Bioassay System Research Articles

Development of a cardiotoxicity evaluation system using a Heart-on-a-Chip Microdevice with aligned fiber device

Abstract Background and Purpose Cardiovascular cells differentiated from human induced pluripotent stem (iPS) cells, such as cardiomyocytes, are expected to be useful for evaluating cardiac pharmacology and toxicity in humans. In recent years, myocardial damage caused by anticancer drugs has begun to attract attention. Being able to evaluate human cardiotoxicity early in the development of novel drugs like anticancer agents confers advantages in terms of cost and development speed. The aim of this study was to develop a highly sensitive bioassay system that can evaluate the physiological function of the human heart, and to validate the efficacy of the system toward detection of cardiotoxicity of drugs. Methods We differentiated human iPS cells into cardiovascular cells (cardiomyocytes, endothelial cells and mural cells) and applied dynamic culture training to generate vascularized microtissues (VCM) with vascular network structures. By integrating this VCM with a microfluidic chip containing microchannels, we developed a heart-on-a-chip microdevice (HMD) specialized for evaluating cardiac function (Figure 1). Furthermore, through a collaborative study with the Stem Cell & Device Laboratory (SCAD), application of their aligned fiber device improved throughput and stability, allowing development of a SCAD-HMD capable of evaluating longer-term cardiotoxicity. Results SCAD-HMD can quantify physiological parameters of VCM such as ejection volume and force based on displacement of particles within the microchannels. Our results showed that SCAD-HMD not only exhibited pharmacological responses to representative circulatory stimulants, but also reproduced clinical events occurring in actual practice, demonstrating that the anticancer drug trastuzumab, commonly used for breast cancer treatment, causes myocardial disfunction when combined with doxorubicin. Conclusions This study demonstrated that SCAD-HMD can stably evaluate the toxicity of drugs on the human heart. In the future, establishing SCAD-HMD using iPS cells derived from individual patients and assessing responses to specific drugs may enable "individualized cardiotoxicity assessment systems" that can preemptively avoid cardiac complications associated with anticancer drugs and other pharmacological agents.

Bone allograft impregnated with tobramycin and vancomycin delivers antibiotics in high concentrations for prophylaxis against bacteria commonly associated with prosthetic joint infections.

Local delivery of antibiotics as prophylaxis for prosthetic joint infections (PJIs) is frequently used during total hip replacement surgery. Morselized bone allograft impregnated with vancomycin and tobramycin (TobraVanc) could provide effective prophylaxis against bacteria commonly associated with PJIs. In this study, the concentrations of antibiotics released by bone allograft impregnated with TobraVanc were determined by using an in vitro bioassay system entailing measuring inhibition zone diameters caused by antibiotic-impregnated bone chips cast in agar against standard curves. The concentrations were determined in samples of TobraVanc-impregnated bone graft taken before and after the application of the bone graft in the patients undergoing acetabular revision surgery. Antibiotic-impregnated bone grafts, sampled prior to application in the patient, delivered antibiotics in the concentration ranges of 730-9,800 mg/L for tobramycin and 1,300-11,000 mg/L for vancomycin. Samples taken after application in the patient released lower concentrations of tobramycin (490-1,900 mg/L; P < 0.01) and vancomycin (3,000-5,100 mg/L; P < 0.05); however, these concentrations remained well above the tobramycin minimum inhibitory concentrations (MICs) for investigated, highly tobramycin-resistant Staphylococcus epidermidis strains (MICs > 256 mg/L). At the tested concentrations, bone graft material mixed with TobraVanc delivered antibiotics in potent concentrations above the MICs for bacteria causing PJIs. Clinical trials are needed to evaluate the efficacy and risk of TobraVanc-impregnated bone graft as a prophylactic agent for patients undergoing hip replacement surgery.IMPORTANCEAntibiotic prophylaxis is the cornerstone of successful joint replacement surgery, reducing the risk for the dreaded complication of prosthetic joint infection (PJI) to roughly 0.5%-2% in standard total hip replacement (THR). In addition to systemic antibiotics, antibiotics added locally have the potential to reduce the PJI risk even further, because of the high concentrations that can be achieved in the joint with limited risk for systemic toxicity. The results in the current study show that bone chips impregnated with a combination of tobramycin and vancomycin (TobraVanc) release antibiotics in concentrations that are potent against common bacteria causing PJIs. Especially in high-risk patients, our results support the prophylactic use of TobraVanc in hip replacement surgery requiring the use of a bone graft. A clinical study testing the efficacy of TobraVanc-impregnated bone graft in reducing the incidence of PJI in hip replacement surgery is currently ongoing (EudraCT: 2021-001708-14).

3D Z-Scheme Conjugated Polymer/Cu2O for Organic Photoelectrochemical Transistor Bioassay

Organic photoelectrochemical transistor (OPECT) is an emerging technology studying photo-electric-biological recognition events. Here, this work reports the three-dimensional (3D) Z-scheme poly(1,4-diethynylbenzene) (pDEB)@Cu2O heterojunction as a high-efficacy photogating module and its application for OPECT bioassay. Specifically, 3D Z-scheme pDEB@Cu2O heterojunction enabled fast charge transport and ion diffusion in the system, achieving remarkable amplification capability with a current gain as high as ca. 9.6×103. By linking with GOx-labeled sandwich immunorecognition, the impact of GOx-generated H2O2 on the OPECT made possible the sensitive bioassay. Exemplified by carcinoembryonic antigen (CEA) as the model target, the OPECT device achieved a linear detection range spanning from 100 fg/mL to 100 ng/mL and coupled with a detection limit as low as 72 fg/mL. This work provided a generic and extensible platform for the designation of novel bioassay systems.

Design of a portable bioassay system based on highly efficent nanozyme and Au-DNA nanocluster: Toward current/colorimetric dual-signal detection of sugarcane smut

Sugarcane smut is a major agricultural pest affecting sugarcane yield and quality, posing a significant threat to the sustainable and healthy development of the global sugar industry. The best strategy for controlling Sugarcane smut is early and precise diagnosis. An intelligent mobile bio-platform is constructed based on Au-Co/Zn ZIF nanozymes and Au-DNA nanoclusters for current/colorimetric dual signal detection of the sugarcane smut pathogen. Efficient and specific cycling of the specific gene fragment (bE4′) for Sugarcane smut is achieved by strand displacement reaction. The output strand then forms Au-DNA nanoclusters with AuNPs-DNA, and the bioconjugate is captured by H2 probe, which results in the formation of more double-stranded structures, causing methylene blue (MB) to adsorb on electrode. The anodic nanozyme efficiently catalyzes the oxidation of glucose, generating a large number of electrons, and MB in the system is therefore reduced to MBH, causing a color change. This results in distinct current/colorimetric dual output signals, significantly enhancing the accuracy. A capacitor is further coupled with the system to improve the sensitivity. The detection signals are read in real-time by a smartphone. The developed method for sugarcane smut genes shows good linearity within a concentration range of 0.0001–10000 pM, with a detection limit as low as 23.59 aM (S/N = 3). This work integrates advanced technologies from various fields to create a portable sensing device with extremely high sensitivity and precision for detecting sugarcane smut. It provides a reliable new option for early and precise diagnosis of the disease and offers valuable theoretical and technical insights for early prevention, intelligent detection, and real-time monitoring of other plant diseases.

IGF-1 and Glucocorticoid Receptors Are Potential Target Proteins for the NGF-Mimic Effect of β-Cyclocitral from Lavandula angustifolia Mill. in PC12 Cells.

In the present study, the PC12 cells as a bioassay system were used to screen the small molecules with nerve growth factor (NGF)- mimic effect from Lavandula angustifolia Mill. The β-Cyclocitral (β-cyc) as an active compound was discovered, and its chemical structure was also determined. Furthermore, we focused on the bioactive and action mechanism of this compound to do an intensive study with specific protein inhibitors and Western blotting analysis. The β-cyc had novel NGF-mimic and NGF-enhancer effects on PC12 cells, while the insulin-like growth factor-1 receptor (IGF-1R)/phosphatidylinositol 3 kinase, (PI3K)/serine/threonine-protein kinase (AKT), and glucocorticoid receptor (GR)/phospholipase C (PLC)/protein kinase C (PKC) signaling pathways were involved in the bioactivity of β-cyc. In addition, the important role of the rat sarcoma (Ras)/protooncogene serine-threonine protein kinase (Raf) signaling pathway was observed, although it was independent of tyrosine kinase (Trk) receptors. Moreover, the non-label target protein discovery techniques, such as the cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS), were utilized to make predictions of its target protein. The stability of IGF-R and GR, proteins for temperature and protease, was dose-dependently increased after treatment of β-cyc compared with control groups, respectively. These findings indicated that β-cyc promoted the neuron differentiation of PC12 cells via targeting IGF-1R and GR and modification of downstream signaling pathways.

Sedative and anxiolytic effects of Capparis sicula Duhamel: in vivo and in silico approaches with phytochemical profiling.

The World Health Organization reports that 30% of adults worldwide suffer from insomnia, while 10% of people worldwide suffer with various forms of anxiety. The significant negative effects of conventional medications used to treat anxiety and insomnia, such as abuse, addiction, amnesia, and cognitive and sexual dysfunction, have led to an increased preference for naturally derived substances with fewer side effects. Accordingly, in this study, the sedative and anxiolytic effects of n-hexane, ethyl acetate (EtOAc), methanol (MeOH) and water extracts of the aerial parts of Capparis sicula Duhamel., which is used for sedative purposes in folk medicine, were evaluated. To evaluate the sedative and anxiolytic effects of each extract, bioassay systems were used including traction and hole-board tests. The MeOH extract of C. sicula was the most active extract on in vivo traction and hole-board tests compared to Diazepam. From the MeOH extract, major components were isolated, and their structures were identified as three flavonoid glycosides [rutin (1), quercetin-3-O-glucoside (2), and quercetin 3-O-rhamnoside (3)] using spectral techniques. The most abundant component was determined to be rutin, comprising 8mg/100mg dry extract in MeOH extract and 76.7mg/100mg dry fraction in fraction C using HPLC. The molecular docking studies evaluated the interaction of isolated flavonoid glycosides with the interaction energies and protein-ligand interaction details of the anxiety-related receptors GABAA and GABAB. For the GABAA receptor, quercetin-3-O-glucoside demonstrated the highest docking score. Quercetin-3-O-rhamnoside and rutin also show promising interactions, particularly with the GABAB receptor, highlighting their potential as modulators of these receptors. In conclusion, the use of C. sicula for sedative purposes in folk medicine has been confirmed for the first time by in vivo studies, and its possible active compounds and sedative-anxiolytic mechanism have been determined through phytochemical and in silico studies.

BAX and DDB2 as biomarkers for acute radiation exposure in the human blood ex vivo and non-human primate models

There are currently no available FDA-cleared biodosimetry tools for rapid and accurate assessment of absorbed radiation dose following a radiation/nuclear incident. Previously we developed a protein biomarker-based FAST-DOSE bioassay system for biodosimetry. The aim of this study was to integrate an ELISA platform with two high-performing FAST-DOSE biomarkers, BAX and DDB2, and to construct machine learning models that employ a multiparametric biomarker strategy for enhancing the accuracy of exposure classification and radiation dose prediction. The bioassay showed 97.92% and 96% accuracy in classifying samples in human and non-human primate (NHP) blood samples exposed ex vivo to 0–5 Gy X-rays, respectively up to 48 h after exposure, and an adequate correlation between reconstructed and actual dose in the human samples (R2 = 0.79, RMSE = 0.80 Gy, and MAE = 0.63 Gy) and NHP (R2 = 0.80, RMSE = 0.78 Gy, and MAE = 0.61 Gy). Biomarker measurements in vivo from four NHPs exposed to a single 2.5 Gy total body dose showed a persistent upregulation in blood samples collected on days 2 and 5 after irradiation. The data indicates that using a combined approach of targeted proteins can increase bioassay sensitivity and provide a more accurate dose prediction.

Biofuel cell-based self-powered immunosensor for detection of 17β-estradiol by integrating the target-induced biofuel release and biogate immunoassay.

A novel biofuel cell (BFC)-based self-powered electrochemical immunosensing platform was developed by integrating the target-induced biofuel release and biogate immunoassay for ultrasensitive 17β-estradiol (E2) detection. The carbon nanocages/gold nanoparticle composite was employed in the BFCs device as the electrode material, through which bilirubin oxidase and glucose oxidase were wired to form the biocathode and bioanode, respectively. Positively charged mesoporous silica nanoparticles (PMSN) were encapsulated with glucose molecules as biofuel and subsequently coated by the negatively charged AuNPs-labelled anti-E2 antibody (AuNPs-Ab) serving as a biogate. The biogate could be opened efficiently and the trapped glucose released once the target E2 was recognized and captured by AuNPs-Ab due to the decreased adhesion between the antigen-antibody complex and PMSN. Then, glucose oxidase oxidized the glucose to produce a large number of electrons, resulting in significantly increased open-circuit voltage (EOCV). Promisingly, the proposed BFC-based self-powered immunosensor demonstrated exceptional sensitivity for the detection of E2 in the concentration range from 1.0pgmL-1 to 10.0ngmL -1, with a detection limit of 0.32pgmL-1 (S/N = 3). Furthermore, the prepared BFC-based self-powered homogeneous immunosensor showed significant potential for implementation as a viable prototype for a mobile and an on-site bioassay system in food and environmental safety applications.

Enzymatic biofuel cells-based self-powered electrochemical biosensor for detection of 17β-estradiol based on dual-enzyme cascade amplification and bioconjugate

Self-powered electrochemical biosensors (SPEB) based on enzymatic biofuel cells (EBFC) have garnered significant attention due to their distinctive characteristic of not necessitating the source of external power supply. This study developed a novel EBFC-based SPEB by incorporating dual-enzyme cascade amplification and bioconjugate for extremely precise detection of 17β-estradiol (E2). The carbon nanocages/gold nanoparticles composite was employed as substrate to immobilize bilirubin oxidase (BOD) and complementary ssDNA (csDNA) to form bioconjugate. The carbon nanotubes/gold nanoparticles composite was used in the EBFC device as the electrode material, through which glucose oxidase (GOD) and E2 aptamer were wired to form the bioanode and biocathode, respectively. The biocathode further linked with bioconjugate via a hybridization reaction between E2 aptamer and csDNA. The bioconjugate integrates the recognition and signal probes for homogeneous electrochemical assay of targets, which released from biocathode once the target E2 was recognized and hindered the transfer of electrons produced by GOD on bioanode. Thus, the open-circuit voltage (EOCV) of the EBFC-based SPEB dramatically decreased, exhibiting exceptional sensitivity for the detection of E2 over a concentration range from 10 pg mL−1 to 20 ng mL−1, with a detection limit of 3.3 pg mL−1 (S/N = 3). Furthermore, the prepared EBFC-based SPEB showed significant potential for implementation as a viable prototype for a mobile and an on-site bioassay system in food and environmental safety applications.

Aortic Valve Stenosis Causes Accumulation of Extracellular Hemoglobin and Systemic Endothelial Dysfunction.

Whether aortic valve stenosis (AS) can adversely affect systemic endothelial function independently of standard modifiable cardiovascular risk factors is unknown. We therefore investigated endothelial and cardiac function in an experimental model of AS mice devoid of standard modifiable cardiovascular risk factors and human cohorts with AS scheduled for transcatheter aortic valve replacement. Endothelial function was determined by flow-mediated dilation using ultrasound. Extracellular hemoglobin (eHb) concentrations and nitric oxide (NO) consumption were determined in blood plasma of mice and humans by ELISA and chemiluminescence. This was complemented by measurements of aortic blood flow using 4-dimensional flow acquisition by magnetic resonance imaging and computational fluid dynamics simulations. The effects of plasma and red blood cell (RBC) suspensions on vascular function were determined in transfer experiments in a murine vasorelaxation bioassay system. In mice, the induction of AS caused systemic endothelial dysfunction. In the presence of normal systolic left ventricular function and mild hypertrophy, the increase in the transvalvular gradient was associated with elevated eryptosis, increased eHb, and increased plasma NO consumption; eHb sequestration by haptoglobin restored endothelial function. Because the aortic valve orifice area in patients with AS decreased, postvalvular mechanical stress in the central ascending aorta increased. This was associated with elevated eHb, circulating RBC-derived microvesicles, eryptotic cells, lower haptoglobin levels without clinically relevant anemia, and consecutive endothelial dysfunction. Transfer experiments demonstrated that reduction of eHb by treatment with haptoglobin or elimination of fluid dynamic stress by transcatheter aortic valve replacement restored endothelial function. In patients with AS and subclinical RBC fragmentation, the remaining circulating RBCs before and after transcatheter aortic valve replacement exhibited intact membrane function, deformability, and resistance to osmotic and hypoxic stress. AS increases postvalvular swirling blood flow in the central ascending aorta, triggering RBC fragmentation with the accumulation of hemoglobin in the plasma. This increases NO consumption in blood, thereby limiting vascular NO bioavailability. Thus, AS itself promotes systemic endothelial dysfunction independent of other established risk factors. Transcatheter aortic valve replacement is capable of limiting NO scavenging and rescuing endothelial function by realigning postvalvular blood flow to near physiological patterns. URL: https://www.clinicaltrials.gov; Unique identifiers: NCT05603520 and NCT01805739.

Pathogens of the oak processionary moth Thaumetopoea processionea: Developing a user-friendly bioassay system and metagenome analyses for microorganisms

The oak processionary moth (OPM) Thaumetopoea processionea is a pest of oak trees and poses health risks to humans due to the urticating setae of later instar larvae. For this reason, it is difficult to rear OPM under laboratory conditions, carry out bioassays or examine larvae for pathogens. Biological control targets the early larval instars and is based primarily on commercial preparations of Bacillus thuringiensis ssp. kurstaki (Btk). To test the entomopathogenic potential of other spore-forming bacteria, a user-friendly bioassay system was developed that (i) applies bacterial spore suspensions by oak bud dipping, (ii) targets first instar larvae through feeding exposure and (iii) takes into account their group-feeding behavior. A negligible mortality in the untreated control proved the functionality of the newly established bioassay system. Whereas the commercial Btk HD-1 strain was used as a bioassay standard and confirmed as being highly efficient, a Bacillus wiedmannii strain was ineffective in killing OPM larvae. Larvae, which died during the infection experiment, were further subjected to Nanopore sequencing for a metagenomic approach for entomopathogen detection. It further corroborated that B.wiedmannii was not able to infect and establish in OPM, but identified potential insect pathogenic species from the genera Serratia and Pseudomonas.

Multiplex analysis platform for evaluation of endocrine disruption of emerging contaminants against human steroid hormone receptors using autobioluminescent yeast bioassay: Application to bisphenols

Emerging synthetic or derivative chemicals have drawn global concern due to their potential endocrine-disrupting effects, either as agonists or antagonists or through indirect effects on enzymes of signal transduction pathways. Currently, in vitro screening models are limited to poor tolerance, robustness, high cost and need for sphisticated equipment or complex procedures that require high levels of expertise. Herein, a multiplex analysis platform based on autobioluminescent yeast strains, including BLYhERαS, BLYhERβS, BLYhPRS, BLYhARS, BLYhMRS, and BLYhGRS, was developed for use in easy, rapid, robust, and sensitive screening for potential modulation of endocrine systems. Methods were applied to assess endocrine-disrupting effects of Bisphenol A (BPA) and its alternatives. Disrupting activities of bisphenols (BPs) varied greatly, and some BPA substitutes exerted more potent activities than those of BPA. Most BPs were primarily agonists of ERα and ERβ, while others were antagonists to AR, PR, GR, and MR. BPP and BPM exhibited non-monotonic dose-response relationships toward ERα and ERβ, and BHPF and BPG displayed converse disrupting activities to ERα and ERβ. Overall, the bioassay system provides an efficient tool for comprehensive evaluation of endocrine-disrupting activities of emerging contaminants.

Host-Guest Recognition-Mediated Supramolecular Aggregation-Induced Electrochemiluminescence of Iridium(III) Complexes for Nucleic Acid Bioassay.

Currently reported aggregation-induced electroluminescence (AIECL) is usually based on the electrostatic integration of luminous monomers, and its application is still limited by the low ECL efficiency and poor structural stability of electrostatic integration-based AIECL emitters. Herein, host-guest recognition-mediated supramolecular AIECL was creatively developed to overcome the defects of electrostatic-integration-based AIECL. Cucurbit[8]uril (CB[8]) as the host recognized tris (2-phenylpyridine) iridium(III) [Ir(ppy)3] as the guest to form a novel supramolecular complex Ir-CB[8]. CB[8] can not only provide a large hydrophobic cavity to efficiently load Ir(ppy)3 and enrich coreactant tripropylamine but also utilize its carbonyl-laced portals to form intramolecular hydrogen bonds to stabilize the supramolecular structure, so Ir-CB[8] revealed excellent AIECL performance. The AIECL emitter Ir-CB[8] coupled the efficient DNA walker to construct a sensing system for miRNA-16 detection. Au nanoparticles@norepinephrine (AuNPs@NE) trapped by single-strand S1 was developed to significantly quench the ECL emission of Ir-CB[8]. When the target microRNA-16 (miRNA-16) existed, H1 was opened and the sequential assembly from H2 to H7 was triggered, forming "windmill"-like DNA walker with six Pb2+-dependent leg DNA. The assembled DNA walker, which was centered on DNA structure, had high efficiency and biocompatibility and can cut S1 to keep the DNA fragment-carrying quencher AuNPs@NE away from the electrode surface, thus restoring the ECL emission of Ir-CB[8] and realizing ultrasensitive detection of miRNA-16. Supramolecular AIECL mediated by host-guest recognition provides a new way for constructing AIECL emitters with excellent structural stability and AIECL efficiency, and an Ir-CB[8] coupling "windmill"-like DNA walker builds a promising ECL-sensing system for bioassay.

Ultrahigh peroxidase-like catalytic performance of Cu–N4 and Cu–N4S active sites-containing reduced graphene oxide for sensitive electrochemical biosensing

Carbon-based nanozymes possessing peroxidase-like activity have attracted significant interest because of their potential to replace native peroxidases in biotechnology. Although various carbon-based nanozymes have been developed, their relatively low catalytic efficiency needs to be overcome to realize their practical utilization. Here, inspired by the elemental uniqueness of Cu and the doped elements N and S, as well as the active site structure of Cu-centered oxidoreductases, we developed a new carbon-based peroxidase-mimicking nanozyme, single-atom Cu-centered N- and S-codoped reduced graphene oxide (Cu-NS-rGO), which preserved many Cu–N4 and Cu–N4S active sites and showed dramatically high peroxidase-like activity without any oxidase-like activity, yielding up to 2500-fold higher catalytic efficiency (kcat/Km) than that of pristine rGO. The high catalytic activity of Cu-NS-rGO might be attributed to the acceleration of electron transfer from Cu single atom as well as synergistic effects from both Cu–N4 and Cu–N4S active sites, which was theoretically confirmed by Gibbs free energy calculations using density functional theory. The prepared Cu-NS-rGO was then used to construct an electrochemical bioassay system for detecting choline and acetylcholine by coupling with the corresponding oxidases. Using this system, both target molecules were selectively determined with high sensitivity that was sufficient to clinically determine their levels in physiological fluids. Overall, this study will facilitate the development of nanocarbon-based nanozymes and their electrochemical biosensing applications, which can be extended to the development of miniaturized devices in point-of-care testing environments.

Oxygen Vacancy Engineering of FeOx toward Oxygen-Tolerant Hydrogen Peroxide Reduction for Reliable Bioassays.

The accurate determination of hydrogen peroxide (H2O2), an important clinical disease relevant biomarker, is of great importance for the diagnosis and management of illnesses. By using the cathodic monitoring approach, H2O2 can be accurately detected because interfering signals from easily oxidizable endogenous and exogenous species in biofluids can be avoided. However, the simultaneous occurrence of the oxygen reduction reaction (ORR) restricts the practical use of this cathodic method. In this study, via oxygen vacancy modulation, we synthesized FeOx catalysts that can selectively reduce H2O2 over O2. The H2O2 detection system based on this catalyst exhibits an outstanding ORR inhibition ability. Furthermore, by integrating this catalyst with glucose oxidase, a model enzyme, a reliable bioassay system was developed that can selectively detect glucose over a wide variety of interferents in artificially simulated tissue fluids. The bioassay system employing this catalyst in conjunction with oxidases is generally applicable to accurate detect a wide range of biomarkers.

Isoquercitrin from Apocynum venetum L. Exerts Antiaging Effects on Yeasts via Stress Resistance Improvement and Mitophagy Induction through the Sch9/Rim15/Msn Signaling Pathway.

With the development of an aging sociality, aging-related diseases, such as Alzheimer's disease, cardiovascular disease, and diabetes, are dramatically increasing. To find small molecules from natural products that can prevent the aging of human beings and the occurrence of these diseases, we used the lifespan assay of yeast as a bioassay system to screen an antiaging substance. Isoquercitrin (IQ), an antiaging substance, was isolated from Apocynum venetum L., an herbal tea commonly consumed in Xinjiang, China. In the present study, we utilized molecular-biology technology to clarify the mechanism of action of IQ. The replicative lifespans of K6001 yeasts and the chronological lifespans of YOM36 yeasts were used to screen and confirm the antiaging effect of IQ. Furthermore, the reactive oxygen species (ROS) and malondialdehyde (MDA) assay, the survival assay of yeast under stresses, real-time polymerase chain reaction (RT-PCR) and Western blotting analyses, the replicative-lifespan assay of mutants, such as Δsod1, Δsod2, Δgpx, Δcat, Δskn7, Δuth1, Δatg32, Δatg2, and Δrim15 of K6001, autophagy flux analysis, and a lifespan assay of K6001 yeast after giving a mitophagy inhibitor and activator were performed. IQ extended the replicative lifespans of the K6001 yeasts and the chronological lifespans of the YOM36 yeasts. Furthermore, the reactive nitrogen species (RNS) showed no change during the growth phase but significantly decreased in the stationary phase after treatment with IQ. The survival rates of the yeasts under oxidative- and thermal-stress conditions improved upon IQ treatment, and thermal stress was alleviated by the increasing superoxide dismutase (Sod) activity. Additionally, IQ decreased the ROS and MDA of the yeast while increasing the activity of antioxidant enzymes. However, it could not prolong the replicative lifespans of Δsod1, Δsod2, Δgpx, Δcat, Δskn7, and Δuth1 of K6001. IQ significantly increased autophagy and mitophagy induction, the presence of free green fluorescent protein (GFP) in the cytoplasm, and ubiquitination in the mitochondria of the YOM38 yeasts at the protein level. IQ did not prolong the replicative lifespans of Δatg2 and Δatg32 of K6001. Moreover, IQ treatment led to a decrease in Sch9 at the protein level and an increase in the nuclear translocation of Rim15 and Msn2. These results indicated that the Sch9/Rim15/Msn signaling pathway, as well as antioxidative stress, anti-thermal stress, and autophagy, were involved in the antiaging effects of IQ in the yeasts.

Dynamic Mapping of Electrochemiluminescence Reactivity in Space: Application to Bead-Based Assays.

As an electrochemical technique offering an optical readout, electrochemiluminescence (ECL) evolved recently into a powerful microscopy technique with the visualization of a wide range of microscopic entities. However, the dynamic imaging of transient ECL events did not receive intensive attention due to the limited number of electrogenerated photons. Here, the reaction kinetics of the model ECL bioassay system was revealed by dynamic imaging of single [Ru(bpy)3]2+-functionalized beads in the presence of the efficient tripropylamine coreactant. The time profile behavior of ECL emission, the variations of the ECL layer thickness, and the position of maximum ECL intensity over time were investigated, which were not achieved by static imaging in previous studies. Moreover, the dynamics of the ECL emission were confronted with the simulation. The reported dynamic ECL imaging allows the investigation of the ECL kinetics and mechanisms operating in bioassays and cell microscopy.

A Druggability study to Probe immunomodulatory Unani plants as active Inhibitors of Pseudotyped Particle Cell Entry

Medicinal herbs, plants and their products show revolutionized effect in modern therapeutic system. Industries used their activity as antioxidant, antitoxin, antimicrobial, immune booster and many others and use them accordingly as the prime agent to produce cosmetics, food, drug synthesis etc. Unani is one of the traditional therapeutic system which use medicinal herbs mostly for making their herbal medicines. There are several immunomodulatory herbs along with their phytochemicals are present to serve their best. Bioassay is the method of analysis to find out the potency or concentration of any substance when effect on living things. Sars Cov-2 virus and its fatal outbreak is a real threat to modern society. To perform in vitro procedure to screened out small molecule inhibitors of this malicious pseudo typed particle cell entry, Dr. Whittaker's group use this bioassay system like PubChem AID: 1479144, PubChem AID: 1479149, PubChem AID: 1479150 and PubChem AID: 1494158. This in silico study is to show the potency of the phytochemicals of traditional herbs of Unani, as if inhibitors by means of machine learning with the help of WEKA. The ADMET study and molecular docking with Mpro (6lu7 chain A) is also done to test the druggability and binding affinity of the qualified phytochemicals.

SCH23390 and a humanized anti-cocaine mAb decrease the latency to cocaine-induced reinstatement of lever pressing behavior in rats that self-administer cocaine

In rats that self-administer cocaine, the latency to the reinstatement of lever pressing behavior induced by a single dose of cocaine is due to the time taken for cocaine levels to fall to the satiety threshold. The D1 dopamine receptor antagonist SCH23390, and the recombinant humanized anti-cocaine mAb h2E2 increase the cocaine satiety threshold and would be expected to alter the latency to reinstatement. Male rats acquired cocaine self-administration behavior on an FR1 schedule. These rats received a single injection of cocaine (12 µmol/kg i.v.) after an i.v. injection of SCH23390 or an infusion of h2E2 or vehicle. The latency to, and the duration of, lever pressing was measured but the presses had no consequence. SCH23390 decreased the latency to lever pressing consistent with dose-dependent increases in satiety threshold. The duration of lever pressing behavior was inversely proportional to the SCH23390 dose suggesting that SCH23390 also increased the cocaine compulsion zone. The mAb h2E2 also produced a similar decrease in latency to responding that gradually reversed over 2 weeks. SCH23390 and h2E2 had an additive effect on the decreased latency to cocaine-induced lever pressing. The single cocaine dose reinstatement paradigm within the context of the compulsion zone theory is a useful pharmacological bioassay system to explore potential pharmacotherapies for relapse prevention in cocaine use disorder.

A Novel and Versatile Microfluidic Device for Cell Assays under Radio Frequency Exposure.

Wound healing is a complex process composed of different stages, which involves extensive communication between the different cellular factors of the extracellular matrix (ECM). The radio frequency electromagnetic field (RF-EMF) has been used to accelerate the wound-healing process and it has been found to enhance cell alignment and mobility. The conventional methods for cell mobility analysis in an electromagnetic field generated by a radiation source are not advisable due to the low-precision, nonuniform distribution of the field, low efficiency of the analysis in batch and the lack of system integration for autonomous on-body operation. Here, a novel and versatile electromagnetic exposure system integrated with a microfluidic chip was fabricated to explore the EMF-induced response. A gradient electromagnetic field in a two-dimensional plane has been successfully established in the microchambers placed along the field line. In this work, by deploying our radiation experiments in vitro, we validated the on-chip monitoring of cell response to exposure. This electromagnetic field was simulated and human amniotic epithelial cells (HAECs) were cultured in different microchambers for continuous exposure to the electromagnetic field excited by a monopole RF antenna (1.8 GHz). New protrusions were generated and an obvious increase in filopodia with the increased field intensity was investigated. Meanwhile, the variation in intracellular Ca2+ concentration under the electromagnetic field was examined. The inhibitory effect of the Ca2+ circulation was further inspected to reveal the potential downstream signaling pathway in the RF-EMF-related bioassay, suggesting that cytoskeletal dynamics of cells under exposure are highly associated with the EGF receptor (EGFR)-cytoskeleton downstream signaling pathway. Finally, the field-induced cell elongation and alignment parallel to the field direction were observed. Additionally, the subsequent recovery (field withdrawal) and re-establishment (field re-exposure) were explored. These results indicated that this reliable and versatile exposure system for bioassay could achieve precise and high-throughput detection of the RF-EMF-induced cytoskeletal reorganization in vitro and evaluate the possible health risk from RF-EMF exposure.