Intein-Mediated Protein Engineering for Cell-Based Biosensors.

Cell-based sensors provide a flexible platform for screening biologically active targets and for monitoring their interactions in live cells. Their applicability extends across a vast array of biological research and clinical applications. Particularly, cell-based sensors are becoming a potent tool in drug discovery and cell-signaling studies by allowing function-based screening of targets in biologically relevant environments and enabling the in vivo visualization of cellular signals in real-time with an outstanding spatiotemporal resolution. In this review, we aim to provide a clear view of current cell-based sensor technologies, their limitations, and how the recent improvements were using intein-mediated protein engineering. We first discuss the characteristics of cell-based sensors and present several representative examples with a focus on their design strategies, which differentiate cell-based sensors from in vitro analytical biosensors. We then describe the application of intein-mediated protein engineering technology for cell-based sensor fabrication. Finally, we explain the characteristics of intein-mediated reactions and present examples of how the intein-mediated reactions are used to improve existing methods and develop new approaches in sensor cell fabrication to address the limitations of current technologies.

Genetically Encoded Sensor Cells for the Screening of Glucocorticoid Receptor (GR) Effectors in Herbal Extracts.

Although in vitro sensors provide facile low-cost ways to screen for biologically active targets, their results may not accurately represent the molecular interactions in biological systems. Cell-based sensors have emerged as promising platforms to screen targets in biologically relevant environments. However, there are few examples where cell-based sensors have been practically applied for drug screening. Here, we used engineered cortisol-detecting sensor cells to screen for natural mimetics of cortisol. The sensor cells were designed to report the presence of a target through signal peptide activation and subsequent fluorescence signal translocation. The developed sensor cells were able to detect known biological targets from human-derived analytes as well as natural product extracts, such as deer antlers and ginseng. The multi-use capability and versatility to screen in different cellular environments were also demonstrated. The sensor cells were used to identify novel GR effectors from medicinal plant extracts. Our results suggest that decursin from dongquai had the GR effector function as a selective GR agonist (SEGRA), making it a potent drug candidate with anti-inflammatory activity. We demonstrated the superiority of cell-based sensing technology over in vitro screening, proving its potential for practical drug screening applications that leads to the function-based discovery of target molecules.

Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells.

Cortisol, a stress hormone, plays key roles in mediating stress and anti-inflammatory responses. As abnormal cortisol levels can induce various adverse effects, screening cortisol and cortisol analogues is important for monitoring stress levels and for identifying drug candidates. A novel cell-based sensing system was adopted for rapid screening of cortisol and its functional analogues under complex cellular regulation. We used glucocorticoid receptor (GR) fused to a split intein which reconstituted with the counterpart to trigger conditional protein splicing (CPS) in the presence of targets. CPS generates functional signal peptides which promptly translocate the fluorescent cargo. The sensor cells exhibited exceptional performance in discriminating between the functional and structural analogues of cortisol with improved sensitivity. Essential oil extracts with stress relief activity were screened using the sensor cells to identify GR effectors. The sensor cells responded to peppermint oil, and L-limonene and L-menthol were identified as potential GR effectors from the major components of peppermint oil. Further analysis indicated L-limonene as a selective GR agonist (SEGRA) which is a potential anti-inflammatory agent as it attenuates proinflammatory responses without causing notable adverse effects of GR agonists.

The Health Status of Informal Waste Collectors in Korea.

Background: A broad, holistic approach was performed among informal waste collectors (IWCs) in Korea to understand their complex multidimensional health and safety problems. Methods: In the quantitative study, a survey of IWCs was conducted at four junk shops in Gangbuk-gu, Seoul, and survey data were used to calculate age-standardized prevalence rates based on comparisons with the general population in Korea. A qualitative study was also performed to provide more details on IWCs' occupational and musculoskeletal injuries and depression. Results: In the quantitative study, the age-standardized prevalence rate (aSPR) of occupational injury was higher than that of the general standard population (aSPR: 10.42, 95% confidence interval (CI) 5.19-18.64) and that of blue-collar workers (aSPR: 4.65, 95% CI 2.32-8.32). Regarding musculoskeletal problems, compared to employed populations, the aSPRs of shoulder pain (aSPR: 2.63, 95% CI 1.60-4.06), wrist pain (aSPR: 3.33, 95% CI 1.33-6.86), knee pain (aSPR: 1.51, 95% CI 1.01-2.17), and ankle pain (aSPR: 3.54, 95% CI 1.14-8.26) were higher. Regarding psychological problems, depression (aSPR: 2.55, 95% CI 1.27-4.56) and suicidal or self-harm ideation (aSPR: 2.09, 95% CI 1.11-3.58) were higher compared to general populations. Through the qualitative study and case study on muscular problems, more details on the work environment problems of IWCs were obtained. Conclusions: IWCs are exposed to various occupational hazards and lack proper protection. They show a high prevalence of occupational injury, musculoskeletal disease, and depression.

Genetically encoded biosensors for the detection of rapamycin: toward the screening of agonists and antagonists.

Biosensors are valuable tools for the rapid screening of biological targets with high sensitivity and specificity. It is important to screen biological events in their native context for pharmacological and toxicological applications. However, in vitro biosensors often require purified probes and targets for screening, thus providing limited information on the biological activities of targets in their native environment. To address this issue, we developed a cell-based sensing system that could detect a biologically active small molecule, rapamycin (Rapa). We designed a reporter system based on fluorescence translocation by signal peptide reconstitution. Herein, signal peptides are activated by conditional protein splicing without the need for refolding into a functional tertiary structure, thus eliminating false positives and negatives due to mere binding or misfolding. The developed biosensor demonstrated excellent sensitivity with a limit of detection of 0.1 nM, and it was able to screen the agonist and antagonist of Rapa. The developed cell-based sensing system could contribute to improving the screening system aimed to identify the natural mimetics of Rapa and potential drug candidates.

Molecular origin of AuNPs-induced cytotoxicity and mechanistic study.

Gold nanoparticles (AuNPs) with diverse physicochemical properties are reported to affect biological systems differently, but the relationship between the physicochemical properties of AuNPs and their biological effects is not clearly understood. Here, we aimed to elucidate the molecular origins of AuNP-induced cytotoxicity and their mechanisms, focusing on the surface charge and structural properties of modified AuNPs. We prepared a library of well-tailored AuNPs exhibiting various functional groups and surface charges. Through this work, we revealed that the direction or the magnitude of surface charge is not an exclusive factor that determines the cytotoxicity of AuNPs. We, instead, suggested that toxic AuNPs share a common structural characteristics of a hydrophobic moiety neighbouring the positive charge, which can induce lytic interaction with plasma membrane. Mechanistic study showed that the toxic AuNPs interfered with the formation of cytoskeletal structure to slow cell migration, inhibited DNA replication and caused DNA damage via oxidative stress to hinder cell proliferation. Gene expression analysis showed that the toxic AuNPs down-regulated genes associated with cell cycle processes. We discovered structural characteristics that define the cytotoxic AuNPs and suggested the mechanisms of their cytotoxicity. These findings will help us to understand and to predict the biological effects of modified AuNPs based on their physicochemical properties.

Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules.

Live-cell-based biosensors have emerged as a useful tool for biotechnology and chemical biology. Genetically encoded sensor cells often use bimolecular fluorescence complementation or fluorescence resonance energy transfer to build a reporter unit that suffers from nonspecific signal activation at high concentrations. Here, we designed genetically encoded sensor cells that can report the presence of biologically active molecules via fluorescence-translocation based on split intein-mediated conditional protein trans-splicing (PTS) and conditional protein trans-cleavage (PTC) reactions. In this work, the target molecules or the external stimuli activated intein-mediated reactions, which resulted in activation of the fluorophore-conjugated signal peptide. This approach fully valued the bond-making and bond-breaking features of intein-mediated reactions in sensor construction and thus eliminated the interference of false-positive signals resulting from the mere binding of fragmented reporters. We could also avoid the necessity of designing split reporters to refold into active structures upon reconstitution. These live-cell-based sensors were able to detect biologically active signaling molecules, such as Ca2+ and cortisol, as well as relevant biological stimuli, such as histamine-induced Ca2+ stimuli and the glucocorticoid receptor agonist, dexamethasone. These live-cell-based sensing systems hold large potential for applications such as drug screening and toxicology studies, which require functional information about targets.

Detection of Metallothionein in Javanese Medaka (Oryzias javanicus), Using a scFv-Immobilized Protein Chip.

Environmental pollution by various industrial chemicals and biological agents poses serious risks to human health. Especially, marine contamination by potentially toxic elements (PTEs) has become a global concern in recent years. Many efforts have been undertaken to monitor the PTE contamination of the aquatic environment. However, there are few approaches available to assess the PTE exposure of aquatic organisms. In this research, we developed a strategy to evaluate the heavy metal exposure of marine organisms, by measuring the expression levels of metallothionein protein derived from Oryzias javanicus (OjaMT). OjaMT is a biomarker of heavy metal exposure because the expression level increases upon heavy metal exposure. The developed assay is based on a real-time, label-free surface plasmon resonance (SPR) measurement. Anti-OjaMT antibody and anti-OjaMT single-chain fragment of variable region (scFv) were used as detection probes. Two types of SPR sensor chips were fabricated, by immobilizing antibody or Cys3-tagged scFv (scFv-Cys3) in a controlled orientation and were tested for in situ label-free OjaMT detection. Compared to the antibody-presenting sensor chips, the scFv-presenting sensor chips showed improved performance, displaying enhanced sensitivity and enabling semi-quantitative detection. The portable SPR system combined with scFv-immobilized sensor chips is expected to provide an excellent point-of-care testing system that can monitor target biomarkers in real time.

Combined silicosis and mixed dust pneumoconiosis with rapid progression: A case report and literature review.

BACKGROUND: Rapidly progressive pneumoconiosis (RPP) occasionally occurs in coal workers, particularly those with high exposure to silica. Here, we report the case of a 64-year-old male miller with RPP. CASE SUMMARY: The patient had a persistent cough for one month and had been clinically diagnosed with pulmonary tuberculosis in 2011. He worked in a stone processing factory from the ages of 20 through 37 and has owned his own mill for the past 25 years. His chest radiograph showed significant increases in the size and number of lung nodules since his last follow-up in 2013. By percutaneous needle lung biopsy, the nodular lesions showed diffuse infiltration of phagocytic macrophages and birefringent crystals by polarizing microscopy. He was finally diagnosed with RPP of mixed dust pneumoconiosis combined with silicosis. CONCLUSION: In this case, mixed dust pneumoconiosis with silicosis might be accelerated by persistent exposure to grain dust from working in a mill environment.

The relationship between shift work and mental health among electronics workers in South Korea: A cross-sectional study.

OBJECTIVE: To determine the relationship between shift work and mental health, particularly insomnia, depression, and suicidal ideation, among electronics production workers. METHODS: A survey was conducted with 14,226 workers from an electronics manufacturer in South Korea. After excluding 112 individuals with incomplete responses, 14,114 respondents were analyzed. As part of a larger project, we collected data on respondents' general characteristics, work-related characteristics, and health status; however, in this study, we focused on the data related to shift work and mental health. Insomnia, depression, and suicidal ideation were set as dependent variables and working schedule as set as the independent variable. We performed multiple logistic regression analysis with daytime workers as the reference group. The model was adjusted for age, gender, body mass index, workplace, educational level, and marital status with or without children under 6 years of age. RESULTS: Relative to daytime workers, shift workers had 2.35, 1.23, and 1.17 greater odds of insomnia, depression, and suicidal ideation, respectively. Within the shift worker group, we found that the odds of depression and suicidal ideation increased dramatically when respondents had insomnia. The ORs for depression and suicidal ideation were 4.899 and 7.934, respectively. CONCLUSIONS: Our findings suggest that shift work is related to an increased risk of mental health problems in production workers, and the sleep disturbance related with shift work is a central mechanism for this relationship. Since these results suggest that proactive management of sleep problems might attenuate their detrimental effects on shift worker's mental health.

Associations of Shift Work and Its Duration with Work-Related Injury among Electronics Factory Workers in South Korea.

This study aimed to explore the association between shift work and work-related injuries. We collected data on workers from an electronics factory. This cross-sectional study included 13,610 subjects, who were assessed based on a self-reported questionnaire about their shift work experiences, work-related injuries, and other covariates. Multiple logistic regression models were used to evaluate the associations between shift work and work-related injuries and were estimated using the odds ratio. We found that the current and past shift workers, compared to non-shift workers, were associated with a 2.7- and 1.7-fold higher risk of work-related injury. There was a dose-response relationship between shift work duration and work-related injury among current female shift workers. Shift work increased the risk of work-related injuries, and the impact could be different depending on gender.

Toxicity of Methylcyclohexane and Its Effect on the Reproductive System in SD Rats.

OBJECTIVES: There is limited data regarding the toxicity of methylcyclohexane, despite its wide use in rubber adhesives, paint diluents, and cleansing agents. This study aimed to verify the toxicity and influence on the reproductive system of methylcyclohexane after its repeated injection in Sprague Dawley (SD) rats. METHODS: Methylcyclohexane was injected subcutaneously into male and female SD rats once a day, five times a week, for 13 weeks at different doses (0, 10, 100, and 1,000 mg/kg/day) for each group. The toxicity of testing material was verified by observing the change in body and organ weight, hematological change, pathological findings, and effect on the reproductive system at each different concentration. RESULTS: In the 1,000 mg/kg/day group, there were cases of animal deaths. In animals that survived, hematological changes, including a decrease in the red blood cell count, were observed. A considerable weight gain or loss and pathological abnormalities in the liver, kidney, and other organs were found. However, the 10 and 100 mg/kg/day groups did not cause deaths or other specific abnormalities. In terms of reproductive toxicity, there were changes in hormone levels, including a significant decrease in hormones such as estradiol and progesterone (p < 0.001) in male animals. Menstrual cycle change for female animals did not show concentration dependency. CONCLUSION: When injected repeatedly for 13 weeks, methylcyclohexane proved to be toxic for the liver, heart, and kidney at a high dose. The absolute toxic dose was 1,000 mg/kg/day, while the no observed adverse effect level was less than 100 mg/kg/day. The substance exerted little influence on the reproductive system.

Isoflavones from phytoestrogens and gastric cancer risk: a nested case-control study within the Korean Multicenter Cancer Cohort.

BACKGROUND: The role of soybean products in gastric cancer risk is not clear in epidemiologic studies due to measurement error from dietary intake questionnaires and due to different degrees of bias according to study design. To examine the association between soybean products and gastric cancer risk, we measured phytoestrogen biological markers in a nested case-control study. METHODS: The study population was composed of 131 cases and 393 matched controls within the Korean Multicenter Cancer Cohort. The concentrations of the four biomarkers in the plasma samples were measured using time-resolved fluoroimmunoassay. Conditional and unconditional logistic regression models were used to compute the odds ratio (OR) and 95% confidence intervals (CI). RESULTS: Median plasma concentrations of genistein (229 nmol/L for controls, 181.8 nmol/L for cases; P=0.07) and daidzein (131.2 nmol/L for controls, 80.5 nmol/L for cases; P=0.04) in cases were lower than in controls, whereas equol concentrations were similar. Compared with the reference group, gastric cancer risk decreased in the highest groups for genistein (OR, 0.54; 95% CI, 0.31-0.93) and daidzein (OR, 0.21; 95% CI, 0.08-0.58). Higher equol concentrations were associated with a decreased risk for gastric cancer (OR, 0.50; 95% CI, 0.27-0.90). The combination of the highest concentrations for each isoflavone category was associated with a 0.09-fold decreased risk for gastric cancer compared with the combination of the lowest concentrations for each category. There was no association between plasma lignan concentrations and gastric cancer. CONCLUSIONS: High serum concentrations of isoflavones were associated with a decreased risk for gastric cancer. IMPACT: These results suggest a beneficial effect of high soybean product intake for gastric cancer risk.