ABSTRACT
Spaceborne radar remote sensing of the earth system is essential to study natural and man-made changes in the ecosystem, water and energy cycles, weather and air quality, sea level, and surface dynamics. A major challenge with current approaches is the lack of broad spectrum tunability due to narrow band microwave electronics, that limit systems to specific science variable retrievals. This results in a significant limitation in studying dynamic coupled earth system processes such as surface and subsurface hydrology from a single compact instrument, where co-located broad spectrum radar remote sensing is needed to sense multiple variables simultaneously or over a short duration. Rydberg atomic sensors are highly sensitive broad-spectrum quantum detectors that can be dynamically tuned to cover micro-to-millimeter waves with no requirement for RF band-specific electronics. Rydberg atomic sensors can use existing transmitted signals such as from navigation and communication satellites to enable remote sensing. We demonstrate remote sensing of soil moisture, an important earth system variable, via ground-based radar reflectometry with Rydberg atomic systems. To do this, we sensitize the atoms to XM satellite radio signals and use signal correlations to demonstrate use of these satellite signals for remote sensing of soil moisture.
ABSTRACT
A defect-passivated photosensor based on cesium lead bromide (CsPbBr3) perovskite quantum dots (QD) was fabricated using parylene films, and the photosensor was applied for the microbial detection. The CsPbBr3 perovskite QDs were synthesized to be homogeneous in size under thermodynamic control, and the perovskite QD-based photosensor was fabricated using MoS2 flakes as the electron transfer layer. In this work, a parylene film with functional groups was deposited on a photosensor for physical protection (waterproof) and defect (halide vacancy) passivation of the perovskite QD. As the first effect of the parylene film, the physical protection of the perovskite QD from water was estimated by comparing the photosensor performance after incubation in water. As the second effect of the parylene, the interaction between the functional groups of the parylene film and the halide vacancies of the perovskite QDs was investigated through the bandgap, crystal structure, and trap-state density analysis. Additionally, density functional theory analysis on Mulliken charges, lattice parameters, and Gibbs free energy demonstrated the effect of the defect passivation by parylene films. Finally, the parylene-passivated QD-based photosensor was applied to the detection of two kinds of food-poisoning and gastroduodenal disease bacteria (Listeria monocytogenes and Helicobacter pylori).
ABSTRACT
The detection of severe acute respiratory syndrome coronavirus (SARS-CoV-1) was demonstrated using screened Fv-antibodies for SPR biosensor and impedance spectrometry. The Fv-antibody library was first prepared on the outer membrane of E. coli using autodisplay technology and the Fv-variants (clones) with a specific affinity toward the SARS-CoV-1 spike protein (SP) were screened using magnetic beads immobilized with the SP. Upon screening the Fv-antibody library, two target Fv-variants (clones) with a specific binding affinity toward the SARS-CoV-1 SP were determined and the Fv-antibodies on two clones were named "Anti-SP1" (with CDR3 amino acid sequence: 1GRTTG5NDRPD11Y) and "Anti-SP2" (with CDR3 amino acid sequence: 1CLRQA5GTADD11V). The binding affinities of the two screened Fv-variants (clones) were analyzed using flow cytometry and the binding constants (KD) were estimated to be 80.5 ± 3.6 nM for Anti-SP1 and 45.6 ± 8.9 nM for Anti-SP2 (n = 3). In addition, the Fv-antibody including three CDR regions (CDR1, CDR2, and CDR3) and frame regions (FRs) between the CDR regions was expressed as a fusion protein (Mw. 40.6 kDa) with a green fluorescent protein (GFP) and the KD values of the expressed Fv-antibodies toward the SP estimated to be 15.3 ± 1.5 nM for Anti-SP1 (n = 3) and 16.3 ± 1.7 nM for Anti-SP2 (n = 3). Finally, the expressed Fv-antibodies screened against SARS-CoV-1 SP (Anti-SP1 and Anti-SP2) were applied for the detection of SARS-CoV-1. Consequently, the detection of SARS-CoV-1 was demonstrated to be feasible using the SPR biosensor and impedance spectrometry utilizing the immobilized Fv-antibodies against the SARS-CoV-1 SP.
Subject(s)
Biosensing Techniques , Severe acute respiratory syndrome-related coronavirus , Viral Envelope Proteins/chemistry , Membrane Glycoproteins , Escherichia coli , Antibodies , Antibodies, ViralABSTRACT
An electrochemical immunoassay based on the redox cycling method was presented using vertically paired electrodes (VPEs), which were fabricated using poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) as an electrode material and parylene-C as a dielectric layer. For the application to immunoassays, different electrochemical properties of PEDOT:PSS were analyzed for the redox reaction of 3,3',5,5'-tetramethylbenzidine (TMB, the chromogenic substrate for enzyme-immunoassays) at different pH conditions, including the conductivity (σ), electron transfer rate constant (kapp), and double-layer capacitance (Cdl). The influencing factors on the sensitivity of redox cycling based on VPE based on PEDOT:PSS were analyzed for the redox reaction of TMB, such as the electrode gap and number of electrode pairs. Computer simulation was also performed for the redox cycling results based on VPEs, which had limitations in fabrication, such as VPEs with an electrode gap of less than 100 nm and more than five electrode pairs. Finally, the redox cycling based on VPE was applied to the medical diagnosis of human hepatitis-C virus (hHCV) using a commercial ELISA kit. The sensitivity of the redox cycling method for the medical diagnosis of hHCV was compared with conventional assay methods, such as TMB-based chromogenic detection, luminol-based chemiluminescence assay, and a rapid test kit (lateral flow immunoassay).
Subject(s)
Computer Simulation , Humans , Electrodes , Oxidation-Reduction , Immunoassay , Immunoenzyme TechniquesABSTRACT
PURPOSE: Programmed cell death protein 1 (PD-1) and ligand programmed death ligand 1 (PD-L1) are important immune-suppressive regulators in the tumor microenvironment. A vaccine-induced immune effect on tumor cells is blunted by the immunosuppressive tumor microenvironment. Therefore, we hypothesized that a dendritic cell (DC) vaccine combined with anti-PD-1 (αPD-1) antibodies could elicit a synergistic anti-tumor immunity in bladder cancer. MATERIALS AND METHODS: We produced a model of subcutaneous transplantation in C3H/HeJ mice by transplanting murine MBT-2 bladder cancer cells. DCs were isolated from normal C3H/HeJ mice, followed by stimulation against MBT-2 lysate before injection. Two weeks later of MBT-2 inoculation, αPD-1 and stimulated DCs were injected two times at one-week interval intraperitoneally and intravenously, respectively. Tumor-infiltrating immune cells and splenocytes were analyzed using flow cytometry. T-cell-mediated anti-tumor responses were measured by interferon (IFN)-γ ELISPOT and lactate dehydrogenase assays. RESULTS: The mice treated with DC+αPD-1 showed a significant decrease in tumor volume compared to the DC-treated mice and IgG-treated group. Survival of the DC+αPD-1-treated group was improved compared with that of the IgG-treated mice. IFN-γ secretion from splenocytes against tumor cells was significantly increased in the DC+αPD-1 group compared with that of αPD-1-treated mice. The frequency of CD8+ and CD4+ T-cells in spleens was statistically increased in the DC+αPD-1-treated mice compared to those receiving monotherapy (DC- or αPD-1-treated group). CONCLUSIONS: Our results support the hypothesis that the combination therapy of a DC vaccine and αPD-1 antibodies could enhance the anti-tumor immune response against bladder cancer.
Subject(s)
Cancer Vaccines , Immunoglobulin G , Urinary Bladder Neoplasms , Animals , Mice , Cell Line, Tumor , Immunoglobulin G/therapeutic use , Mice, Inbred C3H , Tumor Microenvironment , Urinary Bladder Neoplasms/therapyABSTRACT
A one-step immunoassay was developed for five types of food-poisoning-related bacteria using a switching peptide and antibodies isolated from unimmunized horse serum. The one-step immunoassay involves mixing samples and reagents in a homogeneous solution without any washing steps. In this work, a one-step immunoassay configuration was developed using isolated antibodies labelled with an organic fluorescence quencher and a switching-peptide labelled with a fluorescent dye. The fluorescence-labelled switching-peptide was bound to the antigen-binding site of the isolated antibodies before binding to the bacteria (no fluorescence signal), and the switching-peptide dissociated from the antibodies as soon as they bound to the bacteria (fluorescence signal turns on). By quantifying the generated fluorescence signal, the one-step immunoassay presented here allows microbial detection without any washing step.
Subject(s)
Antibodies , Fluorescence Resonance Energy Transfer , Immunoassay , Antibodies/chemistry , Peptides/chemistry , BacteriaABSTRACT
In this study, parylene-C films from plasma deposition as well as thermal deposition were pyrolyzed to prepare a carbon electrode for application in electrochemical immunoassays. Plasma deposition could prepare parylene-C in a faster deposition rate and more precise control over the thickness in comparison with the conventional thermal deposition. To analyze the influence of the deposition method, the crystal and electronic structures of the pyrolyzed parylene-C films obtained via both deposition methods were compared using Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. For application as a carbon electrode in immunoassays, the electrochemical properties of the pyrolyzed carbon films from two both deposition methods were analyzed, including the double layer capacitance (2.10 µF cm-2 for plasma deposition and 2.20 µF cm-2 for thermal deposition), the apparent electron transfer rate (approximately 1.1 × 10-3 cm s-1 for both methods), and the electrochemical window (approximately -1.0 â¼ 2.1 V for both methods). Finally, the applicability of the pyrolyzed carbon electrode from parylene-C was demonstrated for the diagnosis of human hepatitis-C using various amperometric methods, such as cyclic voltammetry, chronoamperometry, square-wave voltammetry and differential pulse voltammetry.
Subject(s)
Carbon , Pyrolysis , Carbon/chemistry , Electrodes , Humans , Immunoassay , Polymers , XylenesABSTRACT
Switching peptides were designed to bind reversibly to the binding pocket of antibodies (IgG) by interacting with frame regions (FRs). These peptides can be quantitatively released when antigens bind to IgG. As FRs have conserved amino acid sequences, switching peptides can be used as antibodies for different antigens and different source animals. In this study, an electrochemical one-step immunoassay was conducted using switching peptides labeled with ferrocene for the quantitative measurement of analytes. For the effective amperometry of the switching peptides labeled with ferrocene, a pyrolyzed carbon electrode was prepared by pyrolysis of the parylene-C film. The feasibility of the pyrolyzed carbon electrode for the electrochemical one-step immunoassay was determined by analyzing its electrochemical properties, such as its low double-layer capacitance (Cdl), high electron transfer rate (kapp), and wide electrochemical window. In addition, the factors influencing the amperometry of switching peptides labeled with ferrocene were analyzed according to the hydrodynamic radius, the number of intrahydrogen bonds, dipole moments, and diffusion coefficients. Finally, the applicability of the electrochemical one-step immunoassay for the medical diagnosis of the human hepatitis B surface antigen (hHBsAg) was assessed.
Subject(s)
Carbon , Peptides , Animals , Carbon/chemistry , Electrodes , Immunoassay , Immunoglobulin GABSTRACT
Vertically paired electrodes (VPEs) with multiple electrode pairs were developed for the enhancement of capacitive measurements by optimizing the electrode gap and number of electrode pairs. The electrode was fabricated using a conductive polymer layer of PEDOT:PSS instead of Ag and Pt metal electrodes to increase the VPE fabrication yield because the PEDOT:PSS layer could be effectively etched using a reactive dry etching process. In this study, sensitivity enhancement was realized by decreasing the electrode gap and increasing the number of VPE electrode pairs. Such an increase in sensitivity according to the electrode gap and the number of electrode pairs was estimated using a model analyte for an immunoassay. Additionally, a computer simulation was performed using VPEs with different electrode gaps and numbers of VPE electrode pairs. Finally, VPEs with multiple electrode pairs were applied for SARS-CoV-2 nucleoprotein (NP) detection. The capacitive biosensor based on the VPE with immobilized anti-SARS-CoV-2 NP was applied for the specific detection of SARS-CoV-2 in viral cultures. Using viral cultures of SARS-CoV-2, SARS-CoV, MERS-CoV, and CoV-strain 229E, the limit of detection (LOD) was estimated to satisfy the cutoff value (dilution factor of 1/800) for the medical diagnosis of COVID-19, and the assay results from the capacitive biosensor were compared with commercial rapid kit based on a lateral flow immunoassay.
Subject(s)
Biosensing Techniques , COVID-19 , Biosensing Techniques/methods , Computer Simulation , Electrodes , Humans , SARS-CoV-2 , Sensitivity and SpecificityABSTRACT
OBJECTIVES: The present study translated the Vocal Tract Discomfort Scale (VTDS) into Korean (K-VTDS) and evaluated its reliability and validity. STUDY DESIGN: This was a prospective study. METHODS: The VTDS was first translated into Korean and validated. One hundred and fifty-nine patients with voice disorders were divided into three different diagnostic groups (functional, structural, and neurologic voice disorder) and 131 vocally healthy adults were also included. All participants completed the K-VTDS and the Korean version of the Voice Handicap Index (K-VHI) and Korean versions of the Voice-Related Quality of Life (K-VRQOL). The internal consistency of the K-VTDS was analyzed through Cronbach's α coefficient. The VTDS score differences related to the diagnostic groups were assessed with t test and analysis of variance. We assessed the correlation between the K-VTDS, the K-VHI, and the K-VRQOL using Pearson's correlation analysis. RESULTS: High internal consistency and the test-retest reliability of the K-VTDS were found. The voice disorder group had significantly higher K-VTDS scores for the subscales and total scores than those in the healthy group (P < 0.001). The K-VTDS scores for the subscale of frequency and total scores were highest in the functional voice disorder group. We found a significant difference in frequency and total score of the K-VTDS between the functional voice disorder group and the structural voice disorder group (P < 0.05). We observed a strong positive correlation among the scores for the subscales of frequency and severity, and total scores in the K-VTDS. The K-VTDS showed moderate correlation with the K-VHI and the K-VRQOL. CONCLUSION: The K-VTDS is a reliable and valid instrument for voice assessment for voice disorders in Korean-speaking patients.
Subject(s)
Dysphonia , Voice Disorders , Adult , Disability Evaluation , Humans , Prospective Studies , Quality of Life , Reproducibility of Results , Republic of Korea , Severity of Illness Index , Surveys and Questionnaires , Voice Disorders/diagnosis , Voice QualityABSTRACT
Vascular occlusive disease is a chronic disease with significant morbidity and mortality. Although a variety of therapies and medications have been developed, the likelihood of disease re-emergence is high and this can be life-threatening. Based on a previous screening experiment related to vascular obstructive diseases using 34 types of essential oils, cold-pressed oil (CpO) from Citrus aurantifolia (lime) has been demonstrated to have the best effect for the inhibition of vascular smooth muscle cells (VSMCs) proliferation. The aim of the present study was to evaluate the effect of lime CpO on the pathological changes of VSMCs. To determine this, the effect of lime CpO on VSMC proliferation, a major cause of vascular disease, was investigated. To determine the safe concentration interval for toxicity of CpO during VSMC culture, a dilution of 1x10-5 was determined using Cell Counting Kit-8 assay, which was confirmed to be non-toxic using a lactate dehydrogenase assay. To examine the effect of lime CpO in cellular signaling pathways, changes in phosphorylation of both the PI3K/AKT/mTOR and extracellular signal-regulated MEK/ERK signaling pathways with serum were investigated. Furthermore, lime CpO with FBS also significantly decreased the expression levels of the cell cycle regulators cyclin D1 and proliferating cell nuclear antigen. Additionally, lime CpO with FBS significantly inhibited the sprouting of VSMCs in an ex vivo culture system. These results suggested that lime CpO inhibited the abnormal proliferation of VSMCs and can be developed as a nature-based therapeutic agent for obstructive vascular disease.
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Neurodegenerative disease is a brain disorder caused by the loss of structure and function of neurons that lowers the quality of human life. Apart from the limited potential for endogenous regeneration, stem cell-based therapies hold considerable promise for maintaining homeostatic tissue regeneration and enhancing plasticity. Despite many studies, there remains insufficient evidence for stem cell tracing and its correlation with endogenous neural cells in brain tissue with three-dimensional structures. Recent advancements in tissue optical clearing techniques have been developed to overcome the existing shortcomings of cross-sectional tissue analysis in thick and complex tissues. This review focuses on recent progress of stem cell treatments to improve neurodegenerative disease, and introduces tissue optical clearing techniques that can implement a three-dimensional image as a proof of concept. This review provides a more comprehensive understanding of stem cell tracing that will play an important role in evaluating therapeutic efficacy and cellular interrelationship for regeneration in neurodegenerative diseases.
ABSTRACT
Clinical nurses have training needs related to the strategies for infection spread prevention. This study aimed to investigate the levels of importance of and performance in the various areas of care for communicable respiratory infections among clinical nurses and to determine the priority of educational needs. Hospitals in which a baseline survey could be conducted were considered, and nurses working in six hospitals that were designated as COVID-19 care centers in Korea were enrolled. The training needs for the care of patients with communicable respiratory infections were analyzed using Borich's needs equation, and the locus for focus model. Among participants with prior COVID-19 patient care experience, according to Borich's equation, the need score was the highest for "initial response to communicable respiratory infection", followed by "management of aerosol-generating procedures in patients with communicable respiratory infection" and "reporting of patients with communicable respiratory infection and death of patient". An item rated highly in both Borich's equation and the locus for focus model among the participants with prior experience was "initial response to communicable respiratory infection". Among participants without prior relevant experience, according to Borich's equation, the need score was highest for "management of aerosol-generating procedures in patients with a communicable respiratory infection", followed by "initial response to communicable respiratory infection" and "reporting of patients with communicable respiratory infection and death of patient". None of the items were rated high in both Borich's equation and the locus for focus model among participants without relevant prior experience.
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The acute demise of stem cells following transplantation significantly compromises the efficacy of stem cell-based cell therapeutics for infarcted hearts. As the stem cells transplanted into the damaged heart are readily exposed to the hostile environment, it can be assumed that the acute death of the transplanted stem cells is also inflicted by the same environmental cues that caused massive death of the host cardiac cells. Pyroptosis, a highly inflammatory form of programmed cell death, has been added to the list of important cell death mechanisms in the damaged heart. However, unlike the well-established cell death mechanisms such as necrosis or apoptosis, the exact role and significance of pyroptosis in the acute death of transplanted stem cells have not been explored in depth. In the present study, we found that M1 macrophages mediate the pyroptosis in the ischemia/reperfusion (I/R) injured hearts and identified miRNA-762 as an important regulator of interleukin 1ß production and subsequent pyroptosis. Delivery of exogenous miRNA-762 prior to transplantation significantly increased the post-transplant survival of stem cells and also significantly ameliorated cardiac fibrosis and heart functions following I/R injury. Our data strongly suggest that suppressing pyroptosis can be an effective adjuvant strategy to enhance the efficacy of stem cell-based therapeutics for diseased hearts.
Subject(s)
MicroRNAs , Myocardial Reperfusion Injury , Pyroptosis , Stem Cell Transplantation , Stem Cells , Animals , Humans , Macrophages/metabolism , Macrophages/pathology , Male , Mice , MicroRNAs/genetics , MicroRNAs/pharmacology , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/therapy , Pyroptosis/drug effects , Pyroptosis/genetics , RAW 264.7 Cells , Rats , Rats, Sprague-Dawley , Stem Cells/metabolism , Stem Cells/pathologyABSTRACT
Chemiluminescence immunoassays have been widely employed for diagnosing various diseases. However, because of the extremely low intensity chemiluminescence signals, highly sensitive transducers, such as photomultiplier tubes and image sensors with cooling devices, are required to overcome this drawback. In this study, a hypersensitive photosensor was developed based on cesium lead bromide (CsPbBr3) perovskite quantum dots (QDs) with sufficient high sensitivity for chemiluminescence immunoassays. First, CsPbBr3 QDs with a highly uniform size, that is, 5 nm, were synthesized under thermodynamic control to achieve a high size confinement effect. For the fabrication of the photosensor, MoS2 nanoflakes were used as an electron transfer layer and heat-treated at an optimum temperature. Additionally, a parylene-C film was used as a passivation layer to improve the physical stability and sensitivity of the photosensor. In particular, the trap states on the CsPbBr3 QDs were reduced by the passivation layer, and the sensitivity was increased. Finally, a photosensor based on CsPbBr3 QDs was employed in chemiluminescence immunoassays for the detection of human hepatitis B surface antigen, human immunodeficiency virus antibody, and alpha-fetoprotein (AFP, a cancer biomarker). When compared with the conventionally used equipment, the photosensor was determined to be feasible for application in chemiluminescence immunoassays.
Subject(s)
Calcium Compounds/chemistry , Immunoassay/methods , Lead/chemistry , Luminescent Measurements/methods , Oxides/chemistry , Quantum Dots/chemistry , Titanium/chemistry , Cesium/chemistry , HIV Antibodies/analysis , Hepatitis B Surface Antigens/analysis , Humans , Polymers/chemistry , Xylenes/chemistryABSTRACT
Herein, we present switching-peptides for a one-step immunoassay, without the need for additional antibody treatment or washing steps to detect antigen-antibody interactions. Fluorescently labeled switching-peptides were dissociated from the immobilized antibody soon after the antigens were bound to the binding pockets. In this study, four different parts of the antibody (IgG) frame regions were chemically synthesized, and these peptides were bound to immobilized antibodies as switching-peptides. We presented the design principle of switching-peptides and used Pymol software, based on the changes in thermodynamic parameters, to study the interaction between antibodies and switching-peptides. The binding properties of switching-peptides were analyzed based on Förster resonance energy transfer between switching-peptides as well as between switching-peptides and antibodies (IgGs) isolated from different animals. The binding constants of the four switching-peptides to antibodies were estimated to be in the range of 1.48-3.29 µM. Finally, the feasibility of using switching-peptides for the quantitative one-step immunoassay was demonstrated by human hepatitis B surface antigen (hHBsAg) detection and statistical comparison of the assay results with those of conventional ELISA. The limit of detection for HBsAg was determined to be 56 ng/mL, and the dynamic range was estimated to be 136 ng/mL-33 µg/mL. These results demonstrate the feasibility of the one-step immunoassay for HBsAg.
Subject(s)
Biosensing Techniques , Hepatitis B , Immunoassay , Peptides , Animals , Hepatitis B/diagnosis , Hepatitis B Surface Antigens , Humans , Peptides/analysisABSTRACT
In this work, we present a novel microbial biosensor for Salmonella based on impedance spectrometry by using isolated antibodies against a specific bacterial strain from human serum. Anti-Salmonella (or BL21(DE3)) antibodies were isolated from human serum using S. enteritidis (or BL21(DE3)) and the mutant strain ClearColi. After the purification steps, the purification yield of the antibodies was calculated to be 0.2 %. From the FACS analysis, the isolated anti-Salmonella antibodies were estimated to have more than 6-fold higher binding affinity for S. enteritidis compared to antibodies against other kinds of Gram-negative bacterial strains, including HB101, ClearColi, JM110, DH5α, and BL21(DE3). Finally, the anti-Salmonella antibodies isolated herein were used for bacterial detection using electrochemical biosensors based on impedance spectrometry and the Rct value of the antibodies was estimated for S. enteritidis from the Nyquist plot. The limit of detection of the isolated anti-Salmonella antibodies was estimated to be 1.0 × 103 cells/mL for S. enteritidis and 1.0 × 106 cells/mL for BL21(DE3), respectively.
Subject(s)
Antibodies, Bacterial , Biosensing Techniques , Humans , Salmonella enteritidisABSTRACT
In this work phospholipid quantification was carried out using an enzymatic assay based on cyclic voltammetry of the condensation product of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS) and 4-aminoantipyrine (4-AP) with a graphite electrode. For the optimization of electrochemical measurement for the product, electrochemical properties such as the electrochemical window, double layer capacitance (Cdl) and electron transfer rate (kapp) were analyzed for a graphite-electrode and Au-electrode. The phospholipid enzymatic assay based the on electrochemical measurement using the graphite electrode was applied to the diagnosis of sepsis for sera from healthy volunteers (nâ¯=â¯16), patients with systemic inflammatory response syndrome (SIRS, nâ¯=â¯16) and severe sepsis patients (nâ¯=â¯24). Finally, the phospholipid quantification results from the electrochemical measurement were statistically compared with the conventional method based on optical density measurement.
Subject(s)
Graphite , Sepsis , Electrochemical Techniques , Electrodes , Enzyme Assays , Humans , Phospholipids , Sepsis/diagnosisABSTRACT
As the shelf life of platelets collected from donated blood is very short, approximately 5 days, the determination of bacterial contamination in platelets has become necessary. In this study, rapid analysis of Gram-positive and Gram-negative bacterial contamination in platelet samples was presented without pre-enrichment using pig serum-derived antibodies against the outer membrane proteins (OMP) of Gram-negative bacteria and antibodies against lipoteichoic acid (LTA) on the surface of Gram-positive bacteria. The anti-OMP antibodies against Gram-negative bacteria were isolated using sequential incubation with (1) the modified Gram-negative bacteria ClearColi, which lacks lipopolysaccharide (LPS) on the outer membrane, and (2) the Gram-positive bacteriaBacillus subtilis to filter away nonspecifically bound proteins from ClearColi. The anti-lipoteichoic acid (LTA) antibodies against Gram-positive bacteria were isolated using sequential incubation with (1) the Gram-positive bacteriaB. subtilis and (2) the Gram-negative bacteria Escherichia coli BL21 to filter away nonspecifically bound proteins fromB. subtilis. The feasibility of using the antibodies isolated from pig serum against Gram-negative and Gram-positive bacteria was demonstrated using flow cytometry. Finally, detection of the contamination of platelets with Gram-negative and Gram-positive bacteria using the impedance immunosensor based on these isolated antibodies was successfully demonstrated.
Subject(s)
Biosensing Techniques , Animals , Antibodies/metabolism , Blood Platelets , Escherichia coli , Gram-Negative Bacteria/metabolism , Gram-Positive Bacteria/metabolism , Immunoassay , Membrane Proteins/metabolism , SwineABSTRACT
Previous studies have reported that Hedyotis diffusa Willdenow extract shows various biological activities on cerebropathia, such as neuroprotection and short-term memory enhancement. However, there has been a lack of studies on the inhibitory activity on neurodegenerative diseases such as Alzheimer's disease (AD) through enzyme assays of H. diffusa. Therefore, H. diffusa extract and fractions were evaluated for their inhibitory effects through assays of enzymes related to AD, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), and on the formation of advanced glycation end-product (AGE). In this study, ten bioactive compounds, including nine iridoid glycosides 1-9 and one flavonol glycoside 10, were isolated from the ethyl acetate and n-butanol fractions of H. diffusa using a bioassay-guided approach. Compound 10 was the strongest inhibitor of cholinesterase, BACE1, and the formation of AGEs of all isolated compounds, while compound 5 had the lowest inhibitory activity. Compounds 3, 6, and 9 exhibited better inhibitory activity than other compounds on AChE, and two pairs of diastereomeric iridoid glycoside structures (compounds 4, 8, and 6, 7) showed higher inhibitory activity than others on BChE. In the BACE1 inhibitory assay, compounds 1-3 were good inhibitors, and compound 10 showed higher inhibitory activity than quercetin, the positive control. Moreover, compounds 1 and 3 were stronger inhibitors of the formation of AGE than aminoguanidine (AMG), the positive control. In conclusion, this study is significant since it demonstrated that the potential inhibitory activity of H. diffusa on enzymes related to AD and showed the potential use for further study as a natural medicine for AD treatment on the basis of the bioactive components isolated from H. diffusa.