Detecting early-stage malignant melanoma using a calcium switch-enriched exosome subpopulation containing tumor markers as a sample.

An exosome species containing CD63 as a marker of melanoma was isolated from bulk exosome population and used as a sample for detecting malignant melanoma. A calcium binding protein (CBP) was produced and then used to raise monoclonal antibody. The antibody was sensitive to a conformational change of CBP caused by Ca2+ binding. Immuno-magnetic beads were prepared by immobilizing the conformation-sensitive binder and subsequent binding of CBP conjugated with the capture antibody specific to CD63. These immuno-beads were used to isolate CD63-positive exosome from a bulk exosome sample (normal or melanoma) based on the 'calcium switch-on/off' mechanism through magnetic separation. After recovery, the subpopulation sample was analyzed by immunoassays for cavelion1 (Cav1), CD81, and CD9 as sub-subpopulation markers. Normalized signals of Cav1 and/or CD81 over CD9 were higher in melanoma samples than in normal samples, depending on clinical stages (I, II, and IV) of patients. This was in contrast to assay results for the bulk exosome population that showed a completely mixed state of melanoma and normal samples. These results showed that an exosome subpopulation sample prepared using a 'Ca2+-dependent switch' technology might be useful for diagnosing malignant melanoma at an early stage to increase 5-year survival rates.

Modeling Hypoxic Stress In Vitro Using Human Embryonic Stem Cells Derived Cardiomyocytes Matured by FGF4 and Ascorbic Acid Treatment.

Mature cardiomyocytes (CMs) obtained from human pluripotent stem cells (hPSCs) have been required for more accurate in vitro modeling of adult-onset cardiac disease and drug discovery. Here, we found that FGF4 and ascorbic acid (AA) induce differentiation of BG01 human embryonic stem cell-cardiogenic mesoderm cells (hESC-CMCs) into mature and ventricular CMs. Co-treatment of BG01 hESC-CMCs with FGF4+AA synergistically induced differentiation into mature and ventricular CMs. FGF4+AA-treated BG01 hESC-CMs robustly released acute myocardial infarction (AMI) biomarkers (cTnI, CK-MB, and myoglobin) into culture medium in response to hypoxic injury. Hypoxia-responsive genes and potential cardiac biomarkers proved in the diagnosis and prognosis of coronary artery diseases were induced in FGF4+AA-treated BG01 hESC-CMs in response to hypoxia based on transcriptome analyses. This study demonstrates that it is feasible to model hypoxic stress in vitro using hESC-CMs matured by soluble factors.

CRISPR-Cas12a-Based Nucleic Acid Amplification-Free DNA Biosensor via Au Nanoparticle-Assisted Metal-Enhanced Fluorescence and Colorimetric Analysis.

Cell-free DNA (cfDNA) has attracted significant attention due to its high potential to diagnose diseases, such as cancer. Still, its detection by amplification method has limitations because of false-positive signals and difficulty in designing target-specific primers. CRISPR-Cas-based fluorescent biosensors have been developed but also need the amplification step for the detection. In this study, for the first time CRISPR-Cas12a based nucleic acid amplification-free fluorescent biosensor was developed to detect cfDNA by a metal-enhanced fluorescence (MEF) using DNA-functionalized Au nanoparticle (AuNP). Upon activating the CRISPR-Cas12a complex by the target cfDNA and subsequent single-strand DNA (ssDNA) degradation between AuNP and fluorophore, MEF occurred with color changes from purple to red-purple. Using this system, breast cancer gene-1 (BRCA-1) can be detected with very high sensitivity in 30 min. This rapid and highly selective sensor can be applied to measure other nucleic acid biomarkers such as viral DNA in field-deployable and point-of-care testing (POCT) platform.

Normal-incidence type solution immersed silicon (SIS) biosensor for ultra-sensitive, label-free detection of cardiac troponin I.

Early diagnosis of acute myocardial infarction (AMI) significantly reduce the mortality rate and can be achieved via high-sensitive detection of AMI specific cardiac troponin I (cTnI) biomarker. Here, we present normal-incident type solution-immersed silicon (NI-SIS) ellipsometric biosensor, designed for ultra-high sensitive, high-throughput, label-free detection of the target protein. The NI-SIS sensors are equipped with a specially designed prism that maintains the angle of incidence close to the Brewster angle during operation, which significantly reduces SIS noise signals induced by the refractive index fluctuations of the surrounding medium, improves the signal-to-noise ratio, in-results lowers the detection limit. We applied NI-SIS biosensor for ultra-sensitive detection of cTnI biomarkers in human serum. The optimized sensor chip fabrication and detection operation procedures are proposed. The wide linear concentration ranges of fg/mL to ng/mL is achieved with the detection limit of 22.0 fg/mL of cTnI. The analytical correlation was assessed by linear regression analysis with the results of the Pathfast reference system. These impressive biosensing capabilities of NI-SIS technology have huge potentials for accurate detection of target species in different application areas, such as diagnosis, drug discovery, and food contaminations.

Biochemical-immunological hybrid biosensor based on two-dimensional chromatography for on-site sepsis diagnosis.

A hybrid-biosensor system that can simultaneously fulfill the immunoassay for protein markers (e.g., C-reactive protein (CRP) and procalcitonin (PCT)) and the enzyme assay for metabolic substances (e.g., lactate) in the same sepsis-based sample has been devised. Such a challenge was pursued through the installation of an enzyme-reaction zone on the signal pad of the typical immuno-strip for the rapid two-dimensional (2-D)-chromatography test. To minimize the mutual interference in the hybrid assays, a pre-determined membrane site was etched in a pattern and mounted with a biochemical-reaction pad, thereby allowing a loaded sample to enter and then stay in the pad for a colored-signal production over the course of an immunoassay. By employing such a constructed system, a serum sample was analyzed according to the vertical direction flowing along the strip, which supplied lactate to the biochemical-reaction zone and then protein markers to the immunological-binding area that was pre-coated with capture antibodies. Thereafter, the enzyme-signal tracers for the immunoassay and the substrate solution were sequentially furnished using a horizontal path for the tracing of the immune complexes that were formed with CRP or PCT. The color signal that was produced from each assay was detected at a pre-determined time and quantified on a smartphone-based detector. Under the optimal conditions, the dynamic ranges for the analytes covered the respective clinical ranges, and the total coefficient of variation was between 8.6% and 13.3%. The hybrid biosensor further showed a high correlation (R2 > 0.95) with the reference systems for the target markers.

Label-free detection of hepatitis B virus using solution immersed silicon sensors.

Highly sensitive solution immersed silicon (SIS) biosensors were developed for detection of hepatitis B virus (HBV) infection in the early stage. The ultrasensitivity for overlayer thickness at the nonreflecting condition for the p-polarized wave is the basis of SIS sensing technology. The change in thickness due to biomolecular interactions and change in refractive index of the surrounding buffer medium were assessed simultaneously using two separate ellipsometric parameters (Ψ and Δ), respectively, from a single sensing spot. A direct antigen-antibody affinity assay was used to detect and quantify hepatitis B surface antigen (HBsAg), which is the early stage biomarker for HBV infection. The detection limit of 10 pg/ml was achieved for HBsAg in the human blood serum, which is comparable with the results of enzyme-linked immunosorbent assay and other hybrid assays. The SIS sensor's response time was less than 10 min. The SIS sensors exhibit excellent stability and high signal-to-noise ratio, and are cost-effective, which makes them a suitable candidate for point-of-care applications.

Semi-continuous, real-time monitoring of protein biomarker using a recyclable surface plasmon resonance sensor.

Although label-free immunosensors based on, for example, surface plasmon resonance (SPR) provide advantages of real-time monitoring of the analyte concentration, its application to routine clinical analysis in a semi-continuous manner is problematic because of the high cost of the sensor chip. The sensor chip is in most cases regenerated by employing an acidic pH. However, this causes gradual deterioration of the activity of the capture antibody immobilized on the sensor surface. To use sensor chips repeatedly, we investigated a novel surface modification method that enables regeneration of the sensor surface under mild conditions. We introduced a monoclonal antibody (anti-CBP Ab) that detects the conformational change in calcium binding protein (CBP) upon Ca2+ binding (>1mM). To construct a regenerable SPR-based immunosensor, anti-CBP Ab was first immobilized on the sensor surface, and CBP conjugated to the capture antibody (specific for creatine kinase-MB isoform (CK-MB); CBP-CAb) then bound in the presence of Ca2+. A serum sample was mixed with the detection antibody to CK-MB, which generated an SPR signal proportional to the analyte concentration. After each analysis, the sensor surface was regenerated using medium (pH 7) without Ca2+, and then adding fresh CBP-CAb in the presence of Ca2+ for the subsequent analysis. Analysis of multiple samples using the same sensor was reproducible at a rate >98.7%. The dose-response curve was linear for 1.75-500.75ng/mL CK-MB, with an acceptable coefficient of variation of <8.8%. The performance of the immunosensor showed a strong correlation with that of the Pathfast reference system (R2>96%), and exhibited analytical stability for 1 month. To our knowledge, this is the first report of a renewal of a sensor surface with fresh antibody after each analysis, providing high consistency in the assay during a long-term use (e.g., a month at least).

Ultrasensitive, label-free detection of cardiac biomarkers with optical SIS sensor.

Acute myocardial infarction (MI) is the leading cause of high mortality and morbidity rate worldwide, early and accurate diagnosis can increase the chances of survival. In this work, we report a simple, ultrasensitive, label-free, and high-throughput solution immersed silicon (SIS) immunosensor based on non-reflection condition (NRC) for p-polarized wave for early diagnosis of MI. SIS sensor chips are just a thin dielectric polymer layer on the silicon surface, which can be functionalized for specific application. At NRC, SIS sensors are extremely sensitive to the growing thickness of a bio-layer on the sensor surface while independent of refractive index change of the surrounding medium. Therefore, SIS signal is free from thermal noise, unlike surface plasmon resonance based sensor. Also, there is no need of reference signal which facilitates fast and accurate interaction measurement. Here, SIS technology is applied to tackle two issues in MI diagnosis: high sensitivity with the direct assay and the ability to measure in human serum. Myoglobin, creatine kinase-MB, and cardiac troponin I (cTnI) proteins were used as the MI biomarkers. We were able to measure over a broad concentration range with the detection limit of 5 and 10pg/ml for cTnI in PBS and blood serum, respectively. The response time is about 5min. This novel technique is a suitable candidate for cost effective point-of-care application.

A Human Serum-Based Enzyme-Free Continuous Glucose Monitoring Technique Using a Needle-Type Bio-Layer Interference Sensor.

The incidence of diabetes is continually increasing, and by 2030, it is expected to have increased by 69% and 20% in underdeveloped and developed countries, respectively. Therefore, glucose sensors are likely to remain in high demand in medical device markets. For the current study, we developed a needle-type bio-layer interference (BLI) sensor that can continuously monitor glucose levels. Using dialysis procedures, we were able to obtain hypoglycemic samples from commercial human serum. These dialysis-derived samples, alongside samples of normal human serum were used to evaluate the utility of the sensor for the detection of the clinical interest range of glucose concentrations (70-200 mg/dL), revealing high system performance for a wide glycemic state range (45-500 mg/dL). Reversibility and reproducibility were also tested over a range of time spans. Combined with existing BLI system technology, this sensor holds great promise for use as a wearable online continuous glucose monitoring system for patients in a hospital setting.

Conformation-sensitive antibody-based point-of-care immunosensor for serum Ca(2+) using two-dimensional sequential binding reactions.

To assess the homeostasis of Ca(2+) metabolism, we have developed a rapid immunosensor for ionic calcium using a membrane chromatographic technique. As calcium-binding protein (CBP) is available for the recognition and undergone conformation change upon Ca(2+) binding, a monoclonal antibody sensitive to the altered structure of CBP has been employed. The sequential binding scheme was mathematically simulated and shown to match with the experimental results. At the initial stage, the rapid analytical system using lateral flow was constructed by immobilizing the antibody on the immuno-strip nitrocellulose membrane and labeling CBP with colloidal gold as a tracer. A major problem with this system in measuring ionic calcium levels was retarded migration of the gold tracer along the immuno-strip. It was conceivable that the divalent cation at a high concentration caused a change in the physical properties of the tracer, resulting in a non-specific interaction with the membrane surface. This problem was circumvented by first eluting a sample containing biotinylated CBP along the immuno-strip and then supplying the gold coupled to streptavidin across the signal generation pad of the strip. The color signal was then generated via biotin-SA linkage and measured using a smartphone-based detector developed in our laboratory. This two-dimensional chromatographic format completed the Ca(2+) analysis within 15min, the analytical performance covered the clinical dynamic range (0.25-2.5mM) and highly correlated with that of the reference system, i-STAT. These results inspired us to eventually investigate a dual-immunoassay system that measures simultaneously ionic calcium and parathyroid hormone, which regulates the ionic calcium level in serum. This will significantly simplify the current diagnostic protocols, which involve separate devices.

A High-Performance Fluorescence Immunoassay Based on the Relaxation of Quenching, Exemplified by Detection of Cardiac Troponin I.

The intramolecular fluorescence self-quenching phenomenon is a major drawback in developing high-performance fluorometric biosensors which use common fluorophores as signal generators. We propose two strategies involving liberation of the fluorescent molecules by means of enzymatic fragmentation of protein or dehybridization of double-stranded DNA. In the former, bovine serum albumin (BSA) was coupled with the fluorescent BODIPY dye (Red BSA), and then immobilized on a solid surface. When the insolubilized Red BSA was treated with proteinase K (10 units/mL) for 30 min, the fluorescent signal was significantly increased (3.5-fold) compared to the untreated control. In the second case, fluorophore-tagged DNA probes were linked to gold nanoparticles by hybridization with capture DNA strands densely immobilized on the surface. The quenched fluorescence signal was recovered (3.7-fold) by thermal dehybridization, which was induced with light of a specific wavelength (e.g., 530 nm) for less than 1 min. We next applied the Red BSA self-quenching relaxation technique employing enzymatic fragmentation to a high-performance immunoassay of cardiac troponin I (cTnI) in a microtiter plate format. The detection limit was 0.19 ng/mL cTnI, and the fluorescent signal was enhanced approximately 4.1-fold compared with the conventional method of direct measurement of the fluorescent signal from a non-fragmented fluorophore-labeled antibody.

A fluorescent immunosensor for high-sensitivity cardiac troponin I using a spatially-controlled polymeric, nano-scale tracer to prevent quenching.

For detection of high-sensitivity cardiac troponin I (hs-cTnI<0.01ng/mL), signal amplification was attained using a rapid immunosensor with a fluorescently-labeled, polymeric detection antibody. As fluorescent molecules tend to quench when they are less than 10nm apart, a synthetic scheme for the labeled antibody was devised to control the molecular distance and so minimize the quenching effect in a single conjugate unit. To this end, we first performed novel polymerization of fluorophore-coupled streptavidin (FL-SA) with biotinylated detection antibody (b-Ab) in a stepwise manner by adding FL-SA to b-Ab five times sequentially. Relative spatial positions of the fluorophore molecules in the polymer were then distally fixed using di-biotinylated oligonucleotides and passed through a 0.45µm filter to obtain a polymer of uniform size (i.e., ~400nm in diameter). We produced polymeric tracers using two different inexpensive fluorophores, Dylight 650 and Alexa 647, and applied it to the detection of hs-cTnI spiked in human serum using a two-dimensional chromatography-based immunosensor. The tracers showed a limit of detection of 0.002ng/mL for Dylight 650 and 0.007ng/mL for Alexa 647. The standard curves linearized via log-logit transformation exhibited regression lines with correlation coefficients (R(2))>0.97. The total coefficient of variation for the overall standard curve was 3.4±3.3% for the Dylight fluorophore and 5.9±1.5% for the Alexa dye. Such performances were comparable to those of the reference systems employing sophisticated technologies, Pathfast (Mitsubishi, Japan) and i-STAT (Abbott, US), with a strong correlation (R(2)>0.91) for the concentration range <100pg/mL.

Construction of an antimyoglobin single-chain variable fragment with rapid reaction kinetics.

Antibodies with rapid reaction kinetics (high association and dissociation rates), named reversible antibodies, are used to perform continuous monitoring of sensitive disease biomarkers. In cases of acute myocardial infarction (AMI), continuous monitoring and early diagnosis are important. Human myoglobin (Myo) is a useful biomarker for AMI during the early stage after the onset of symptoms. In this study, a single-chain variable fragment (scFv) specific to Myo was derived from an IgG antibody that has rapid reaction kinetics. Enzyme-linked immunosorbent assay revealed that recombinant scFv exhibited 3.8-fold reduced affinity compared with the parent IgG antibody based on the antibody concentration necessary for 50% of the maximum signal. The scFv retained the rapid reaction kinetic mode with average kon and koff of 2.63 × 10(5) M(-1) Sec(-1) and 3.25 × 10(-3) Sec(-1) , respectively, which were reduced to 10- and 2.3-fold compared with those of the parent antibody. The equilibrium constant for the association of the scFv (KA = 8.09 × 10(7) M(-1) ) was 4.6-fold lower than that of its parent IgG antibody. This scFv may be a starting point for further mutagenesis/kinetic and structural analyses providing valuable insight into the mechanism of reversible antibodies.

Chemiluminometric Immunosensor for High-Sensitivity Cardiac Troponin I Employing a Polymerized Enzyme Conjugate as a Tracer.

To detect high-sensitivity cardiac troponin I (hs-cTnI; <0.01 ng/mL) at points of care, we developed a rapid immunosensor by using horseradish peroxidase polymerized in 20 molecules on average (Poly-HRP) as a tracer conjugated with streptavidin (SA-Poly-HRP). As shown in the conventional system, enhanced sensitivity could be achieved by using a sequential binding scheme for the complex formation to contain the huge molecular tracer. We used a 2-dimensional chromatographic technology to carry out the sequential bindings in cross-flow directions. After the complex formation of antigen-antibody with analyte in a vertical direction, SA-Poly-HRP was horizontally supplied across the membrane strip for additional binding via a biotin-SA linkage. The HRP substrate was subsequently supplied along the same direction to produce a chemiluminometric signal, which was measured by a cooled charge-coupled device. Hs-cTnI analysis was completed in this format within 25 min, and the results showed a high correlation with those of the CentaurXP® reference system (R(2) > 0.99). The detection limit of the rapid immunosensor was 0.003 ± 0.001 ng/mL cTnI, corresponding to a 10-fold improvement compared to results using the plain enzyme tracer. This demonstrated the measurement of hs-cTnI in a much more cost-effective manner compared to the automated versions currently available.

An innate immune system-mimicking, real-time biosensing of infectious bacteria.

An animal cell-based biosensor was investigated to monitor bacterial contamination in an unattended manner by mimicking the innate immune response. The cells (RAW 264.7 cell line) were first attached onto the solid surfaces of a 96-well microtiter plate and co-incubated in the culture medium with a sample that might contain bacterial contaminants. As Toll-like receptors were present on the cell membrane surfaces, they acted as a sentinel by binding to pathogen-associated molecular patterns (PAMPs) of any contaminant. Such biological recognition initiates signal transmission along various pathways to produce different proinflammatory mediators, one of which, tumor necrosis factor-α (TNF-α) was measured using an immunosensor. To demonstrate automated bacterium monitoring, a capture antibody specific for TNF-α was immobilized on an optical fiber sensor tip and then used to measure complex formation in a label-free sensor system (e.g., Octet Red). The sensor response time depended significantly on the degree of agitation of the culture medium, controlling the biological recognition and further autocrine/paracrine signaling by cytokines. The response, particularly under non-agitated conditions, was also influenced by the medium volume, revealing a local gradient change of the cytokine concentration and also acidity, caused by bacterial growth near the bottom surfaces. A biosensor system retaining 50 µL medium and not employing agitation could be used for the early detection of bacterial contamination. This novel biosensing model was applied to the real-time monitoring of different bacteria, Shigella sonnei, Staphylococcus aureus, and Listeria monocytogenes. They (<100 CFU mL(-1)) could be detected automatically within the working time. Such analysis was carried out without any manual handling regardless of the bacterial species, suggesting the concept of non-targeted bacterial real-time monitoring. This technique was further applied to real sample testing (e.g., with milk) to exemplify, for example, the food quality control process without using any additional sample pretreatment such as magnetic concentration.

Crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae.

Nitroreductases are flavoenzymes that catalyze nitrocompounds and are widely utilized in industrial applications due to their detoxification potential and activation of biomedicinal prodrugs. Type I nitroreductases are classified into subgroups depending on the use of NADPH or NADH as the electron donor. Here, we report the crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae, one of the uncharacterized subgroups of proteins, to reveal its minimal architecture previously observed in bacterial nitroreductases such as CinD and YdjA. The structure lacks protruding helical motifs that form part of the cofactor and substrate binding site, resulting in an open and wide active site geometry. Arg82 is uniquely conserved in proximity to the substrate binding site in Frm2 homologues and plays a crucial role in the activity of the active site. Frm2 primarily utilizes NADH to reduce 4-NQO. Because missing helical elements are involved in the direct binding to the NAD(P)H in group A or group B in Type I family, Frm2 and its homologues may represent a distinctive subgroup with an altered binding mode for the reducing compound. This result provides a structural basis for the rational design of novel prodrugs with the ability to reduce nitrogen-containing hazardous molecules.

Purification and characterization of Fab fragments with rapid reaction kinetics against myoglobin.

Myoglobin is an early biomarker for acute myocardial infarction. Recently, we isolated the antibody IgG-Myo2-7ds, which exhibits unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid dissociation kinetics are thought to be premature IgG forms that are produced during the early stage of in vivo immunization. In the present study, we identified the epitope region of the IgG-Myo2-7ds antibody to be the C-terminal region of myoglobin, which corresponds to 144-154 aa. The Fab fragment was directly purified by papain cleavage and protein G affinity chromatography and demonstrated kinetics of an association constant of 4.02 × 10(4) M(-1) s(-1) and a dissociation constant of 2.28 × 10(-2) s(-1), which retained the unique reaction kinetics of intact IgG-Myo2-7ds antibodies. Because a rapid dissociation antibody can be utilized for antibody recycling, the results from this study would provide a platform for the development of antibody engineering in potential diagnostic areas such as a continuous monitoring system for heart disease.

Refolded scFv antibody fragment against myoglobin shows rapid reaction kinetics.

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the V(H)-V(L) sequence with a (Gly4Ser)3 linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10⁻4 M⁻¹·s⁻¹ and 6.29 × 10⁻³ s⁻¹, respectively, with an affinity value exceeding 107 M⁻¹ (k(on)/k(off)), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor.

A simple and low-cost biofilm quantification method using LED and CMOS image sensor.

A novel biofilm detection platform, which consists of a cost-effective red, green, and blue light-emitting diode (RGB LED) as a light source and a lens-free CMOS image sensor as a detector, is designed. This system can measure the diffraction patterns of cells from their shadow images, and gather light absorbance information according to the concentration of biofilms through a simple image processing procedure. Compared to a bulky and expensive commercial spectrophotometer, this platform can provide accurate and reproducible biofilm concentration detection and is simple, compact, and inexpensive. Biofilms originating from various bacterial strains, including Pseudomonas aeruginosa (P. aeruginosa), were tested to demonstrate the efficacy of this new biofilm detection approach. The results were compared with the results obtained from a commercial spectrophotometer. To utilize a cost-effective light source (i.e., an LED) for biofilm detection, the illumination conditions were optimized. For accurate and reproducible biofilm detection, a simple, custom-coded image processing algorithm was developed and applied to a five-megapixel CMOS image sensor, which is a cost-effective detector. The concentration of biofilms formed by P. aeruginosa was detected and quantified by varying the indole concentration, and the results were compared with the results obtained from a commercial spectrophotometer. The correlation value of the results from those two systems was 0.981 (N = 9, P < 0.01) and the coefficients of variation (CVs) were approximately threefold lower at the CMOS image-sensor platform.

Pseudomonas aeruginosa-induced IL-1ß production is inhibited by Sophora flavescens via the NF-κB/inflammasome pathways.

The proinflammatory cytokine interleukin-1ß plays an important role in protecting the host against airway infection; however, it can also trigger a massive influx of neutrophils into the airways, causing tissue damage. Anti-inflammatory treatments are particularly in demand for patients suffering from chronic inflammatory diseases. Sophora flavescens is a traditional herbal medicine used to reduce inflammation, but no study has examined its ability to block IL-1ß production. Here, we show that S. flavescens reduced the Pseudomonas aeruginosa-induced expression of IL-1ß by lung epithelial cells and macrophages. S. flavescens was also effective at reducing IL-1ß production induced by either Staphylococcus aureus or phorbol 12-myristate 13-acetate, indicating that the effect is generalizable to diverse inflammatory stimuli. In addition, S. flavescens blocked the phosphorylation of IKKα/ß, key upstream kinases involved in the degradation of IκBα, and the cleavage of caspase-1, a key component of the inflammasome. Thus, this study demonstrates that S. flavescens exerts its anti-inflammatory effects by blocking P. aeruginosa-mediated NF-κB/inflammasome activation and the subsequent production of IL-1ß.