Quick Assay Research Articles

Rapid molecular identification of Trichogramma (Hymenoptera: Trichogrammatidae) parasitizing the eggs of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae) in Serbia

BackgroundThe European corn borer (ECB), Ostrinia nubilalis (Hübner, 1796) (Lepidoptera: Crambidae), is the major pest of maize (Zea mays Linnaeus, 1753) in Serbia. One potential method for managing this pest is the augmentative release of naturally occuring egg parasitoids of the genus Trichogramma. The first step in this process is accurately identifying the naturally occuring species and estimating their natural distribution and abundance. Molecular identification, based on differences in DNA sequences, has commonly been employed for the identification of Trichogramma species. A simple, quick, and accurate molecular assay is urgently required for the identification of two common Trichogramma species, associated with ECB in Serbia: T. brassicae Bezdenko, 1968 and Trichogramma evanescens Westwood, 1833. Such an assay will facilitate an expansive survey of resident populations of Trichogramma associated with ECB across agricultural growing regions of Vojvodina province.ResultsA species-specific multiplex PCR assay for the 2 species was developed and validated that assay using a sample of 79 parasitoid wasps reared from ECB egg masses collected from sample sites across Vojvodina province. Trichogramma brassicae was confirmed as the dominant egg parasitoid of ECB in this region, accounting for 77 of the 79 wasps (97.47%). The remaining 2 were confirmed as T. evanescens. Trichogramma brassicae was detected at all 12 sample sites, while T. evanescens was detected at only 2 plots, Mokrin and Nakovo.ConclusionsThe species-specific multiplex PCR assay presented herein can provide the basis of a quick, cheap, and reliable means for identifying the species of Trichogramma that parasitize ECB egg masses in Serbia. Two currently documented species, T. brassicae and T. evanescens, are readily diagnosed by the size of the PCR product they produce in the assay. Any additional species are expected to not produce a band of a diagnostic size. Such species would subsequently be identified by sequencing, which may also allow them to be promptly incorporated into a revised assay.

Standard operating procedure for fluorescent thermal shift assay (FTSA) for determination of protein–ligand binding and protein stability

A standard operating procedure for a fluorescence-based thermal shift assay (FTSA) is provided describing its typical applications, advantages and limitations. FTSA is a simple, robust, universal and quick assay to determine protein-ligand binding affinities and protein stabilities in the presence of various excipients and solution conditions. Therefore, the assay is very useful for the straightforward characterization of new recombinantly produced proteins. The assay has a wide dynamic range enabling simultaneous determination of affinities in the milimolar to picomolar range. The assay could be used for essentially any protein that is sufficiently soluble and stable in the studied aqueous solution. Here we provide examples and typical experimental protocols for both affinity and stability determinations.

A novel one-step quick assay for detection of SARS-COV2 antibodies across mammalian species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has so far infected almost a hundred of millions of people and caused more than a million of death across the world. Many serological tests have been developed to track down virus infection in community via identification of antibodies against SARS-CoV2 virus. However, the tests vary in sensitivity, specificity, complexity, and speed. Here, I developed a simple, one-step, quick test to detect antibodies against SARS-CoV2 N (scN) nucleocapsid protein via direct visualization of antigen-antibody reaction. A total of 40 serum samples of SARS-CoV2 patients were purchased from RayBiotech. A total of 50 pre-pandemic human serum samples from San Diego Blood Bank were used as negative controls. After performing the one-step quick test of these 90 serum samples, I found that 39 samples are positive for anti-scN antibodies. All of the 39 positives are from the 40 SARS-CoV2 patients, suggesting that the one-step test is more sensitive than the lateral flow immunoassay (LFIA), the most widely used rapid antibody test. None of the 50 pre-pandemic samples is positive for anti-scN antibodies, indicating that the one-step test has an excellent specificity. The one-step test takes only ~5 min to detect the antibodies; and 1 ml of Escherichia coli culture can produce reagent proteins sufficient for thousands of the tests. Since the one-step test does not need a secondary antibody, it can be used as a universal test for anti-scN antibodies across different mammalian species to track down both human infection and the animal reservoir of SARS-CoV2 virus.

The prediction values of carbapenemase detection methods and carbapenem susceptibility testing for clinical outcomes of patients with Acinetobacter bacteremia under carbapenem treatment.

Carbapenem-resistant Acinetobacter species have emerged as notorious pathogens causing nosocomial infections. Several phenotypic methods have been developed for detecting carbapenemase production in Enterobacteriaceae. The accuracy of these methods in the prediction of carbapenemase production in Acinetobacter species has not been studied well. This retrospective study enrolled adult patients with Acinetobacter bacteremia from four medical centers in Taiwan between 2012 and 2016. Their demographics and clinical outcomes were recorded. The carbapenem susceptibility of the Acinetobacter species was determined using the agar diffusion method. The carbapenemase genes were detected by PCR. Four phenotypic methods, including the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), Carba NP test, and CarbAcineto NP test were carried out to determine the production of carbapenemase. We analyzed 257 adults who received initial carbapenem monotherapy for the treatment of Acinetobacter bacteremia. Shock within three days of bacteremia and acquisition of carbapenem non-susceptible isolates were independently associated with a higher 14-day and 30-day mortality in patients with Acinetobacter bacteremia. Among the four phenotypic tests for carbapenemase detection, MHT using the imipenem disc displayed the greatest sensitivity (94%; 95% confidence interval [CI], 89-97%) and specificity (81%; 95% CI, 73-88%) for predicting imipenem non-susceptibility. Carbapenem non-susceptibility and shock were independent risk factors for mortality in patients with Acinetobacter bacteremia. The MHT could predict the carbapenem susceptibility of Acinetobacter isolates. It is a cheap and quick assay, which could be applied in clinical practice.

An innovative and user-friendly smartphone-assisted molecular diagnostic approach for rapid detection of canine vector-borne diseases.

Present-day diagnostic tools and technologies for canine diseases and other vector-borne parasitic diseases hardly meet the requirements of an efficient and rapid diagnostic tool, which can be suitable for use at the point-of-care in resource-limited settings. Loop-mediated isothermal amplification (LAMP) technique has been always a method of choice in the development and validation of quick, precise, and sensitive diagnostic assays for pathogen detection and to reorganize point-of-care (POC) molecular diagnostics. In this study, we have demonstrated an efficient detection system for parasitic vector-borne pathogens like Ehrlichia canis and Hepatozoon canis by linking the LAMP assay to a smartphone via a simple, inexpensive, and a portable “LAMP box,” All the components of the LAMP box were connected to each other wirelessly. This LAMP box was made up of an isothermal heating pad mounted below an aluminum base which served as a platform for the reaction tubes and LAMP assay. The entire setup could be connected to a smartphone via an inbuilt Wi-Fi that allowed the user to establish the connection to control the LAMP box. A 5 V USB power source was used as a power supply. The sensitivity of the LAMP assay was estimated to be up to 10−6 dilution limit using the amplified, purified, and quantified specific DNA templates. It can also serve as an efficient diagnostic platform for many other veterinary infectious or parasitic diseases of zoonotic origin majorly towards field-based diagnostics.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00436-021-07077-z.

Improving evaluation of potato resistance to Rhizoctonia solani infection by optimizing inoculum-based method combined with toxin-based assay

Rhizoctonia solani causes stem canker and significantly impacts potato production. Conventional assay to evaluate potato resistance using R. solani inoculum is time consuming. To establish an effective and fast assay, 20 potato cultivars/lines were examined using both R. solani AG2-1 as an inoculum and its derived toxin (RS toxin). In field trials of 2009 and 2010, wheat-bran-mediated inoculum of R. solani AG2-1 was incorporated at either 0, 2, 3, 4 or 5 g per seed piece in the soil before sowing in the furrow. Stem canker was evaluated sixty-five days after planting. Inoculation at 2, 3, or 4 g R. solani AG2-1 well distinguished resistance of potato cultivars/lines. For a quick assay of resistance screening, a toxin-based method was established by treating potato seedlings with RS toxin of R. solani AG2-1. RS toxin was prepared by heating R. solani AG2-1 culture. Potato seedlings were obtained through tissue culture and grown in Murashige and Skoog medium. Seedlings at the stage of 12 cm in height were transferred into agar amended with RS toxin and incubated for eight days. The level of inhibition caused by RS toxin was positively correlated with toxin concentration. By evaluating various potato cultivars/liens that have different sensitivities to toxin, the inhibition was from 33% to 100% on potato stems sections and from 32% to 148% on seedlings. The toxin-based evaluation was highly correlated with field data using pathogen inoculum (r = 0.731, P < 0.01). Inoculation with wheat bran-mediated R. solani AG2-1 of 2, 3 or 4 g per seed piece was effective in field evaluations. The toxin-based assay improved the efficiency of disease resistance evaluation. Both assays showed that none of the 20 potato cultivars/liens was completely resistant to R. solani AG2-1. However, cultivar ‘Desiree’ had the lowest level of disease, whereas ‘Atlantic’, ‘Favorita’, and ‘Shepody’ were the most susceptible cultivars.

A new voltammetric approach for the determination of β-carotene in vegetables and pharmaceutical capsules using a gold electrode

Significant improvements in the voltammetric determination of β-carotene (BCA) have been achieved, mainly by the replacement of toxic dichloromethane with acetone and using non-mercury electrode. The respective procedure is based on anodic oxidation of BCA at a gold electrode in the disc configuration, when using square-wave voltammetry in pure acetone (99.8%) with 0.1 mol L−1 LiClO4 as the supporting electrolyte. The method comprises extraction of the analyte from the sample with acetone, thus avoiding the usually used highly toxic solvents. Analytically, it can be characterized by a linear range from 6.0 × 10−6 to 5.9 × 10−4 mol L−1 with regression equation Ipa = 0.0184c −0.1631 and correlation coefficient, R2 = 0.9998, limits of detection and quantification LOD = 1.6 × 10−6 mol L−1 and LOQ = 5.4 × 10−6 mol L−1, respectively; both being obtained at a potential step of 5 mV, with the pulse amplitude of 25 mV, and a frequency of 80 Hz. After optimization, the method was evaluated in series of analyses; namely, with two samples of vegetables and two pharmaceutical preparations (capsules), when the results could be compared to those of a reference spectrophotometric method. Due to a simple instrumentation, including sample preparation, the voltammetric method for the determination of BCA can be recommended as a quick screening assay in food and pharmaceutical analysis.

Insights into Plant Programmed Cell Death Induced by Heavy Metals-Discovering a Terra Incognita.

Programmed cell death (PCD) is a process that plays a fundamental role in plant development and responses to biotic and abiotic stresses. Knowledge of plant PCD mechanisms is still very scarce and is incomparable to the large number of studies on PCD mechanisms in animals. Quick and accurate assays, e.g., the TUNEL assay, comet assay, and analysis of caspase-like enzyme activity, enable the differentiation of PCD from necrosis. Two main types of plant PCD, developmental (dPCD) regulated by internal factors, and environmental (ePCD) induced by external stimuli, are distinguished based on the differences in the expression of the conserved PCD-inducing genes. Abiotic stress factors, including heavy metals, induce necrosis or ePCD. Heavy metals induce PCD by triggering oxidative stress via reactive oxygen species (ROS) overproduction. ROS that are mainly produced by mitochondria modulate phytotoxicity mechanisms induced by heavy metals. Complex crosstalk between ROS, hormones (ethylene), nitric oxide (NO), and calcium ions evokes PCD, with proteases with caspase-like activity executing PCD in plant cells exposed to heavy metals. This pathway leads to very similar cytological hallmarks of heavy metal induced PCD to PCD induced by other abiotic factors. The forms, hallmarks, mechanisms, and genetic regulation of plant ePCD induced by abiotic stress are reviewed here in detail, with an emphasis on plant cell culture as a suitable model for PCD studies. The similarities and differences between plant and animal PCD are also discussed.

Evaluation of a commercially-available surrogate virus neutralization test for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)

There remains an urgent need for assays to quantify humoral protective immunity to SARS-CoV-2 to understand the immune responses of COVID-19 patients, evaluate efficacy of vaccine candidates in clinical trials, and conduct large-scale epidemiological studies. The plaque-reduction neutralization test (PRNT) is the reference-standard for quantifying antibodies capable of neutralizing SARS-CoV-2. However, the PRNT is logistically demanding, time-consuming, and requires containment level-3 facilities to safely work with live virus. In contrast, a surrogate virus neutralization test (sVNT) manufactured by Genscript is a quick and simple assay that detects antibodies that inhibit the RBD-ACE2 interaction, crucial for virus entry into host cells. In this study, we evaluate the sensitivity, specificity, and cross-reactivity of the sVNT compared with the PRNT using both 50% and 90% SARS-CoV-2 neutralization as a reference-standard. We found that the sVNT provides a high-throughput screening tool prior to confirmatory PRNT testing for the evaluation of SARS-CoV-2 neutralizing antibodies.

Covariation of Ergot Severity and Alkaloid Content Measured by HPLC and One ELISA Method in Inoculated Winter Rye across Three Isolates and Three European Countries.

Ergot caused by Claviceps purpurea is a problem for food and feed security in rye due to the occurrence of toxic ergot alkaloids (EAs). For grain elevators and breeders, a quick, easy-to-handle, and cheap screening assay would have a high economic impact. The study was performed to reveal (1) the covariation of ergot severity (= percentage of sclerotia in harvested grain) and the content of 12 EAs determined by high performance liquid chromatography (HPLC) and (2) the covariation between these traits and results of one commercial enzyme linked immunosorbent assays (ELISA). In total, 372 winter rye samples consisting of a diverse set of genotypes, locations from Germany, Austria, and Poland over two years, and three isolates were analyzed. Ergocornine and α-ergocryptine were detected as major EAs. Ergocristinine occurred as a minor component. Claviceps isolates from different countries showed a similar EA spectrum, but different quantities of individual EAs. A moderate, positive covariation between ergot severity and EA content determined by HPLC was observed across two years (r = 0.53, p < 0.01), but large deviation from the regression was detected. ELISA values did neither correlate with the HPLC results nor with ergot severity. In conclusion, a reliable prediction of the EA content based on ergot severity is, at present, not possible.

Tutorial: design and fabrication of nanoparticle-based lateral-flow immunoassays

Lateral-flow assays (LFAs) are quick, simple and cheap assays to analyze various samples at the point of care or in the field, making them one of the most widespread biosensors currently available. They have been successfully employed for the detection of a myriad of different targets (ranging from atoms up to whole cells) in all type of samples (including water, blood, foodstuff and environmental samples). Their operation relies on the capillary flow of the sample throughout a series of sequential pads, each with different functionalities aiming to generate a signal to indicate the absence/presence (and, in some cases, the concentration) of the analyte of interest. To have a user-friendly operation, their development requires the optimization of multiple, interconnected parameters that may overwhelm new developers. In this tutorial, we provide the readers with: (i) the basic knowledge to understand the principles governing an LFA and to take informed decisions during lateral flow strip design and fabrication, (ii) a roadmap for optimal LFA development independent of the specific application, (iii) a step-by-step example procedure for the assembly and operation of an LF strip for the detection of human IgG and (iv) an extensive troubleshooting section addressing the most frequent issues in designing, assembling and using LFAs. By changing only the receptors, the provided example procedure can easily be adapted for cost-efficient detection of a broad variety of targets.

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases.

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)-two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 ± 3.6 µM and a Ki of 23.2 ± 3.2 µM. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Triplex qRT-PCR with specific probe for synchronously detecting Bovine parvovirus, bovine coronavirus, bovine parainfluenza virus and its applications.

Bovine parvovirus (BPV), bovine coronavirus (BCoV) and bovine parainfluenza virus (BPIV) are common etiologies causing gastrointestinal and respiratory diseases in dairy herds. However, there are few reports on the synchronous detection of BPV, BCoV and BPIV. The present article aimed to develop a quick and accurate RT-PCR assay to synchronously detect BPV, BCoV and BPIV based on their specific probes. One pair universal primers, one pair specific primers and one specific probe was designed and synthesized. After the concentrations of primer and probe and annealing temperature were strictly optimized, the specificity, sensitivity and repeatability of the established triplex probe qRT-PCR were evaluated, respectively. The results showed the recombinant plasmids of pMD18-T-BPV, pMD18-T-BCoV and pMD18-T-BPIV were 554bp, 699bp and 704bp, respectively. The optimal annealing temperature was set at 45.0°C for triplex qRT-PCR. The triplex probe qRT-PCR can only synchronously detect BPV, BCoV and BPIV. Detection sensitivities were 2.0×102, 2.0×102 and 2.0×101 copies/μL for BPV, BCoV and BPIV, being 1000-fold greater than that in the conventional PCR. Detection of clinical samples demonstrated that triplex probe qRT-PCR had a higher sensitivity and specificity. The intra-assay and inter-assay coefficient of variation were lower than 2.0%. Clinical specimens verified that the triplex qRT-PCR had a higher sensitivity and specificity than universal PCR. In conclusion, this triplex probe qRT-PCR could detect only BPV, BCoV and BPIV. Minimum detection limits were 2.0×102 copies/μL for BPV and BCoV, and 2.0×101 copies/μL for BPIV. The sensitivity of this triplex probe qRT-PCR was 1000-fold greater than that in the conventional PCR. The newly qRT-PCR could be used to monitor or differentially diagnose virus infection.

Computational Evaluation and In Vitro Validation of New Epidermal Growth Factor Receptor Inhibitors.

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein that acts as a receptor of extracellular protein ligands of the epidermal growth factor (EGF/ErbB) family. It has been shown that EGFR is overexpressed by many tumours and correlates with poor prognosis. Therefore, EGFR can be considered as a very interesting therapeutic target for the treatment of a large variety of cancers such as lung, ovarian, endometrial, gastric, bladder and breast cancers, cervical adenocarcinoma, malignant melanoma and glioblastoma. We have followed a structure-based virtual screening (SBVS) procedure with a library composed of several commercial collections of chemicals (615,462 compounds in total) and the 3D structure of EGFR obtained from the Protein Data Bank (PDB code: 1M17). The docking results from this campaign were then ranked according to the theoretical binding affinity of these molecules to EGFR, and compared with the binding affinity of erlotinib, a well-known EGFR inhibitor. A total of 23 top-rated commercial compounds displaying potential binding affinities similar or even better than erlotinib were selected for experimental evaluation. In vitro assays in different cell lines were performed. A preliminary test was carried out with a simple and standard quick cell proliferation assay kit, and six compounds showed significant activity when compared to positive control. Then, viability and cell proliferation of these compounds were further tested using a protocol based on propidium iodide (PI) and flow cytometry in HCT116, Caco-2 and H358 cell lines. The whole six compounds displayed good effects when compared with erlotinib at 30 μM. When reducing the concentration to 10μM, the activity of the 6 compounds depends on the cell line used: the six compounds showed inhibitory activity with HCT116, two compounds showed inhibition with Caco-2, and three compounds showed inhibitory effects with H358. At 2 μM, one compound showed inhibiting effects close to those from erlotinib. Therefore, these compounds could be considered as potential primary hits, acting as promising starting points to expand the therapeutic options against a wide range of cancers.

Performance of rapid tests in the management of dengue fever imported cases in Lazio, Italy 2014-2019

BackgroundIn Italy, dengue virus is the most frequent agent of imported viral infections. The use of rapid diagnostic tests (RDTs) may be of help as a preliminary user-friendly quick assay to facilitate dengue diagnosis, as ordinary laboratory diagnosis of dengue fever may require special efforts in terms of tools availability, interpretation of results, and skilled personnel. The performance of RDTs, however, may vary according to different epidemiological and laboratory background. MethodsWe reviewed five years of laboratory records of two dengue RDT results (Colorimetric SD-Bioline Dengue-Duo-RDT and Fluorimetric SD-Biosensor-STANDARD-F-Dengue-RDT), able to detect viral NS1 antigen and specific IgM and IgG. Diagnostic parameters were calculated using as reference the results of molecular (RT-PCR) and serological (immunofluorescence, IFA) tests. Overall performance, calculated considering the final case definition, was included in the accuracy assessment of RDTs. ResultsThe combined use of NS1 and IgM/IgG RDT for the detection of acute dengue cases resulted in an overall sensitivity and specificity of 87.2% and 97.9% for Colorimetric RDT, 96.2% and 96.2% for Fluorimetric RDT. NS1 was the most reliable marker of acute infection, while IgM resulted falsely positive in nine samples, including sera derived from 2 Zika and 4 non-arbovirus infected patients. ConclusionsThe inclusion of RDT in the diagnostic algorithm is of undeniable help in the prompt management and surveillance of dengue infection in non-endemic areas. Confirmatory tests are, however, necessary to rule in or rule out dengue fever diagnosis.

Schisandra rubriflora Plant Material and In Vitro Microshoot Cultures as Rich Sources of Natural Phenolic Antioxidants.

Schisandra rubriflora is a dioecious, underestimated medicinal plant species known from traditional Chinese medicine. The present study was aimed at characterising the polyphenolic profile composition and the related antioxidant capacity of S. rubriflora fruit, stem and leaf and in vitro microshoot culture extracts. Separate analyses of material from female and male specimens were carried out. This study was specifically aimed at detailed characterisation of the contribution of phenolic compounds to overall antioxidant activity using ultra-high-performance liquid chromatography with a photodiode array detector coupled to electrospray ionization ion trap mass spectrometry (UHPLC-DAD-ESI-MS3) and a high-performance liquid chromatography-diode array detector (HPLC-DAD). Using UHPLC-DAD-ESI-MS3, twenty-seven phenolic compounds from among phenolic acids and flavonoids were identified. Concentrations of three phenolic acids (neochlorogenic, chlorogenic and cryptochlorogenic acids) and eight flavonoids (hyperoside, rutoside, isoquercitrin, guaijaverin, trifolin, quercetin, kaempferol, and isorhamnetin) were determined using HPLC-DAD using reference standards. The highest total phenolic content was confirmed for the stem and leaf extracts collected in spring. The contents of phenolic compounds of in vitro biomasses were comparable to that in the fruit extracts. The methanolic extracts from the studied plant materials were evaluated for their antioxidant properties using various in vitro assays, namely free radicals scavenging estimation using 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), ferric-reducing antioxidant power (FRAP) and cupric-reducing antioxidant capacity (CUPRAC) as well as QUick, Easy, New, CHEap, and Reproducible CUPRAC (QUENCHER-CUPRAC) assays. A close relationship between the content of polyphenolic compounds in S. rubriflora and their antioxidant potential has been documented.

A cell suspension based uptake method to study high affinity glucosinolate transporters

BackgroundGlucosinolates are an important class of secondary metabolites characteristic to the order Brassicales. They are known to play a major role in plant defense and from the human perspective, can be anticarcinogenic or antinutritive. GTRs are plasma-membrane localized high affinity glucosinolate transporters, which are important components of the source (leaf) to sink (seed) translocation of intact glucosinolates in members of Brassicaceae family. GTRs are identified as major candidates for Brassica crop improvement, thus dictating a need for their functional characterization. However, currently there are limitations in availability of heterologous assay systems for functional characterization of plant secondary metabolite transporters. To date, the animal-based Xenopus oocyte system is the best established heterologous system for functional characterization of these transporters. Inherent biochemical and physiological attributes unique to the plant membranes necessitate the need for developing plant-based transporters assay systems as well.MethodsIn this study, Agrobacterium mediated transformation was used to develop GTR expressing cotton cell lines (CCL-1) for functional characterization of the Arabidopsis high affinity glucosinolate transporters, AtGTR1 and AtGTR2. Following sub-cellular localization of AtGTRs, we standardized the glucosinolate uptake assays using cell suspension cultures of AtGTR expressing CCL-1 its requirement of pH, salt, and time based glucosinolate uptake. Using the GTR expressing CCL-1, we subsequently performed kinetic analysis of AtGTR1 and AtGTR2 for different glucosinolate substrates, sinigrin, gluconapin and sinalbin.ResultsSeveral clones expressing each of AtGTR1 and AtGTR2 were obtained showing high level of GTR expression and were maintained through regular sub-culturing. Both AtGTR1 and AtGTR2 are predominantly plasma-localized proteins when overexpressed in CCL-1 cells. Uptake assays were standardized, suggesting that glucosinolate uptake of GTR expressing CCL-1 is robust within the physiological pH range 5–6, and at lower concentration of nitrate salts. GTR expressing CCL-1 cells show increasing glucosinolate accumulation in time course experiment. Kinetic studies over a wide glucosinolate concentrations (10–800 µM) revealed that our novel assay system displayed robust GTR-mediated uptake of different glucosinolates and unambiguously helps elucidate the saturable kinetics of GTRs. Our system confirms the high affinity of AtGTRs for both aliphatic and aromatic glucosinolates.ConclusionThe transporter assay system described in this study holds potential for studying sub-functionalization amongst GTR homologs present across Brassicaceae family. The fast growing CCL-1 cells, confer the benefits of an in vitro system for quick assays and is plant based thus enabling optimal expression without sequence modifications. The efficient functioning of the GTR transporters in the heterologous CCL-1 opens the possibility of using this plant cell suspension system for functional characterization of other metabolite transporters.

Development of UPLC–MS/MS method for studying the pharmacokinetic interactions of pexidartinib with antifungal drugs in rats

Pexidartinib was approved in the USA for targeted therapy of adult patients with symptomatic tenosynovial giant cell tumour (TGCT) by the FDA. The purpose of our study was to develop and establish a quick assay based on ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) for the measurement of pexidartinib concentrations in plasma and to survey whether antifungal drugs (isavuconazole, posaconazole, fluconazole and itraconazole) could change the pharmacokinetic parameters of pexidartinib in rats. After the quick protein crash with acetonitrile, the chromatographic separation of pexidartinib and upadacitinib (used as the internal standard in this study, IS) were conducted on an Acquity BEH C18 (2.1 × 50 mm, 1.7 μm) column, and the detection of the analyte was also accomplished with a Xevo TQ-S triple quadrupole tandem mass spectrometer in the positive ion electro-spray ionization (ESI) interface. The assay showed good linearity in the range of 1−7000 ng/mL. The accuracy and precision were all within the acceptable limits in the bioanalytical method, and the results of recovery, matrix effect, stability, and carry-over were also met the requirements. The application of the validated UPLC–MS/MS bioanalytical method was further successfully involved in the drug-drug interactions study from rats. It was found that fluconazole and itraconazole significantly increased the concentration of pexidartinib and had the inhibitory effect on the metabolism of pexidartinib, while not isavuconazole and posaconazole. Thus, more attention should be paid to the concurrent use of pexidartinib with fluconazole or itraconazole to reduce the risk of unexpected clinical outcomes.

A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems.

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here, we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate-NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanolitre scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed a split NanoLuc-based assay that enables the real-time monitoring of type III secretion-dependent injection of effector-HiBiT fusions into host cells stably expressing the complementing NanoLuc-LgBiT.

A Variety of Bio-nanogold in the Fabrication of Lateral Flow Biosensors for the Detection of Pathogenic Bacteria.

Pathogenic bacteria constitute one of the most serious threats to human health. This has led to the development of technologies for the rapid detection of bacteria. Bio-nanogold-based lateral flow biosensors (LFBs) are a promising assay due to their low limit of detection, high sensitivity, good selectivity, robustness, low cost, and quick assay performance ability. The aim of this review is to provide a critical overview of the current variety of bio-nanogold LFBs and their targets, with a special focus on whole-cell and DNA detection of pathogenic bacteria. The challenges of bio-nanogold-based LFBs in improving their performance and accessibility are also comprehensively discussed.