Biosensor Assay Research Articles

Electrochemical biosensor for highly sensitive detection of cTnI based on a dual signal amplification strategy of ARGET ATRP and ROP

Cardiac troponin I (cTnI), a gold biomarker for the diagnosis of acute myocardial infarction (AMI), plays a vital role in the early diagnosis, treatment and prognosis analysis of AMI. In this paper, an electrochemical biosensor for the highly sensitive determination of cTnI was fabricated based on the dual signal amplification strategy of electron transfer atom transfer radical polymerization (ARGET ATRP) and ring-opening polymerization (ROP) for the first time. Briefly, the thiolate cTnI-aptamer 1, which was bonded to the electrode via Au–S bonds, specifically captured cTnI to the electrode surface. Then, cTnI-aptamer 2 (Apt2) was successfully introduced to the electrode surface to form Apt-cTnI-Apt sandwich structure. Subsequently, the initiator BIBB was connected to Apt2 through bromination reaction, and then the resulting ATRP polymer was employed as a macromolecular initiator for the succeeding reaction. Next, the monomers, α-amino acid-N-carboxylic acid anhydride ferrocene derivatives (NCA-Fc), used for the ROP reaction produced numerous electroactive polymers on the electrode surface. The dual action of ARGET ATRP and ROP significantly improved sensitivity of cTnI biosensor assay, the prepared biosensor displayed a wide linear detection range from 100 fg mL−1 to 100 ng mL−1, with a detection limit of 32.24 fg mL−1. The method exhibited favorable selectivity, simple operation and excellent stability. Furthermore, the biosensor still rendered satisfactory analytical performance in the detection of clinical serum samples, indicating the application potential in clinical diagnosis.

Machine learning methods for endocrine disrupting potential identification based on single-cell data

Humans are continuously exposed to a variety of toxicants and chemicals which is exacerbated during and after environmental catastrophes such as floods, earthquakes, and hurricanes. The hazardous chemical mixtures generated during these events threaten the health and safety of humans and other living organisms. This necessitates the development of rapid decision-making tools to facilitate mitigating the adverse effects of exposure on the key modulators of the endocrine system, such as the estrogen receptor alpha (ERα), for example. The mechanistic stages of the estrogenic transcriptional activity can be measured with high content/high throughput microscopy-based biosensor assays at the single-cell level, which generates millions of object-based minable data points. By combining computational modeling and experimental analysis, we built a highly accurate data-driven classification framework to assess the endocrine disrupting potential of environmental compounds. The effects of these compounds on the ERα pathway are predicted as being receptor agonists or antagonists using the principal component analysis (PCA) projections of high throughput, high content image analysis descriptors. The framework also combines rigorous preprocessing steps and nonlinear machine learning algorithms, such as the Support Vector Machines and Random Forest classifiers, to develop highly accurate mathematical representations of the separation between ERα agonists and antagonists. The results show that Support Vector Machines classify the unseen chemicals correctly with more than 96% accuracy using the proposed framework, where the preprocessing and the PCA steps play a key role in suppressing experimental noise and unraveling hidden patterns in the dataset.

Polysaccharide-based nano-engineered multilayers for controlled cellular adhesion in label-free biosensors

Controlling cellular adhesion is a critical step in the development of biomaterials, and in cell- based biosensing assays. Usually, the adhesivity of cells is tuned by an appropriate biocompatible layer. Here, synthetic poly(diallyldimethylammonium chloride) (PDADMAC), natural chitosan, and heparin (existing in an extracellular matrix) were selected to assembly PDADMAC/heparin and chitosan/heparin films. The physicochemical properties of macroion multilayers were determined by streaming potential measurements (SPM), quartz crystal microbalance (QCM-D), and optical waveguide lightmode spectroscopy (OWLS). The topography of the wet films was imaged using atomic force microscopy (AFM).The adhesion of preosteoblastic cell line MC3T3-E1 on those well-characterized polysaccharide-based multilayers was evaluated using a resonant waveguide grating (RWG) based optical biosensor and digital holographic microscopy. The latter method was engaged to investigate long-term cellular behavior on the fabricated multilayers.(PDADMAC/heparin) films were proved to be the most effective in inducing cellular adhesion. The cell attachment to chitosan/heparin–based multilayers was negligible. It was found that efficient adhesion of the cells occurs onto homogeneous and rigid multilayers (PDADMAC/heparin), whereas the macroion films forming “sponge-like” structures (chitosan/heparin) are less effective, and could be employed when reduced adhesion is needed.Polysaccharide-based multilayers can be considered versatile systems for medical applications. One can postulate that the presented results are relevant not only for modeling studies but also for applied research.

Directed evolution of a neutrophilic and mesophilic methanol dehydrogenase based on high-throughput and accurate measurement of formaldehyde

Methanol is a promising one-carbon feedstock for biomanufacturing, which can be sustainably produced from carbon dioxide and natural gas. However, the efficiency of methanol bioconversion is limited by the poor catalytic properties of nicotinamide adenine dinucleotide (NAD+)-dependent methanol dehydrogenase (Mdh) that oxidizes methanol to formaldehyde. Herein, the neutrophilic and mesophilic NAD+-dependent Mdh from Bacillus stearothermophilus DSM 2334 (MdhBs) was subjected to directed evolution for enhancing the catalytic activity. The combination of formaldehyde biosensor and Nash assay allowed high-throughput and accurate measurement of formaldehyde and facilitated efficient selection of desired variants. MdhBs variants with up to 6.5-fold higher Kcat/KM value for methanol were screened from random mutation libraries. The T153 residue that is spatially proximal to the substrate binding pocket has significant influence on enzyme activity. The beneficial T153P mutation changes the interaction network of this residue and breaks the α-helix important for substrate binding into two short α-helices. Reconstructing the interaction network of T153 with surrounding residues may represent a promising strategy to further improve MdhBs, and this study provides an efficient strategy for directed evolution of Mdh.

Biosensor Assays Types and Their Roles Toward Ligand-Receptor Interactions in Drug Discovery.

Ligand-receptor interactions (LRIs) are the basis for all the biological processes taking place in living cells and have been exploited to develop and implement in medical field a number of highly sensitive biosensors for the detection of various biomarkers in complex biological fluids. Drug-target interactions, one of the LRIs, are important to understand the biological processes that further help in developing new and better therapeutic molecules. Biosensors based on these interactions give us an idea for the need of modification of existing drugs or to develop new drugs. Common approach to develop biosensors requires the labeling; however, label-free systems provide advantages in avoiding the chances of conformational changes, off-site labeling, and labeling-based hindrances, thus saving time and effort toward assay development. Preliminary drug screening assays are carried out in two-dimensional (2D) models, followed by animal models, which require huge capital investment to reach from bench-top to clinical trials, where only 21% of new compounds make way to phase-1 clinical trials. Three-dimensional culture or organoid culture or organ-on-chip technology has made way for predictive and complex in vitro approach that recapitulates human physiology and represents more similar in vivo behavior than 2D. Multiplexing and nanotechnology have remarkably enhanced the efficacy of biosensors and might lead to a generation of miniaturized biosensors and more than just point-of-care kits. This review provides in-depth analysis of different types of biosensor assays based on drug-target interactions, their advantages, and limitations based on cost, sensitivity, and selectivity and industrial applications.

Tracking and blocking interdependencies of cellular BRAF-MEK oncokinase activities.

The selective targeting of mutated kinases in cancer therapies has the potential to improve therapeutic success and thereby the survival of patients. In the case of melanoma, the constitutively active MAPK pathway is targeted by a combinatorial inhibition of BRAF and MEK activities. These MAPK pathway players may display patient-specific differences in the onco-kinase mutation spectrum, which needs to be considered for the design of more efficient personalized therapies. Here, we extend a bioluminescence-based kinase conformation biosensor (KinCon) to allow for live-cell tracking of interconnected kinase activity states. First, we show that common MEK1 patient mutations promote a structural rearrangement of the kinase to an opened and active conformation. This effect was reversible by the binding of MEK inhibitors to mutated MEK1, as shown in biosensor assays and molecular dynamics simulations. Second, we implement a novel application of the KinCon technology for tracking the simultaneous, vertical targeting of the two functionally linked kinases BRAF and MEK1. Thus, we demonstrate that, in the presence of constitutively active BRAF-V600E, specific inhibitors of both kinases are efficient in driving MEK1 into a closed, inactive conformation state. We compare current melanoma treatments and show that combinations of BRAFi and MEKi display a more pronounced structural change of the drug sensor than the respective single agents, thereby identifying synergistic effects among these drug combinations. In summary, we depict the extension of the KinCon biosensor technology to systematically validate, anticipate, and personalize tailored drug arrangements using a multiplexed setup.

A Rapid Method to Screen for Multi Drug Resistance (MDR) Trait in Clinical Isolates of Acinetobacter baumannii-Acyl Homoserine Lactone (AHL) Screening in CRAB and CSAB Isolates

Aims: Our proposal aimed to evaluate Acyl Homoserine Lactones (AHL) as a functional marker for Multi drug resistant (MDR) potential in clinical isolates of Acinetobacter baumannii. We investigated the AHL production potential of clinical isolates using a biosensor assay directly on a commonly used agar media.
 Place and Duration of Study: Department of Molecular Diagnostics and Biomarkers, Gleneagles Global Hospitals, Lakdikapul, Hyderabad-500004.
 Methodology: Antimicrobial drug sensitivity testing (AST) was performed on 72 clinical isolates of A. baumannii against two front-line antibiotics, Imipenem (10µg) and Meropenem (10µg), by Kirby-Bauer disk diffusion method. Production of long chain Acyl Homoserine lactone (AHLs) in the clinical isolates of A. baumannii was tested by cross streaking with the biosensor Chromobacterium violaceum mutant strain CV026 and Agrobacterium tumefaciens (NTL4pZLR4) by agar plate diffusion assay. Screening and identification of the quorum sensing mediator gene abaI was done by PCR to confirm its presence in all the 72 clinical isolates.
 Results: Out of the 72 clinical isolates, 58 were Carbapenem resistant Acinetobacter baumannii (CRAB) and 14 were Carbapenem sensitive Acinetobacter baumannii (CSAB) for AST by agar disc diffusion method. None of our isolates produced short chain AHLs whereas all the isolates could produce varying amounts of long chain AHLs. Genotypic confirmation of AHL gene was obtained by abaI gene PCR.
 Conclusion: Carbapenems are the front-line antibiotics used to treat gram negative bacterial infections in emergencies and in the critical care units of hospitals. Clinical isolates A. baumannii has innate resistance to several antibiotics due to various mechanisms, biofilms forming the first line of defense against antibiotics for the bacterium. Our study used AST to carbapenem as the leading marker for MDR, assuming the innate resistance of A. baumannii to other beta lactam antibiotics. Our study brought out certain important observations namely: a) All clinical isolates of A. baumannii produced Quorum Sensing signal molecules, the AHLs b) the clinical isolates of A. baumannii did not produce any short chain AHLs b) All the clinical isolates of A. baumannii produced long chain AHLs c) AHL production is not specific to carbapenem drug resistance because even CSAB isolates produced AHL d) AHL production is inherent to all clinical isolates of A. baumannii and it apparently indicates an underlying biofilm potential and MDR trait in these A. baumannii isolates. e) AHLs could be a universal marker for revealing MDR trait and biofilm potential in clinical microbiology AST profiling protocols.

In vitro sepsis up-regulates Nociceptin/Orphanin FQ receptor expression and function on human T- but not B-cells.

In animal models of sepsis, increased activation of the Nociceptin/Orphanin FQ (N/OFQ) receptor NOP is associated with mortality and NOP antagonists improved survival. We have explored the role of the N/OFQ-NOP system in freshly isolated volunteer human B- and T-cells incubated with lipopolysaccharide (LPS) and peptidoglycan G (PepG) as a model of in vitro sepsis. B- and T-cell NOP expression was measured using the NOP fluorescent probe N/OFQATTO594 , N/OFQ content was measured using immunofluorescence, N/OFQ release was tracked using a CHOhNOPGαiq5 biosensor assay and NOP function was measured using transwell migration and cytokine/chemokine release using a 25-plex assay format. Cells were challenged with LPS/PepG. CD19-positive B-cells bound N/OFQATTO594 ; they also contain N/OFQ. Stimulation with CXCL13/IL-4 increased N/OFQ release. N/OFQ trended to reduced migration to CXCL13/IL-4. Surface NOP expression was unaffected by LPS/PepG, but this treatment increased GM-CSF release in an N/OFQ sensitive manner. CD3-positive T-cells did not bind N/OFQATTO594 ; they did contain N/OFQ. Stimulation with CXCL12/IL-6 increased N/OFQ release. When incubated with LPS/PepG, NOP surface expression was induced leading to N/OFQATTO594 binding. In LPS/PepG-treated cells, N/OFQ reduced migration to CXCL12/IL-6. LPS/PepG increased GM-CSF release in an N/OFQ sensitive manner. We suggest both a constitutive and sepsis-inducible N/OFQ-NOP receptor autocrine regulation of B- and T-cell function, respectively. These NOP receptors variably inhibit migration and reduce GM-CSF release. These data provide mechanistic insights to the detrimental role for increased N/OFQ signalling in sepsis and suggest a potential role for NOP antagonists as treatments.

Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin2 G protein–coupled receptor

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.

Feasibility of a DNA biosensor assay based on loop-mediated isothermal amplification combined with a lateral flow dipstick assay for the visual detection of Ascaridia galli eggs in faecal samples

ABSTRACT Ascaridia galli is an important nematode that causes ascaridiasis in free-range and indoor system chicken farms. Infection with A. galli may damage the intestinal mucosa and inhibit nutrient absorption, leading to a reduced growth rate, weight loss and a decreased egg production. Consequently, A. galli infection is a significant health problem in chickens. In this study, we developed a loop-mediated isothermal amplification coupled with a lateral flow dipstick (LAMP-LFD) assay for the visual detection of A. galli eggs in faecal samples. The LAMP-LFD assay consists of six primers and one DNA probe that recognize the internal transcribed spacer 2 (ITS2) region; it can be performed within 70 min and the results can be interpreted with the naked eye. Using the LAMP-LFD assay developed in this study, A. galli DNA was specifically amplified without any cross-reactions with other related parasites (Heterakis gallinarum, Raillietina echinobothrida, R. tetragona, R. cesticillus, Cotugnia sp., Echinostoma miyagawai) and definitive hosts (Gallus gallus domesticus, Anas platyrhynchos domesticus). The minimum detectable DNA concentration was 5 pg/μl, and the detectable egg count was 50 eggs per reaction. The assay can be performed in a water bath, without the need for post-mortem morphological investigations and laboratory instruments. It is therefore a viable alternative for the detection of A. galli in chicken faeces and can replace classical methods in field screening for epidemiological investigations, veterinary health and poultry farming management. RESEARCH HIGHLIGHTS This is the first study using the LAMP-LFD assay for Ascaridia galli detection. The results can be observed by the naked eye. The developed assay can be used to detect Ascaridia galli eggs in faecal samples.

Conductive Ink-Coated Paper-Based Supersandwich DNA Biosensor for Ultrasensitive Detection of Neisseria gonorrhoeae

Herein, we report results of the studies relating to the development of an impedimetric, magnetic bead-assisted supersandwich DNA hybridization assay for ultrasensitive detection of Neisseria gonorrhoeae, the causative agent of a sexually transmitted infection (STI), gonorrhea. First, a conductive ink was formulated by homogenously dispersing carboxylated multiwalled carbon nanotubes (cMWCNTs) in a stable emulsion of terpineol and an aqueous suspension of carboxymethyl cellulose (CMC). The ink, labeled C5, was coated onto paper substrates to fabricate C5@paper conductive electrodes. Thereafter, a magnetic bead (MB)-assisted supersandwich DNA hybridization assay was optimized against the porA pseudogene of N. gonorrhoeae. For this purpose, a pair of specific 5' aminated capture probes (SCP) and supersandwich detector probes (SDP) was designed, which allowed the enrichment of target gonorrheal DNA sequence from a milieu of substances. The SD probe was designed such that instead of 1:1 binding, it allowed the binding of more than one T strand, leading to a 'ladder-like' DNA supersandwich structure. The MB-assisted supersandwich assay was integrated into the C5@paper electrodes for electrochemical analysis. The C5@paper electrodes were found to be highly conductive by a four-probe conductivity method (maximum conductivity of 10.1 S·cm-1). Further, the biosensing assay displayed a wide linear range of 100 aM-100 nM (109 orders of magnitude) with an excellent sensitivity of 22.6 kΩ·(log[concentration])-1. The clinical applicability of the biosensing assay was assessed by detecting genomic DNA extracted from N. gonorrhoeae in the presence of DNA from different non-gonorrheal bacterial species. In conclusion, this study demonstrates a highly sensitive, cost-effective, and label-free paper-based device for STI diagnostics. The ink formulation prepared for the study was found to be highly thixotropic, which indicates that the paper electrodes can be screen-printed in a reproducible and scalable manner.

A lab-on-chip solution for the detection and suppression of COVID-19 severity

At present, the conventional method for detecting COVID-19 infection is reverse transcription-polymerase chain reaction (RT-PCR) performed on nasopharyngeal and pharyngeal swabs. In addition, other detection techniques such as isothermal nucleic acid amplification, enzyme-linked immunosorbent assay (ELISA) have also been proposed and are being used. However, these techniques are limited to central hospitals and pathologies. Point-of-care solutions may play an important role in rapid and timely detection by an individual at their doorstep. In addition, the disease adversely affects the functioning of various constituents of human blood. For example, it reduces the platelets count, increases production of proinflammatory cytokine which results in cytokine storm, and increases level of various plasma proteins with the severity of the disease. Therefore, monitoring and transfusion of blood plasma and platelets may play an essential role in detection and suppression of severity of COVID-19 infection. In this regard, we propose evolution of our existing microdevice for the detection and suppression of severity of COVID-19 infection. We propose modification of our microdevice in the following ways (i) as a platelet-rich plasma separation unit, (ii) as an on-chip device to study inter-cellular properties of platelets, and (iii) for on-chip detection of infection by separating plasma. The integration of these microdevices with lateral flow assays, flow-virometry reader (FVR), direct serological biosensor assay along with proprietary technology based on plasmonic, place these microdevices in an advantageous position. Moreover, coupling of the microdevices with machine leaning application for rapid detection of the severity of COVID-19 and platelets related disorders makes these microdevices as a complete unit for point-of-care application.

Dual-Mode Logic Gate for Intelligent and Portable Detection of MicroRNA Based on Gas Pressure and Lateral Flow Assay.

Molecular logic gate provides an intelligent option for simultaneous detection of biomarkers. Herein, a dual-mode DNA logic gate was proposed to portably and intelligently detect multiple microRNAs (miRNAs) by gas pressure biosensing and lateral flow assay (LFA). A platinum-coated gold nanoparticle (Au@PtNP) with catalase-like activity was used as a signal reporter to achieve a dual-signal readout. MiRNAs as the input initiated the cyclic strand displacement reaction (SDR) to enrich a large amount of Au@PtNPs. Thus, miRNA can be visually detected by a lateral flow strip (LFS) using the grayish-brown color of Au@PtNPs as output 1. Furthermore, Au@PtNP-catalyzed decomposition of H2O2 resulted in gas pressure as output 2, which was measured by a digital and handheld gas pressure meter. As a consequence, microRNA 21 (miR-21) was sensitively and reliably detected with the limit of detection (LOD) of 7.2 pM. The selectivity and real sample analysis were both satisfactory. Significantly, two-input and three-input AND logic gates were successfully developed to realize multiple detection of two miRNAs and three miRNAs, which provide a promising way for intelligent multi-input analysis. Predictably, with the advantages of portability, simplicity, and affordability, the dual-mode logic gate based on gas pressure biosensing and LFA offers a new perspective on the field of intelligent and portable biosensing and bioanalysis.

Genetic fusion of mussel foot protein to ZZ protein improves target detection in solid-phase immunoassays

In the process of a solid-phase immunoassay, the stability and binding orientation between the antibody and the solid matrix can substantially influence the results. ZZ protein is a modified peptide of the B domain of Staphylococcus aureus protein A, which can bind to the Fc fragment of an antibody. It is often used for oriented immobilization of antibodies during solid-phase immunoassay. However, the conjugate is often not retained during the process, for example during washing steps. The resulting low stability detracts from reproducibility and sensitivity. Mfp-5 protein comes from mussel, is one of the components of mussel foot silk protein, and has good adhesion and biocompatibility. In this paper, the fusion protein of ZZ and Mfp-5 was constructed and expressed in Escherichia coli. In this method, the ZZ domain was firmly attached to the solid-phase support by Mfp-5, the directional fixation of IgG was realized by binding the ZZ protein to an Fc fragment, and then a Fab fragment was bound to the antigen to realize the solid-phase immunoassay. In addition, a protein adsorption assay confirmed that the adhesion of ZZ-Mfp-5 was significantly higher than that of ZZ protein, and the presence of Mfp-5 improved the ability of ZZ protein to capture antibodies. In conclusion, compared with the passively immobilized ZZ protein, the ZZ-Mfp-5 protein had stronger immobilization and antibody capture, a 10-fold increase in sensitivity and wider linear range, and better stability of detection. This may be an attractive strategy for solid-phase immunoassays or biosensing assays.

Highly sensitive and rapid identification of coxsackievirus A16 based on reverse transcription multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor assay.

One of the main pathogens responsible for human hand, foot, and mouth disease (HFMD), coxsackievirus A16, has put young children's health at danger, especially in countries in the Asia-Pacific region. Early quick identification is essential for the avoidance and control of the disorder since there are no vaccinations or antiviral medications available to prevent and manage CVA16 infection. Here, we describe the creation of an easy, speedy, and accurate CVA16 infection detection approach using lateral flow biosensors (LFB) and reverse transcriptionmultiple cross displacement amplification (RT-MCDA). A group of 10 primers was developed for the RT-MCDA system in order to amplify the genes in an isothermal amplification device while targeting the highly conserved region of the CVA16 VP1 gene. Then, without requiring any extra tools, RT-MCDA amplification reaction products might well be detected by visual detection reagent (VDR) and LFB. The outcomes showed that 64°C within 40 min was the ideal reaction setting for the CVA16-MCDA test. Target sequences with <40 copies might be found using the CVA16-MCDA. There was no cross-reaction among CVA16 strains and other strains. The findings demonstrated that the CVA16-MCDA test could promptly and successfully identify all of the CVA16-positive (46/220) samples identified by the traditional real-time quantitative polymerase chain reaction (qRT-PCR) assays for 220 clinical anal swab samples. The whole process, such as the processing of the sample (15 min), the MCDA reaction (40 min), and the documenting of the results (2 min), could be finished in 1 h. The CVA16-MCDA-LFB assay, which targeted the VP1 gene, was an efficient, simple, and highly specific examination that might be used extensively in rural regions' basic healthcare institutions and point-of-care settings.

The angiotensin AT2-receptor agonist compound 21 is an antagonist for the thromboxane TP-receptor – Implications for preclinical studies and future clinical use

Since the AT2-receptor (AT2R) agonist C21 has structural similarity to the AT1-receptor antagonists Irbesartan and Losartan, which are antagonists not only at the AT1R, but also at thromboxane TP-receptors, we tested the hypothesis that C21 has TP-receptor antagonistic properties as well. Isolated mouse mesenteric arteries from C57BL/6 J and AT2R-knockout mice (AT2R-/y) were mounted in wire myographs, contracted with either phenylephrine or the thromboxane A2 (TXA2) analogue U46619, and the relaxing effect of C21 (0.1 nM - 10 µM) was investigated. The effect of C21 on U46619-induced platelet aggregation was measured by an impedance aggregometer. Direct interaction of C21 with TP-receptors was determined by an β-arrestin biosensor assay. C21 caused significant, concentration-dependent relaxations in phenylephrine- and U46619-contracted mesenteric arteries from C57BL/6 J mice. The relaxing effect of C21 was absent in phenylephrine-contracted arteries from AT2R-/y mice, whereas it was unchanged in U46619-contracted arteries from AT2R-/y mice. C21 inhibited U46619-stimulated aggregation of human platelets, which was not inhibited by the AT2R-antagonist PD123319. C21 reduced U46619-induced recruitment of β-arrestin to human thromboxane TP-receptors with a calculated Ki of 3.74 µM. We conclude that in addition to AT2R-agonistic properties, C21 also acts as low-affinity TP-receptor antagonist, and that – depending on the constrictor – both mechanisms can be responsible for C21-induced vasorelaxation. Furthermore, by acting as a TP-receptor antagonist, C21 inhibits platelet aggregation. These findings are important for understanding potential off-target effects of C21 in the preclinical and clinical context and for the interpretation of C21-related myography data in assays with TXA2-analogues as constrictor.

7-N-Substituted-3-oxadiazole Quinolones with Potent Antimalarial Activity Target the Cytochrome bc1 Complex.

The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.

N-acetylcysteine (NAC) attenuates quorum sensing regulated phenotypes in Pseudomonas aeruginosa PAO1

The expression of many virulence genes in bacteria is regulated by quorum sensing (QS), and the inhibition of this mechanism has been intensely investigated. N-acetylcysteine (NAC) has good antibacterial activity and is able to interfere with biofilm-related respiratory infections, but little is known whether this compound has an effect on bacterial QS communication. This work aimed to evaluate the potential of NAC as a QS inhibitor (QSI) in Pseudomonas aeruginosa PAO1 through in silico and in vitro analyses, as well as in combination with the antibiotic tobramycin. Initially, a molecular docking analysis was performed between the QS regulatory proteins, LasR and RhlR, of P. aeruginosa with NAC, 3-oxo-C12-HSL, C4-HSL, and furanone C30. The NAC sub-inhibitory concentration was determined by growth curves. Then, we performed in vitro tests using the QS reporter strains P. aeruginosa lasB-gfp and rhlA-gfp, as well as the expression of QS-related phenotypes. Finally, the synergistic effect of NAC with the antibiotic tobramycin was calculated by fractional inhibitory concentrations index (FICi) and investigated against bacterial growth, pigment production, and biofilm formation. In the molecular docking study, NAC bound to LasR and RhlR proteins in a similar manner to the AHL cognate, suggesting that it may be able to bind to QS receptor proteins in vivo. In the biosensor assay, the GFP signal was turned down in the presence of NAC at 1000, 500, 250, and 125 μM for lasB-gfp and rhlA-gfp (p < 0.05), suggesting a QS inhibitory effect. Pyocyanin and rhamnolipids decreased (p < 0.05) up to 34 and 37%, respectively, in the presence of NAC at 125 μM. Swarming and swimming motilities were inhibited (p < 0.05) by NAC at 250 to 10000 μM. Additionally, 2500 and 10000 μM of NAC reduced biofilm formation. NAC-tobramycin combination showed synergistic effect with FICi of 0.8, and the best combination was 2500–1.07 μM, inhibiting biofilm formation up to 60%, besides reducing pyocyanin and pyoverdine production. Confocal microscopy images revealed a stronger, dense, and compact biofilm of P. aeruginosa PAO1 control, while the biofilm treated with NAC-tobramycin became thinner and more dispersed. Overall, NAC at low concentrations showed promising anti-QS properties against P. aeruginosa PAO1, adding to its already known effect as an antibacterial and antibiofilm agent.

Label-Free Multiplexed Microfluidic Analysis of Protein Interactions Based on Photonic Crystal Surface Mode Imaging.

High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.

Surface plasmon resonance assays for the therapeutic drug monitoring of infliximab indicate clinical relevance of anti-infliximab antibody binding properties.

The therapeutic antibody infliximab (IFX) has improved the life quality of numerous autoinflammatory disease patients. However, IFX can trigger the generation ofanti-drug antibodies (ADA), whose optimal evaluation andmanagement are currently subject of controversial discussions. We present two novel surface plasmon resonance (SPR) biosensor assays for therapeutic drug monitoring of IFX and characterization of ADA and investigated the diagnostic value of ADA binding properties. IFX and ADA were quantified via developed SPR biosensor assays (IFXmon and ADAmon, respectively) and diagnostics-approved ELISA in sera from inflammatory bowel disease patients. Pre-analytic ADA enrichment with magnetic beads enabled analytical drug tolerance of the ADAmon assay. The dissociation ratio (DissR) as an index forADA:IFX binding stability was calculated from the SPR sensorgrams of ADA quantification runs. IFX levels determined by IFXmon assay and ELISA showed high agreement, whereas ADA quantification concordance between ADAmon assay and ELISA was poor. In patients, DissR was predominantly constant over time and differed significantly between therapy outcomes. A DissR cut-off of 1.524 indicated undetectable IFX levels with 71.4% sensitivity and 88.9% specificity. Additionally, the SPR reference surface was exploited as serum-individual negative control to check result plausibility within multi-sample run sequences. Overall, both SPR biosensor assays exhibited reliable quantitative performance with accuracies superior to their ELISA counterparts and precision inferior to ELISA only for ADAmon. DissR presented itself as promising ADA binding parameter and could contribute to both earlier and more tailored therapeutic decisions.