Reliable Assay Research Articles

Comparison between monotest and traditional batch-based ELISA assays for therapeutic drug monitoring of infliximab and adalimumab levels and anti-drug antibodies.

To compare a new ready-to-use monotest immunoassay, CHORUS Promonitor, for the quantification of serum biological drug levels and anti-drug antibodies of anti-TNF agents, against the reference batch-based ELISA test, Promonitor. Blood samples were collected from patients treated with anti-TNF agents, infliximab (IFX) or adalimumab (ADL). IFX and ADL levels, as well as anti-IFX and anti-ADL antibodies were quantified and compared between the standard ELISA reference test, Promonitor, and the automated monotest ELISA assay, CHORUS Promonitor. Data analysis included both qualitative and quantitative comparison between both tests. For the qualitative comparison, overall percent agreement (OPA) was calculated. For the quantitative comparison, Passing-Bablok regression analysis and Bland-Altman analysis were used. For IFX and ADL levels, the qualitative overall agreement between methods was 100 % (Cohen's coefficient=1). For anti-IFX and anti-ADL antibodies, OPA was 98.8 % and 97.3 %, respectively. Quantitative comparison indicated a very strong correlation between both assays: IFX (r=0.97, n=74), ADL (r=0.95, n=54), anti-IFX (r=0.93, n=72), and anti-ADL (r=0.97, n=61). The regression analysis determined an excellent comparability of drug levels between methods. Bland-Altman analysis showed a bias difference between assays of 6 % for IFX, 0 % for ADL, 24 % for anti-IFX, and 14 % for anti-ADL. Monotest CHORUS Promonitor was a reliable assay to quantify IFX, ADL, anti-IFX and anti-ADL in samples with comparable results to those obtained with the reference batch-based ELISA technique.

Vector induced humoral responses after rVSVΔG-ZEBOV-GP immunization identify vaccinated individuals and correlate with Ebola virus glycoprotein antibodies

Abstract Background The robustness and persistence of vaccine antigen-induced antibodies are often used as proxy indicators of vaccine efficacy, but immune responses to vaccine vectors are typically less well-defined. Our study considered the kinetics of immunoglobulin (IgG) responses against the vector (vesicular stomatitis Indiana virus [VSIV]) nucleoprotein (N) and the inserted antigen (Ebola virus [EBOV]) glycoprotein (GP1,2) components of the rVSVΔG-ZEBOV-GP (rVSV-ZEBOV) vaccine and evaluated their use as biomarkers to confirm self-reported vaccination status. Methods From the Partnership for Research on Ebola Virus in Liberia (PREVAIL) I clinical trial (NCT02344407), we randomly selected 212 participants who received rVSV-ZEBOV (n=107) or placebo (n=105). Levels of IgG antibodies to EBOV GP1,2 or VSIV N were measured using the Filovirus Animal Non-Clinical Group (FANG) ELISA and a newly developed single-molecule array (Simoa) immunoassay, respectively. Results Anti-EBOV GP1,2 IgG and anti-VSIV N IgG were first detected 10–14 d post-vaccination, further increased at 28 d, and remained stable through 360 d. Antibody titers were significantly higher in women compared to men. Anti-EBOV GP1,2 and anti-VSIV N IgG titers were significantly correlated (p<0.001) at 28 d (r=0.47), 180 d (r=0.45), and 360 d (r=0.59). At 28 d, the area under the curve (AUC) of receiver operating characteristic (ROC) curves discriminated vaccinated from unvaccinated patients with high accuracy (AUC=0.965 for anti-VSIV N IgG; AUC=0.945 for anti-EBOV GP1,2 IgG [p<0.001]). Conclusions We report a reliable assay to measure vector-induced humoral responses after rVSV-ZEBOV vaccination and demonstrate the assay’s utility to confirm vaccination status.

A Rapid and Reliable Absorbance Assay to Identify Drug-Drug Interactions with Thiopurine Drugs.

Thiopurine methyltransferase (TPMT) plays a crucial role in the detoxification of thiopurine drugs, including the antimetabolites azathioprine and 6-mercaptopurine (6-MP) used to treat autoimmune diseases and various cancers. These drugs interfere with DNA synthesis by inhibiting the production of purine-containing nucleotides, leading to the death of rapidly dividing cells. TPMT inactivates thiopurine drugs by methylating at the thiol group. The activity of TPMT can vary significantly between individuals, and its activity is impacted by co-administered drugs, altering the effectiveness and toxicity of thiopurine drugs. TPMT is inhibited by many drugs that are co-administered to treat symptoms associated with diseases treated with thiopurines. For example, aspirin and other anti-inflammatory drugs, including olsalazine, sulfasalazine, and balsalazide, inhibit TPMT. The impact of TPMT genotypes on its methylating activity is well defined, and genotyping patients to identify TPMT metabolizer status is common clinical practice. Unfortunately, there has been no concerted effort to comprehensively identify drugs on the market that impact TPMT activity. The inhibition of TPMT by co-administered drugs could in part be responsible for idiosyncratic toxicities associated with thiopurine drug therapy. Here, we report a facile approach to produce large quantities of recombinant TPMT and a high-throughput assay that utilizes the shift in absorbance due to the methylation of thiopurines to report on TPMT activity. With purified TPMT on hand and the absorbance activity assay, we confirmed several compounds that inhibit TPMT, and the results were comparable to a mass spectral assay that measured 6-MP methylation. Understanding the impact of co-administered drugs on TPMT activity will improve the safety and efficacy of thiopurine-based treatment regimens.

Longitudinal determination of seroprevalence and immune response to SARS-CoV-2 in a population of food and retail workers through decentralized testing and transformation of ELISA datasets.

Since the onset of the global COVID-19 pandemic in early 2020, numerous studies have been conducted worldwide to understand our immune response to the virus and to vaccination. This study investigates the humoral response elicited by SARS-CoV-2 infection and by vaccination in the poorly studied population of food and retail workers. These occupations were classified as essential by the Public Health Agency of Canada, potentially placing this population at greater risk of infection. Such a risk requires access to reliable and adaptable serological assays that can be rapidly deployed to guide public health strategies. Here we investigate the benefits and limitations of applying adaptable, decentralized tests for population-level immune surveillance in response to a pandemic, even before centralized testing is available. The 1.5-year study period spans from early 2021, when vaccination became available in this region, to mid-2022, following the emergence of the first Omicron variants. The cohort of 304 food and retail workers was recruited in the Québec City area. Participants attended five evenly spaced visits, providing blood samples as well as information on SARS-CoV-2 symptoms or risk factors, prior antigen or PCR test results and vaccination status, as well as work-related risk factors and protective measures. Parallel COVID-19 serological assays were performed using both a standardized chemiluminescent ELISA assay at the centralized platform operated in partnership with the Public Health Agency of Canada, and a semi-automated in-house colorimetric ELISA assay developed at our decentralized site. The YES/NO determination of SARS-CoV-2 vaccine seroconversion and/or infection events using the SARS-CoV-2 ancestral spike protein and nucleocapsid protein validated coherence of the centralized and decentralized assays. The flexibility of the decentralized assays allowed broadening the study to determine cross-reactivity of IgG directed against the spike protein of the SARS-CoV-2 Delta and Omicron VOCs, and IgM directed against the ancestral spike and nucleocapsid proteins. The nature of the data obtained in the decentralized assays allowed treatment with a recently developed mathematical transformation to obtain normal distribution, enabling ANOVA-Welsh statistical analysis. Although no significant differences were observed in humoral response as related to BMI, age, level of education, or chronic illnesses in this cohort of workers, statistically higher levels of vaccine-induced antibodies were observed for restaurant workers and hardware store workers in the early stages of the study, compared to workers in bars and grocery stores and in non-smokers versus smokers. This work highlights the importance of developing adaptable, decentralized tests for population-level immune surveillance in response to a pandemic, even before centralized testing is available. To our knowledge, no other study has reported such an extensive longitudinal investigation during key periods of the COVID-19 pandemic in a cohort of food and retail workers to analyze two types of immunoglobulin, three epitopes and antigens to three VOC. This study will inform strategies and measures to be implemented in the event of a future pandemic.

Direct TaqMan assay for the detection and genotyping of bovine viral diarrhea virus types 1 and 2

Bovine viral diarrhea (BVD), caused by bovine viral diarrhea virus (BVDV), has a significant economic impact on affected farms worldwide. For effective disease control, it is crucial to select an appropriate vaccine based on the specific genotype of BVDV. Therefore, developing a rapid and reliable assay to detect and genotype BVDV is imperative for controlling the spread of disease. In this study, we developed a TaqMan assay to detect and genotype BVDV types 1 and 2 directly in bovine serum without extraction of RNA. The direct BVDV TaqMan assay effectively detected both BVDV1 and BVDV2 with confirmed specificity and showed no cross-reactivity with any of the other viruses tested, including bovine respiratory syncytial virus, bovine coronavirus, Akabane virus, bovine herpesvirus 1, bovine parainfluenza virus 3, bovine immunodeficiency virus, and bovine leukemia virus. The assay could detect the virus in serum samples with a titer as low as 102 TCID50/mL in two out of three trials for BVDV1 and all three trials for BVDV2, indicating that its sensitivity is equivalent to that of virus isolation. Our findings represent a significant advancement in BVDV detection and typing directly from bovine serum.

Machine Learning and Metabolomics Predict Mesenchymal Stem Cell Osteogenic Differentiation in 2D and 3D Cultures.

Stem cells have been widely used to produce artificial bone grafts. Nonetheless, the variability in the degree of stem cell differentiation is an inherent drawback of artificial graft development and requires robust evaluation tools that can certify the quality of stem cell-based products and avoid source-tissue-related and patient-specific variability in outcomes. Omics analyses have been utilised for the evaluation of stem cell attributes in all stages of stem cell biomanufacturing. Herein, metabolomics in combination with machine learning was utilised for the benchmarking of osteogenic differentiation quality in 2D and 3D cultures. Metabolomics analysis was performed with the use of gas chromatography-mass spectrometry (GC-MS). A set of 11 metabolites was used to train an XGboost model which achieved excellent performance in distinguishing between differentiated and undifferentiated umbilical cord blood mesenchymal stem cells (UCB MSCs). The model was benchmarked against samples not present in the training set, being able to efficiently capture osteogenesis in 3D UCB MSC cultures with an area under the curve (AUC) of 82.6%. On the contrary, the model did not capture any differentiation in Wharton's Jelly MSC samples, which are well-known underperformers in osteogenic differentiation (AUC of 56.2%). Mineralisation was significantly correlated with the levels of fumarate, glycerol, and myo-inositol, the four metabolites found most important for model performance (R2 = 0.89, R2 = 0.94, and R2 = 0.96, and p = 0.016, p = 0.0059, and p = 0.0022, respectively). In conclusion, our results indicate that metabolomics in combination with machine learning can be used for the development of reliable potency assays for the evaluation of Advanced Therapy Medicinal Products.

An adjusted digital droplets PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products

Background and PurposeGenetically engineered T cell therapy holds immense promise in cancer immunotherapy. These T cell products are typically engineered by vectors that permanently integrate into the T cell genome, thus raising concerns about potential risks of insertional mutagenesis. Therefore, it becomes imperative to assess the integrated vector copy number (VCN) as a critical safety parameter for gene-engineered cell products. Materials and MethodsIn this study, we developed a robust assay for assessing the VCN of CAR-T cell and TCR T cell products, based on the digital droplet polymerase chain reaction (ddPCR) method. To provide accurate representation of the VCN in gene-engineered cells, we implemented a calculation that factors in the putative transduction efficiency based on Poisson distribution statistics. The adjusted VCN value (VCNadj) was also compared with VCN value from sorted transgene-positive cell populations, to validate its accuracy. ResultsThis assay consistently and accurately determines the average VCN for cell products. By comparing the VCN in sorted transgene-positive cell populations, we validated the refinement calculation provides a closer approximation to the actual VCN within transduced cells, offering a more realistic representation of the VCN for engineered cell products. ConclusionIn summary, we present a reliable and robust ddPCR-based assay for quantification of vector copy number in gene-engineered cell products.

Validation and TaqMan Conversion of a Molecular Chronotype Assessment Approach.

The present study aimed to develop a TaqMan genotyping card for molecular chronotype assessment based on a predictive panel of 35 previously identified genetic variants. A reliable TaqMan assay was successfully developed for 33 out of the 35 chronotype-predictive variants. The resulting TaqMan genotyping card was utilized to genetically characterize 196 new individuals (in addition to the previously studied 96) and the Morningness-Eveningness Questionnaire was utilized for their phenotypical chronotype assessment. The predictive panel performance was validated on (a) a group of morning and evening individuals (logistic regression model), (b) a representative sample of the original study population also including intermediate chronotypes (linear regression model) and, (c) 25,986 individuals from the Estonian Biobank, for whom Munich Chronotype Questionnaire scores were available. The validation of the morningness-eveningness logistic regression model on 25 morning and 21 evening types resulted in a predictive value of 72%, confirming the reliability of the predictive panel and the success of its conversion into a TaqMan genotyping card. By contrast, the inclusion of intermediate individuals in the model led to a significant decrease in predictive performance (45% on 100 individuals [25 morning, 54 intermediate, and 21 evening]), with intermediate types being the most affected. No significant associations were observed between the genotype panel and chronotype in the Estonian Biobank sample. In conclusion, our genotyping card might represent a promising molecular chronotyping tool for the Italian population. Its performance in other populations is worthy of further study.

Development of RT-qPCR assay for assessing the expression of ACTB and SDHA housekeeping genes in the cell cultures of mammalian hosts of zoonotic infections

Background. The molecular mechanisms behind the maintenance of zoonotic pathogens in nature can be better understood by examining the gene expression in host cells in response to the infection. Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) is a powerful method to study gene expression, especially with the use of endogenous reference genes (RG) to normalize the data. Therefore, it is critical to develop the reliable qRT-PCR assay and validate that selected RGs are stably expressed in the studied organism or cell culture.The aim. In this work, we aimed to develop the real-time qRT-PCR method for detecting mRNA of two candidate RGs, succinate dehydrogenase subunit A (SDHA) and beta-actin (ACTB), in the cell lines of mammalian hosts of zoonotic infections.Materials and methods. The SPEV (porcine embryonic kidney cell line) and ApnK (Korean field mouse kidney cell line) cells were grown in 24-well culture plates. Total RNA/DNA was isolated from trypsin-detached cell monolayers. Genomic DNA in the samples was removed with RNase-free DNase I, and one-step RT-qPCR was performed using primers for SDHA and ACTB gene fragments and the corresponding TaqMan hydrolysis probes. The experiment was performed in 4 independent replicates.Results. In the Korean field mouse cells, the linearity, efficiency, repeatability, and reproducibility of the RT-qPCR for ACTB gene mRNA corresponded to the modern requirements. However, RT-qPCR for SDHA exhibited good linearity and efficiency of the reaction, but CV values for repeatability and reproducibility slightly exceeded the recommended standards. In porcine cells, both assays had acceptable parameters. Thus, to use the SDHA as RG for ApnK cells, a detailed study of the stability of its expression in this particular model is required.Conclusions. New qRT-PCR assay was developed to assess the expression of housekeeping genes ACTB and SDHA in the cells of the Korean field mouse and domestic pig. Further research is necessary to validate these genes as references for quantitative assessment of gene expression in the cells of mammalian hosts of zoonotic infections.

Development of a Fluorescence Probe for High-Throughput Screening of Allosteric Inhibitors Targeting TRAP1.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a molecular chaperone implicated in pro-tumorigenic pathways by regulating the folding of substrate proteins (clients) within cancer cells. Recent research has pinpointed a potentially druggable allosteric site within the client binding site (CBS) of TRAP1, suggesting this site might offer a more effective strategy for developing potent and selective TRAP1 inhibitors. However, the absence of reliable assay systems has hindered quantitative evaluation of inhibitors. In this study, we have developed a fluorescent probe, Rho6TPP, designed to target the CBS. Utilizing fluorescence polarization-based high-throughput screening assays, Rho6TPP exhibits excellent signal-to-noise ratio (>20), Z factor (>0.6), and Z' factor (>0.6). Additionally, it facilitates comparative analysis of existing small molecules and discovery of novel binders. MitoTam, a mitochondria-targeted tamoxifen, emerges as a potent CBS-targeting TRAP1 inhibitor. Our findings highlight the potential of Rho6TPP as a crucial tool for advancing the development of CBS-targeting TRAP1 inhibitors.

The oxidative potential of nanomaterials: an optimized high-throughput protocol and interlaboratory comparison for the ferric reducing ability of serum (FRAS) assay

Successful implementation of Safe and Sustainable by Design (SSbD) and grouping approaches requires simple, reliable, and cost-effective assays to facilitate hazard screening at early stages of product development. Especially for nanomaterials (NMs), which exist in many different forms, efficient hazard screening is of utmost importance. Oxidative potential (OP), which is the ability of a substance to induce reactive oxygen species (ROS), is an important indicator of the potential to induce oxidative damage and oxidative stress. A frequently used assay to measure OP of NMs is the ferric reducing ability of serum (FRAS) assay. Although the widely used cuvette-based FRAS protocol is considered a robust assay, its low throughput makes the screening of multiple materials challenging. Here, we adapt the original cuvette-based FRAS assay protocol, into a 96-well format and thereby improve its user-friendliness, simplicity, and screening capacity. The adapted protocol allows for the screening of multiple NMs per plate, and multiple plates per day, where the original protocol allows for the screening of one NM dose-range per day. When comparing the two protocols, the adapted protocol showed slightly decreased assay precision as compared to the original protocol. The results obtained with the adapted protocol were compared using eight reference NMs in an interlaboratory study and showed acceptably low intra- and interlaboratory variation. We conclude that the adapted FRAS assay protocol is suitable to be used for hazard screening to facilitate SSbD and grouping approaches.

Dual-mode, signal-amplified DNA biosensor for label-free, reliable assay of gestational diabetes mellitus-related miRNA (miR-135a)

miR-135a is highly expressed in patients with gestational diabetes mellitus, and its target genes are also involved in insulin signaling pathway, so it is one biomarker for gestational diabetes mellitus. Herein we designed a dual-mode DNA biosensor for reliable assay of miR-135a based on the fluorescence and colorimetric signals. Several experiments were carried out to demonstrate the assay feasibility and mechanism for this dual-mode DNA biosensor. With optimum parameters, this proposed dual-mode biosensor has been realized sensitive and quantitative assay of miR-135a. For the fluorescence and colorimetric signals, the working ranges are 0.56–61 and 8.3–74 nM, while limits of detection are 0.18 and 3.7 nM respectively. This dual-mode strategy allows two independent signals for miR-135a assay, so it can verify each other to show more accurate results with good fidelity. Furthermore, there is a good selectivity in the biosensor for target miR-135a over other nucleotide variants, as well as good anti-interference ability in complex samples. This dual-mode DNA biosensor provides a new approach for miR-135a assay and miRNA expression profiling in gestational diabetes mellitus.

Key Amino Acid Residues of the Agt1 Transporter for Trehalose Transport by Saccharomyces cerevisiae.

Trehalose is crucial for the stress resistance of Saccharomyces cerevisiae, primarily through its stabilization of proteins and membranes. The Agt1 transporter, a member of the Major Facilitator Superfamily, mediates trehalose uptake, a key process for maintaining cellular integrity under stress. Despite its importance, the molecular mechanisms of Agt1-mediated trehalose transport remain underexplored. In this study, we expressed and purified the trehalase enzyme TreA from E. coli to develop reliable trehalose assays. We screened 257 wild S. cerevisiae isolates, identifying strains with enhanced trehalose transport capacities. Comparative analyses, including structural modeling and molecular docking, revealed that specific Agt1 variants exhibited significantly higher transport efficiency, influenced by key residues in the transporter. Molecular dynamics simulations and steered molecular dynamics provided further insights, particularly into the role of the Agt1 channel head region in substrate recognition and binding. Site-directed mutagenesis validated these findings, showing that mutations at critical residues, such as 156Q, 164L, 256Q, 395E, 396R, and 507Y significantly reduced transport activity, while 137Q, 230T, and 514 N increased efficiency under certain conditions.

Design, development, and validation of a strand-specific RT-qPCR assay to differentiate replicating versus nonreplicating Rabies virus

The Rabies virus, a single-strand RNA virus with a negative-sense polarity, is responsible for causing encephalitis and is a zoonotic disease. If not promptly treated after infection, it has a close to 100 % fatality rate. Similar to other negative-sense polarity single-strand RNA viruses, the Rabies virus requires the creation of a positive-strand RNA intermediate for replication. One approach to identify this replication activity is to detect the complementary strand of the viral RNA genome in suspected infected cells or tissues. The reported Rabies virus RT-qPCR detection methods are designed to detect total viral load in samples without distinguishing between positive- and negative-strand for RNA viruses. As such, in this study, a sensitive Taqman-based strand-specific RT-qPCR assay has been developed to quantitatively detect both the positive- and negative-strand of the Rabies virus. This method demonstrates good reproducibility across a wide dynamic range and exhibits linearity of 8 logs with a lower limit of detection of 103 copies/μL for the positive-strand and 9 logs with a lower limit of detection of 102 copies/μL for the negative-strand. Notably, it can accurately detect a specific viral RNA strand even in the presence of high levels of the opposite strand, confirming the method’s specificity. In summary, a reliable strand-specific RT-qPCR assay has been developed and validated to differentiate replicating from non-replicating Rabies virus.

Antibiotic susceptibility testing using minimum inhibitory concentration (MIC) assays

Antimicrobial resistance is due to genetic changes that allow bacteria to evade antibiotic treatment. Antimicrobial susceptibility testing is critical for the detection of antibiotic-resistant strains, the selection of effective therapeutic strategies against bacterial infections, and the evaluation of the efficacy of novel antimicrobials. Among the variety of clinical microbiology methods used for antibiotic susceptibility testing, minimum inhibitory concentration (MIC) assays have become the gold standard in clinical practice. MIC assays determine the lowest concentration of an antimicrobial agent that is required to inhibit visible bacterial growth in vitro. Here, we outline MIC assay protocols, in strict accordance with European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines that aim to assess the susceptibility of non-fastidious organisms to antimicrobial agents. The protocols described in this methods paper are intended to aid the performance of reliable and informative MIC assays for research purposes that are in line with clinical microbiology practices.

High Content Screening Assay of Inhibitors of the Legionella Pneumophila Histone Methyltransferase RomA in Infected Cells.

Resistance to anti-microbial agents is a world-wide health threat. Thus, there is an urgent need for new treatments. An alternative approach to disarm pathogens consists in developing drugs targeting epigenetic modifiers. Bacterial pathogens can manipulate epigenetic regulatory systems of the host to bypass defences to proliferate and survive. One example is Legionella pneumophila, a Gram-negative intracellular pathogen that targets host chromatin with a specific, secreted bacterial SET-domain methyltransferase named RomA. This histone methyltransferase specifically methylates H3 K14 during infection and is responsible for changing the host epigenetic landscape upon L. pneumophila infection. To inhibit RomA activity during infection, we developed a reliable high-content imaging screening assay, which we used to screen an in-house chemical library developed to inhibit DNA and histone methyltransferases. This assay was optimised using monocytic leukemic THP-1 cells differentiated into macrophages infected with L. pneumophila in a 96- or 384-well plate format using the Opera Phenix (Perkin Elmer) confocal microscope, combined with Columbus software for automated image acquisition and analysis. H3 K14 methylation was followed in infected, single cells and cytotoxicity was assessed in parallel. A first pilot screening of 477 compounds identified a potential starting point for inhibitors of H3 K14 methylation.

Evaluating stress resilience of cyanobacteria through flow cytometry and fluorescent viability assessment.

Cyanobacteria are prokaryotic organisms characterised by their complex structures and a wide range of pigments. With their ability to fix CO2, cyanobacteria are interesting for white biotechnology as cell factories to produce various high-value metabolites such as polyhydroxyalkanoates, pigments, or proteins. White biotechnology is the industrial production and processing of chemicals, materials, and energy using microorganisms. It is known that exposing cyanobacteria to low levels of stressors can induce the production of secondary metabolites. Understanding of this phenomenon, known as hormesis, can involve the strategic application of controlled stressors to enhance the production of specific metabolites. Consequently, precise measurement of cyanobacterial viability becomes crucial for process control. However, there is no established reliable and quick viability assay protocol for cyanobacteria since the task is challenging due to strong interferences of autofluorescence signals of intercellular pigments and fluorescent viability probes when flow cytometry is used. We performed the screening of selected fluorescent viability probes used frequently in bacteria viability assays. The results of our investigation demonstrated the efficacy and reliability of three widely utilised types of viability probes for the assessment of the viability of Synechocystis strains. The developed technique can be possibly utilised for the evaluation of the importance of polyhydroxyalkanoates for cyanobacterial cultures with respect to selected stressor-repeated freezing and thawing. The results indicated that the presence of polyhydroxyalkanoate granules in cyanobacterial cells could hypothetically contribute to the survival of repeated freezing and thawing.

Development and validation of an LC‒MS/MS method for the determination of cyclocreatine phosphate and its related endogenous biomolecules in rat heart tissues

The cardioprotective drug cyclocreatine phosphate has been awarded Food and Drug Administration-orphan drug designation for the prevention of ischemic injury to enhance cardiac graft recovery and survival in heart transplantation. Cyclocreatine phosphate is the water-soluble derivative of cyclocreatine. Estimating the levels of Cyclocreatine phosphate, Adenosine triphosphate, Creatine Phosphate, Creatine and Cyclocreatine helps us in understanding the energy state as well as evaluating the heart cells’ function. The quantification of endogenous compounds imposes a challenging task for analysts because of the absence of a true blank matrix, whose use is required according to international guidelines. Recently, the International Council for Harmonization issued a new guideline that contains guidance on the validation of methods used to quantify endogenous components, such as the background subtraction approach that was employed in our current study. Specifically, we developed and validated a sensitive, reliable and accurate liquid chromatography-tandem mass spectrometry assay to determine simultaneously the levels of mentioned endogenous compounds in rat heart tissue. Tissue samples were prepared by protein precipitation extraction using water: methanol (1:1). Using Ultra Performance Liquid Chromatography, Chromatographic separation was achieved with ZORBAX Eclipse Plus C18 4.6 × 100 mm,3.5 μm column and conditions as following: ammonium acetate (pH 8.5): acetonitrile, 70:30 mobile phase, 0.7 mL/min flow rate and 25 °C temperature. Electrospray ionization mass detector with Multiple reaction monitoring mode was then employed, using both positive and negative modes, Analysis was carried out using 5.00–2000.00 ng/mL linear concentration range within 2 min for each analyte. According to Food and Drug Administration guidelines for bioanalytical methods, validation was carried out. We investigated the matrix effect, recovery efficiency and process efficiency for the analyte in neat solvent, postextraction matrix and tissue. The results stated mean percentage recoveries higher than 99%, accuracy 93.32–111.99%, and Relative Standard Deviation (RSD) below 15% within the concentration range of our study which indicated that target analytes’ stability in their real matrix is sufficient under the employed experimental conditions.

Development of a Reliable Assay of Eco-friendly Fe3O4@Ag Nanocomposite-Based Giant Magnetoresistance Sensor

For the development of green magnetic-based immunoassay devices, the rapid and reliable assay method of eco-friendly magnetic labels with a lower energy requirement is vital. This work proposes a green-synthesized Fe3O4@Ag magnetic label assay system using GMR chips and a simple microcontroller-based data acquisition tool. Optical analysis shows the successful synthesis of Fe3O4@Ag with the assistance of Moringa oleifera (MO) extract as a reducing and stabilizing agent. Meanwhile, according to characterization, MO-assisted green-synthesized Fe3O4@Ag nanocomposites feature cubic inverse spinel structures and ferromagnetic characteristics that possess multi-domain structures. The sensor system generates an intense signal, varying from tens to hundreds of millivolts, allowing for its detection using a simple microcontroller system. The sensor exhibits a stable and reliable response to the increase in the concentration of Fe3O4@Ag nanocomposite, even though it is subjected to weak magnetic field induction. Furthermore, the introduction of Ag on the surface of Fe3O4 nanoparticles succeeded in optimizing the detection features, as evidenced by the lower limit of detection compared to detecting the bare Fe3O4. The GMR-based sensor, featuring a simple microcontroller structure and an eco-friendly Fe3O4@Ag nanocomposite as a magnetic label, exhibits significant potential as a rapid and reliable green biosensor that is power-efficient.

The first report of cotton leafroll dwarf virus infecting upland cotton plants in Arizona.

Cotton leafroll dwarf virus (CLRDV) represents a persistent threat to cotton production in the United States (U.S.) (Edula et al. 2023). Initially detected in Alabama (Avelar et al. 2019), CLRDV occurs in almost all the states in the U.S. cotton belt, extending from Virginia through Texas. The symptoms associated with CLRDV includes interveinal chlorosis, leaf rolling, stunting, and reduced boll sets. However, asymptomatic CLRDV-infected plants have also been reported (Edula et al. 2023). In the 2023 growing season, upland cotton plants (Gossypium hirsutum) presenting terminal splitting symptoms in the blooming stage were observed in a commercial field in Pinal County, Arizona, at 60% incidence. To evaluate if CLRDV may be associated with these symptoms, young fully expanded terminal leaves were collected from 20 symptomatic and ten asymptomatic plants. Moreover, ten samples were collected from a second asymptomatic block in the same field. Samples were shipped on dry ice to Cornell University in hermetic bags under APHIS PPQ permit 526-23-256-14384. Midribs and petioles were used to extract total nucleic acids from each sample using OPS Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics) per the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using an iScript Reverse transcriptase kit (BioRad) and used for the detection of a partial sequence of the coat protein (CP) of CLRDV using PCR assays as described previously (Mahas et al. 2022). The expected 309-bp product was obtained from only one symptomatic sample. CLRDV presence in the samples was further evaluated using CLRDV-specific primers targeting the RNA-dependent RNA polymerase (RdRp) gene. Primers CLRDV-Pol_innerF1 (5'- ACCCTCCAAGGAACAGAG -3') / R1 (5'- CGAATAATCTGATYGGGTCAC -3') and CLRDV-Pol_outerF1 (5'- AACGCGCCCAGTCCGCACAAATACC-3') / R1 (5'-ACCGGGTTTACTGGGGATTGCACGC-3'), designed based on virus isolates available in GenBank as of September 2022, were used to implement a single-tube nested RT-PCR as detailed by Dey et al. (2012) and to index the presence of CLRDV in all the samples. Two additional symptomatic samples and three asymptomatic samples (two from field one and one from field two) were positive for the virus. Direct Sanger sequencing of the one CP and two RdRp amplicons from symptomatic and asymptomatic samples (PP482918-20) demonstrated they shared >99% nucleotide identity to an isolate from South Carolina (OQ300129). To further evaluate the presence of CLRDV in Arizona, we performed double antibody sandwich (DAS)-ELISA on midrib and petiole samples using camelid single-chain antibodies against the CP as the capture antibody (Filed Patent 18/436,287) and the commercially available Anti-PLRV conjugate (Agdia, Elkhart, IN) as the secondary antibody. DAS-ELISA detected CLRDV in eight symptomatic and three asymptomatic samples, with the positive and negative controls testing positive and negative, respectively. Considering all the diagnostic approaches, ten symptomatic and five asymptomatic samples were positive for CLRDV (Table S1). To the best of our knowledge, this is the first report of CLRDV in Arizona. Future studies are required to evaluate the possible incidence and impact on the crop yield in the state and develop a reliable diagnostic assay to detect the virus in all infected samples. Since our study did not associate CLRDV with the terminal abortion or splitting symptoms, ongoing efforts are underway to elucidate if other viruses may be associated with the symptoms.