency was mitigated as the proportion of EG increased. Citral and its EG mixtures were all positive but DDAC and its EG mixtures were all negative by the DPRA, indicating that the DPRA method is not suitable for chemicals with pro-hapten characteristics. Since humans can be occupationally or environmentally exposed to mixtures of excipients with active ingredients, the present study may give insights into further investigations of the SS potentials of various chemical mixtures.

Pro- and Anti-oxidant Properties of Redox-Active Catechol-Chitosan Films.

Catechols are abundant in nature and are believed to perform diverse biological functions that include photoprotection (e.g., melanins), molecular signaling (e.g., catecholamine neurotransmitters), and mechanical adhesion (e.g., mussel glue). Currently, the structure-property-function relationships for catechols remain poorly resolved, and this is especially true for redox-based properties (e.g., antioxidant, pro-oxidant, and radical scavenging activities). Importantly, there are few characterization methods available to probe the redox properties of materials. In this review, we focus on recent studies with redox-active catechol-chitosan films. First, we describe film fabrication methods to oxidatively-graft catechols to chitosan through chemical, enzymatic, or electrochemical methods. Second, we discuss a new experimental characterization method to probe the redox properties of catechol-functionalized materials. This mediated electrochemical probing (MEP) method probes the redox-activities of catechol-chitosan films by: (i) employing diffusible mediators to shuttle electrons between the electrode and grafted catechols; (ii) imposing tailored sequences of input voltages to "tune" redox probing; and (iii) analyzing the output current response characteristics to infer properties. Finally, we demonstrate that the redox properties of catechol-chitosan films enable them to perform antioxidant radical scavenging functions, as well as a pro-oxidant (reactive oxygen-generation) antimicrobial functions. In summary, our increasing knowledge of catechol-chitosan films is enabling us to better-understand the functions of catechols in biology as well as enhancing our capabilities to create advanced functional materials.

Validation of oxidative stress assay for schizophrenia.

Accumulating evidence implicates oxidative stress in a range of diseases, yet no objective measurement has emerged that characterizes the global nature of oxidative stress. Previously, we reported a measurement that employs the moderately strong oxidant iridium (Ir) to probe the oxidative damage in a serum sample and reported that in a small study (N = 15) the Ir-reducing capacity assay could distinguish schizophrenia from healthy control groups based on their levels of oxidative stress. Here, we used a larger sample size to evaluate the Ir-reducing capacity assay to assess its ability to discriminate the schizophrenia (N = 73) and healthy control groups (N = 45). Each serum sample was measured (in triplicate) at three different times that were separated by several weeks. The Intraclass Correlation Coefficient (ICC = 0.69) for these repeated measurements indicates the assay detects stable components in the sample (i.e., it is not detecting transient reactive species or air-oxidizable serum components). Correlations between the Ir-reducing capacity assay and independently-measured total serum protein levels (r = +0.74, p < 2.2 × 10-16) suggest the assay is detecting information in the protein pool. For cross-validation of the discrimination ability, we used machine learning and receiver operating characteristic (ROC) analysis. After adjusting for potential confounders (age and smoking status), an area under the curve (AUC) of ROC curve was calculated to be 0.89 (p = 9.3 × 10-5). In conclusion, this validation indicates the Ir-reducing capacity assay provides a simple global measure of oxidative stress, and further supports the hypothesis that oxidative stress is linked with schizophrenia.

Redox Is a Global Biodevice Information Processing Modality.

Biology is well-known for its ability to communicate through (i) molecularly-specific signaling modalities and (ii) a globally-acting electrical modality associated with ion flow across biological membranes. Emerging research suggests that biology uses a third type of communication modality associated with a flow of electrons through reduction/oxidation (redox) reactions. This redox signaling modality appears to act globally and has features of both molecular and electrical modalities: since free electrons do not exist in aqueous solution, the electrons must flow through molecular intermediates that can be switched between two states - with electrons (reduced) or without electrons (oxidized). Importantly, this global redox modality is easily accessible through its electrical features using convenient electrochemical instrumentation. In this review, we explain this redox modality, describe our electrochemical measurements, and provide four examples demonstrating that redox enables communication between biology and electronics. The first two examples illustrate how redox probing can acquire biologically relevant information. The last two examples illustrate how redox inputs can transduce biologically-relevant transitions for patterning and the induction of a synbio transceiver for two-hop molecular communication. In summary, we believe redox provides a unique ability to bridge bio-device communication because simple electrochemical methods enable global access to biologically meaningful information. Further, we envision that redox may facilitate the application of information theory to the biological sciences.

Successful genetic modification of porcine spermatogonial stem cells via an electrically responsive Au nanowire injector.

Transgenic pigs are quite useful in many biomedical fields, such as xenotransplantation research and the production of biopharmaceutical materials. The genetic transformation of porcine spermatogonial stem cells (pSSCs) followed by differentiation into mature spermatozoa enables the effective production of transgenic pigs. Improving the transfection efficiency of pSSCs, however, has been much desired. Herein, we report the efficient genetic modification of pSSCs by using an electrically responsive Au nanowire injector (E-R Au NWI). This is the first study that shows an exogenous gene is directly delivered into the nucleus of a pSSC by using a 1-dimensional nanomaterial and then successfully expressed to produce a protein. The E-R Au NWI interfaced noninvasively with the nucleus of the pSSC, and the pEGFP-N1 plasmid was delivered by the application of an electrical stimulus without cell damage. Compared to the results of conventional nonviral vector-based gene delivery methods such as jetPEI, Lipofectamine, and electroporation, the E-R Au NWI-based method improved the pSSC transfection efficiency by at least 6.7-fold and even up to 46.7-fold. Furthermore, we successfully obtained transgenic pSSCs containing the human bone morphogenetic protein 2 gene by using E-R Au NWIs. This result suggests that the E-R Au NWI enables the efficient genetic modification of pSSCs and can be employed to produce diverse kinds of transgenic pigs.

Me-too validation study for in vitro eye irritation test with 3D-reconstructed human cornea epithelium, MCTT HCETM.

The need for in vitro eye irritation test replacing in vivo is steadily increasing. The MCTT HCE™ eye irritation test (EIT) using 3D reconstructed human cornea-like epithelium, was developed to identify ocular irritants from non-irritants those that are not requiring classification and labelling for eye irritation. Here, we report the results of me-too validation study, which was conducted to evaluate the reliability and relevance of the MCTT HCETM EIT, according to performance standards (PS) of OECD TG 492. The optimal cutoff to determine irritation in the prediction model was preliminarily established at 45% with the receiver operation characteristics (ROC) curve for 141 reference substances. To demonstrate the reproducibility of within- and between-laboratory (WLR and BLR), a set of 30 PS reference chemicals were tested in three laboratories three times. The WLR and BLR concordance with the binary decision of whether non-irritant or irritant was estimated to be 90-100% and 90%, respectively, and both met the PS requirements. The predictive capacity of the respective laboratories for the 30 reference chemicals were evaluated based on three different estimation methods, and the results were comparable, with sensitivity ranging from 89.6 to 93.3%, the specificity ranging from 62.2 to 66.7%, and the accuracy ranging from 75.9 to 80.0%. Additional test with the new set of 30 PS substances in the revised OECD GD 216 yielded a performance of sensitivity ranging from 92.6-93.3%, the specificity 62.2-66.7% and the accuracy 77.4-80.0%. 95.0% sensitivity, 67.2% specificity, and 83.0% accuracy were obtained for 141 reference substances in total. Furthermore, separate cutoffs for liquids and solids, 35% and 60%, respectively, produced better predictivity, which was established as a final prediction model. Collectively, our study demonstrated that MCTT HCETM EIT meets the reproducibility and predictivity criteria stated in OECD TG 492 PS.