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Acid-sensing ion channels (ASICs) play a key role in the perception and response to extracellular acidification changes. These proton-gated cation channels are critical for neuronal functions, like learning and memory, fear, mechanosensation and internal adjustments like synaptic plasticity. Moreover, they play a key role in neuronal degeneration, ischemic neuronal injury, seizure termination, pain-sensing, etc. Functional ASICs are homo or heterotrimers formed with (ASIC1–ASIC3) homologous subunits. ASIC1a, a major ASIC isoform in the central nervous system (CNS), possesses an acidic pocket in the extracellular region, which is a key regulator of channel gating. Growing data suggest that ASIC1a channels are a potential therapeutic target for treating a variety of neurological disorders, including stroke, epilepsy and pain. Many studies were aimed at identifying allosteric modulators of ASIC channels. However, the regulation of ASICs remains poorly understood. Using all available crystal structures, which correspond to different functional states of ASIC1, and a molecular dynamics simulation (MD) protocol, we analyzed the process of channel inactivation. Then we applied a molecular docking procedure to predict the protein conformation suitable for the amiloride binding. To confirm the effect of its sole active blocker against the ASIC1 state transition route we studied the complex with another MD simulation run. Further experiments evaluated various compounds in the Enamine library that emerge with a detectable ASIC inhibitory activity. We performed a detailed analysis of the structural basis of ASIC1a inhibition by amiloride, using a combination of in silico approaches to visualize its interaction with the ion pore in the open state. An artificial activation (otherwise, expansion of the central pore) causes a complex modification of the channel structure, namely its transmembrane domain. The output protein conformations were used as a set of docking models, suitable for a high-throughput virtual screening of the Enamine chemical library. The outcome of the virtual screening was confirmed by electrophysiological assays with the best results shown for three hit compounds. [ABSTRACT FROM AUTHOR]

Numerous studies reported an association between GABAAR subunit genes and epilepsy, eating disorders, autism spectrum disorders, neurodevelopmental disorders, and bipolar disorders. This study was aimed to find some potential positive allosteric modulators and was performed by combining the in silico approach with further in vitro evaluation of its real activity. We started from the GABAAR‐diazepam complexes and assembled a lipid embedded protein ensemble to refine it via molecular dynamics (MD) simulation. Then we focused on the interaction of α1β2γ2 with some Z‐drugs (non‐benzodiazepine compounds) using an Induced Fit Docking (IFD) into the relaxed binding site to generate a pharmacophore model. The pharmacophore model was validated with a reference set and applied to decrease the pre‐filtered Enamine database before the main docking procedure. Finally, we succeeded in identifying a set of compounds, which met all features of the docking model. The aqueous solubility and stability of these compounds in mouse plasma were assessed. Then they were tested for the biological activity using the rat Purkinje neurons and CHO cells with heterologously expressed human α1β2γ2 GABAA receptors. Whole‐cell patch clamp recordings were used to reveal the GABA induced currents. Our study represents a convenient and tunable model for the discovery of novel positive allosteric modulators of GABAA receptors. A High‐throughput virtual screening of the largest available database of chemical compounds resulted in the selection of 23 compounds. Further electrophysiological tests allowed us to determine a set of 3 the most outstanding active compounds. Considering the structural features of leader compounds, the study can develop into the MedChem project soon. [ABSTRACT FROM AUTHOR]

The transportome of Saccharomyces cerevisiae comprises approximately 340 membrane-bound proteins, of which very few are well-characterized. Elucidating transporter proteins' function is essential not only for understanding central cellular processes in metabolite exchange with the external milieu but also for optimizing the production of value-added compounds in microbial cell factories. Here, we describe the application of 13C-labeled stable isotopes and detection by targeted LC-MS/MS as a screening tool for identifying Saccharomyces cerevisiae metabolite transporters. We compare the transport assay's sensitivity, reproducibility, and accuracy in yeast transporter mutant cell lines and Xenopus oocytes. As proof of principle, we analyzed the transport profiles of five yeast amino acid transporters. We first cultured yeast transporter deletion or overexpression mutants on uniformly labeled 13C-glucose and then screened their ability to facilitate the uptake or export of an unlabeled pool of amino acids. Individual transporters were further studied by heterologous expression in Xenopus oocytes, followed by an uptake assay with 13C labeled yeast extract. Uptake assays in Xenopus oocytes showed higher reproducibility and accuracy. Although having lower accuracy, the results from S. cerevisiae indicated the system's potential for initial high-throughput screening for native metabolite transporters. We partially confirmed previously reported substrates for all five amino acid transporters. In addition, we propose broader substrate specificity for two of the transporter proteins. The method presented here demonstrates the application of a comprehensive screening platform for the knowledge expansion of the transporter-substrate relationship for native metabolites in S. cerevisiae. [ABSTRACT FROM AUTHOR]

JAK2 transmits signals of several important cytokines, such as growth hormone and erythropoietin. The interest toward the therapeutic targeting of JAK2 was boosted in 2005, when the somatic JAK2 V617F mutation, responsible for the majority of myeloproliferative neoplasms (MPNs) was discovered. JAK2 inhibitors have been approved for MPN therapy and they are effective in alleviating symptoms and improving the quality of life of the patients, but they do not lead to molecular remission. This calls for the discovery of new compounds for JAK2-targeted therapeutic approaches. Here we describe the development of a fluorescence-based activity assay for the screening of versatile inhibitor types against JAK2. The assay was utilized to screen a diverse set of small molecule weight natural products and the assay performance was compared to that of differential scanning fluorimetry. We identified 37 hits and further analysis of the most potent hits revealed that most of them displayed non-ATP competitive binding modes. The hits were profiled against other JAK family members and showed distinctive selectivity profiles. The developed assay is consistent, simple and inexpensive to use, and can be utilized for inhibitor screening of diverse compound classes against all JAK family members.

Due to a lack of endogenous phytase enzymes in monogastric animals, exogenous phytases are employed in the animal feed industry. Phytases catalyze the hydrolysis of phytic acid and its salts (phytate) from plant-based animal feed, to supply phosphorus to monogastric animals and decrease the anti-nutritional effects of phytic acid. This study aimed to discover the new phytase with optimum activity at acidic pH range and 40 °C for the poultry feed industry. In the current investigation, phytase-producing microorganisms were isolated from different sources and locations, demonstrating that ten of the isolates are attributed to bacterial strains, and one of which is a yeast strain. Phytase-producing microorganisms were screened based on qualitative and quantitative assays. Additionally, molecular characterization was carried out based on sequencing of amplified 16S rRNA and nuclear ribosomal transcribed spacer (ITS) genes. Then, degenerate primers were designed to amplify the histidine acid phosphatase gene from potential isolates to find the new natural variant of phytase. Quantitative assays were carried out to find pH and temperature profiles at pH ranges between 2 and 7, with a 0.5 interval, and a range of temperatures from 30 °C to 90 °C, with a 10 °C interval. The results from crude enzymes demonstrated the extracellular phytase activities of isolates with different optimum pH and temperatures ranging from 4 to 6.5 and 40 °C–60 °C, respectively. Moreover, the range of optimum phytase activities of isolates was between 194.21 mU/mL and 381 mU/mL. Sequencing analysis of 16S rDNA and nuclear ribosomal transcribed spacer (ITS) genes revealed that six out of the eleven isolates are attributed to the Acinetobacter genus, two of which are affiliated with Enterobacter genus. One is affiliated with the Pseudomonas genus; one of them is affiliated with Escherichia, and another is affiliated with Saccharomyces. Finally, a new native histidine acid phosphatase gene (PhySc) was detected and amplified from the SPA isolate by designing degenerate primers. This showed a 99.50% identity with PHO5 from Saccharomyces cerevisiae YJM993 with an accession number of CP004601.2. [ABSTRACT FROM AUTHOR]

Kratom (Mitragyna speciosa Korth) has been used traditionally in Southeast Asia for its therapeutic properties. The major alkaloid of kratom, mitragynine, binds to opioid receptors to give opioid‐like effects that causes addiction. In our previous study, we have identified AZ122 as a unique biomarker in habitual or regular kratom users through analysis of their urinary protein profiles. We aimed to develop and validate a screening method by means of enzyme‐linked immunosorbent assay (ELISA) for detection of kratom habitual users. An ELISA approach was applied for the development of a screening method using urinary AZ122 as biomarker. Method validation was carried out using three quality control materials at different concentration of AZ122. The data was analyzed statistically using SPSS (Version 25). The ELISA was presented with Pearson correlation coefficient of 0.9993. The repeatability and reproducibility were presented at CV <7%, while the accuracy ranged from 78 to 96% at various AZ112 concentrations. Upon testing on 176 male respondents (n = 88 regular kratom users and n = 88 healthy controls), the specificity and sensitivity of the assay were both 100%. The ELISA has been validated and can be potentially used as a reliable screening test for detection of kratom habitual users. [ABSTRACT FROM AUTHOR]

Rapid, human relevant assays are needed to assess potential hazards of the many chemicals in commerce. An assay of thyroid peroxidase (TPO) inhibition, using the substrate Amplex Ultra Red, was recently adapted for human TPO (AUR-hTPO). We tested a large number (788) of chemicals through this AUR-hTPO assay and compared performance with published results from an assay using enzyme from rat thyroid microsomes (AUR-rTPO). Coded chemicals, from the US EPA ToxCast Inventory, were tested in a tiered approach: 1) Initial screening at a single concentration; 2) Potency estimation for active chemicals with multiple concentrations; 3) Screening active chemicals for the non-specific activity. The assay gave consistent results for positive chemical methimazole and several positive and negative reference chemicals. hTPO inhibition was observed for 190 chemicals reported as positive in rTPO. Of these, 158 showed no confounding activity (interference due to fluorescence or non-specific protein inhibition). Comparison of all result with rTPO data and with evidence of TPO inhibition found in the literature suggest that the current assay has a higher rate of false negative but a much lower rate of false positive compared with the rTPO screen. These findings underscore the effectiveness of the AUR assay, using hTPO enzyme from engineered cell lines, to identify moderate to strong inhibitors but some improvements may be needed to detect weak TPO inhibitors. [Display omitted] • The performance of a rapid, cell-free assay of human thyroid peroxidase, was assessed by testing 788 unique chemicals. • The high throughput assay using a tiered strategy gave consistent results for positive and negative reference chemicals. • Comparison of results from other TPO assay data suggest a reasonable consistency in positive and negative prediction. [ABSTRACT FROM AUTHOR]

The effective antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed around the world. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a pivotal role in virus replication; it also has become an important therapeutic target for the infection of SARS-CoV-2. In this work, we have identified Darunavir derivatives that inhibit the 3CLpro through a high-throughput screening method based on a fluorescence resonance energy transfer (FRET) assay in vitro. We found that the compounds 29# and 50# containing polyphenol and caffeine derivatives as the P2 ligand, respectively, exhibited favorable anti-3CLpro potency with EC50 values of 6.3 μM and 3.5 μM and were shown to bind to SARS-CoV-2 3CLpro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CLpro through molecular docking. Importantly, 29# and 50# exhibited a similar activity against the protease in Omicron variants. The inhibitory effect of compounds 29# and 50# on the SARS-CoV-2 3CLpro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19. [ABSTRACT FROM AUTHOR]

Many SARS-CoV-2 variants have emerged during the course of the COVID-19 pandemic. These variants have acquired mutations conferring phenotypes such as increased transmissibility or virulence, or causing diagnostic, therapeutic, or immune escape. Detection of Alpha and the majority of Omicron sublineages by PCR relied on the so-called S gene target failure due to the deletion of six nucleotides coding for amino acids 69–70 in the spike (S) protein. Detection of hallmark mutations in other variants present in samples relied on whole genome sequencing. However, whole genome sequencing as a diagnostic tool is still in its infancy due to geographic inequities in sequencing capabilities, higher cost compared to other molecular assays, longer turnaround time from sample to result, and technical challenges associated with producing complete genome sequences from samples that have low viral load and/or high background. Hence, there is a need for rapid genotyping assays. In order to rapidly generate information on the presence of a variant in a given sample, we have created a panel of four triplex RT-qPCR assays targeting 12 mutations to detect and differentiate all five variants of concern: Alpha, Beta, Gamma, Delta, and Omicron. We also developed an expanded pentaplex assay that can reliably distinguish among the major sublineages (BA.1–BA.5) of Omicron. In silico, analytical and clinical testing of the variant panel indicate that the assays exhibit high sensitivity and specificity. This panel can help fulfill the need for rapid identification of variants in samples, leading to quick decision making with respect to public health measures, as well as treatment options for individuals. Compared to sequencing, these genotyping PCR assays allow much faster turn-around time from sample to results—just a couple hours instead of days or weeks.

Human acid-sensing ion channels (ASIC) are ligand-gated ionotropic receptors expressed widely in peripheral tissues as well as sensory and central neurons and implicated in detection of inflammation, tissue injury, and hypoxia-induced acidosis. This makes ASIC channels promising targets for drug discovery in oncology, pain and ischemia, and several modulators have progressed into clinical trials. We describe the use of hASIC1a as a case study for the development and validation of low, medium and high throughput automated patch clamp (APC) assays suitable for the screening and mechanistic profiling of new ligands for this important class of ligand-gated ion channel. Initial efforts to expand on previous manual patch work describing an endogenous hASIC1a response in HEK cells were thwarted by low current expression and unusual pharmacology, so subsequent work utilized stable hASIC1a CHO cell lines. Ligand-gated application protocols and screening assays on the Patchliner, QPatch 48, and SyncroPatch 384 were optimized and validated based on pH activation and nM-ÎM potency of reference antagonists (e.g., Amiloride, Benzamil, Memantine, Mambalgin-3, A-317567, PcTx1). By optimizing single and stacked pipette tip applications available on each APC platform, stable pH-evoked currents during multiple ligand applications enabled cumulative EC50 and IC50 determinations with minimized receptor desensitization. Finally, we successfully demonstrated for the first time on an APC platform the ability to use current clamp to implement the historical technique of input resistance tracking to measure ligand-gated changes in membrane conductance on the Patchliner platform. [ABSTRACT FROM AUTHOR]

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including β2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner. [ABSTRACT FROM AUTHOR]

Introduction: The reliable diagnosis of paroxysmal nocturnal haemoglobinuria (PNH) by flow cytometry is based on mandatory analysis of the erythroid, neutrophilic and monocytic lineages. In this study, we have evaluated the performance characteristics of a recently published immature red blood cell (iRBC) assay as a potential screening test for PNH by flow cytometry. Methods: Intra‐ and inter‐assay imprecision were determined in five replicates of small, medium and large PNH iRBC clones. Analytical and functional sensitivity was assessed by performing spiking tests for five replicates. Thirty healthy donors and 441 PNH patients were tested for evaluation of clinical specificity, sensitivity, positive and negative predictive values. Results: Coefficients of variation (CV) for intra‐/inter‐assay imprecision analyses were 1.31/1.50, 3.19/2.61 and 3.99/1.58 for the big, medium and small clone sizes, respectively. Absolute values (100%) were found for both clinical specificity and sensitivity as well as for both positive and negative predictive values. The CV from 5 replicate results for 10 clustered events was 15.7%. The coefficient of determination (r2), Pearson's correlation coefficient (r) and Bland–Altman mean bias were 0.9436/0.9234/1.7 for PNH iRBC compared to PNH neutrophils and 0.9553/0.9387/2.1 for PNH iRBCs compared to PNH monocytes. Conclusion: Our results confirm very good performance characteristics, high analytical and functional sensitivity, absolute clinical specificity and sensitivity as well as favourable correlation between PNH iRBCs and both PNH neutrophils and monocytes, suggesting that this cost‐effective 3‐colour iRBC assay can be used as a reliable screening test for evaluation of small, medium and large PNH clones by flow cytometry. [ABSTRACT FROM AUTHOR]

Human acid-sensing ion channels (ASIC) are ligand-gated ionotropic receptors expressed widely in peripheral tissues as well as sensory and central neurons and implicated in detection of inflammation, tissue injury, and hypoxia-induced acidosis. This makes ASIC channels promising targets for drug discovery in oncology, pain and ischemia, and several modulators have progressed into clinical trials. We describe the use of hASIC1a as a case study for the development and validation of low, medium and high throughput automated patch clamp (APC) assays suitable for the screening and mechanistic profiling of new ligands for this important class of ligand-gated ion channel. Initial efforts to expand on previous manual patch work describing an endogenous hASIC1a response in HEK cells were thwarted by low current expression and unusual pharmacology, so subsequent work utilized stable hASIC1a CHO cell lines. Ligand-gated application protocols and screening assays on the Patchliner, QPatch 48, and SyncroPatch 384 were optimized and validated based on pH activation and nM-μM potency of reference antagonists (e.g., Amiloride, Benzamil, Memantine, Mambalgin-3, A-317567, PcTx1). By optimizing single and stacked pipette tip applications available on each APC platform, stable pH-evoked currents during multiple ligand applications enabled cumulative EC50 and IC50 determinations with minimized receptor desensitization. Finally, we successfully demonstrated for the first time on an APC platform the ability to use current clamp to implement the historical technique of input resistance tracking to measure ligand-gated changes in membrane conductance on the Patchliner platform.

Background and Objectives: Screening for transfusion transmissible infections in donated blood can be done by either rapid diagnostic tests (RDTs) or enzyme-linked immunosorbent assay (ELISA) or any other available sensitive immunoassay such as chemiluminescence immunoassay (CLIA). The aim of this assay was to evaluate performance of two commercially available RDTs and CLIA against ELISA for serological screening of hepatitis B surface antigen (HBsAg) and anti hepatitis C virus (HCV). In addition, we also tested the seropositive samples by RDTs, CLIA, and/or ELISA by molecular assays (transcription-mediated amplification, transcription-mediated amplification, and quantitative polymerase chain reaction). Methods: In this prospective study, 1000 consecutive blood donors were screened from September 2017 to March 2018. All blood samples collected during the study period were tested by RDTs, CLIA, and ELISA, and the results obtained were compared. Results: On considering ELISA as a reference standard, low analytical sensitivity was noted for both RDTs (64.29%) and CLIA (71.43%) for HBsAg detection. Similar results were also noted for both RDTs (25%) and CLIA (75%). The positive predictive value of CLIA was found to be lower (HBsAg 31.25%; anti-HCV 50%) as compared to RDTs (HBsAg 90%–100%; anti-HCV 100%). High false positivity was noted with CLIA, while RDTs missed seropositive samples. The viral load for HBsAg and anti-HCV seropositive samples ranged from 29.7 IU/ml to 3.4 × 104 IU/ml and 7.56 × 106 IU/ml, respectively. Conclusions: Performance of CLIA as screening assay was better compared to RDTs. CLIA seems to be a suitable screening assay for emergency situations and predonation apheresis donor screening. RDTs may be used as supplemental assay prior to donor notification.

Background: It is often difficult to accurately predict how a melanoma will progress because melanomas can be so diverse in their genetic and histological makeup.Objective: We sought to characterize the current state and progression of biomedical markers towards their utilization as prognostic indicators for patients with melanoma.Methods: A literature search of the research repository databases PubMed and GoogleScholar was conducted using the following inclusion criteria: (1) published within the last 10 years, and (2) use of overall survival, disease progression, or clinical outcome as primary endpoints. Search terms included various permutations of "biomarkers," "prognostic," "immunologic," "serologic," "visual," and "melanoma." Results were evaluated for statistical power, results significance, and experimental design integrity.Results: The prognostic capabilities of clinical tests for malignant melanoma have made great strides in the last few years, with several serologic and immunohistochemical biomarkers being preliminarily linked to various measures of clinical prognosis. While clinical feasibility of a single sensitive and specific biomarker remains unfeasible, use of select combinations of tested biomarkers remain viable.Conclusion: Diagnostic and prognostic genetic assays have begun to cross over from research to commercial application, giving physicians additional tools during the early stages of diagnosis to optimize and individualize treatments. [ABSTRACT FROM AUTHOR]

A number of small molecule and protein therapeutic candidates have been developed in the last four years against SARS-CoV-2 spike. However, there are hardly a few molecules that have advanced through the subsequent discovery steps to eventually work as a therapeutic agent. This is majorly because of the hurdles in determining the affinity of potential therapeutics with live SARS-CoV-2 virus. Furthermore, affinity determined for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, at times, fails to mimic physiological conditions of the host-virus interaction. To bridge this gap between in vitro and in vivo methods of therapeutic agent screening, we report an improved screening protocol for therapeutic candidates using SARS-CoV-2 virus like particles (VLPs). To minimise the interference from the bulkier reporters like GPF in the affinity studies, a smaller hemagglutinin (HA) tag has been fused to one of the proteins of VLP. This HA tag serves as readout, when probed with fluorescent anti-HA antibodies. Outcome of this study sheds light on the lesser known virus neutralisation capabilities of AM type miniprotein mimics. Further, to assess the stability of SARS-CoV-2 spike - miniprotein complex, we have performed molecular dynamic simulations on the membrane embedded protein complex. Simulation results reveal extremely stable intermolecular interactions between RBD and one of the AM type miniproteins, AM1. Furthermore, we discovered a robust network of intramolecular interactions that help stabilise AM1. Findings from our in vitro and in silico experiments concurrently highlight advantages and capabilities of mimic based miniprotein therapeutics. • Screening potential therapeutic candidates using SARS-CoV-2 virus-like particles. • Determining virus neutralisation capabilities of AM type miniprotein mimics. • Mimic-spike trimer complex interaction stability in molecular dynamics. • Stability of ACE2 mimic while in complex with spike receptor binding domain. [ABSTRACT FROM AUTHOR]

Background and Objectives: Screening for transfusion transmissible infections in donated blood can be done by either rapid diagnostic tests (RDTs) or enzyme-linked immunosorbent assay (ELISA) or any other available sensitive immunoassay such as chemiluminescence immunoassay (CLIA). The aim of this assay was to evaluate performance of two commercially available RDTs and CLIA against ELISA for serological screening of hepatitis B surface antigen (HBsAg) and anti hepatitis C virus (HCV). In addition, we also tested the seropositive samples by RDTs, CLIA, and/or ELISA by molecular assays (transcription-mediated amplification, transcription-mediated amplification, and quantitative polymerase chain reaction). Methods: In this prospective study, 1000 consecutive blood donors were screened from September 2017 to March 2018. All blood samples collected during the study period were tested by RDTs, CLIA, and ELISA, and the results obtained were compared. Results: On considering ELISA as a reference standard, low analytical sensitivity was noted for both RDTs (64.29%) and CLIA (71.43%) for HBsAg detection. Similar results were also noted for both RDTs (25%) and CLIA (75%). The positive predictive value of CLIA was found to be lower (HBsAg 31.25%; anti-HCV 50%) as compared to RDTs (HBsAg 90%-100%; anti-HCV 100%). High false positivity was noted with CLIA, while RDTs missed seropositive samples. The viral load for HBsAg and anti-HCV seropositive samples ranged from 29.7 IU/ml to 3.4 × 104 IU/ml and 7.56 × 106 IU/ml, respectively. Conclusions: Performance of CLIA as screening assay was better compared to RDTs. CLIA seems to be a suitable screening assay for emergency situations and predonation apheresis donor screening. RDTs may be used as supplemental assay prior to donor notification. [ABSTRACT FROM AUTHOR]

Alopecia arises due to inadequate hair follicle (HF) stem cell activation or proliferation, resulting in prolongation of the telogen phase of the hair cycle. Increasing therapeutic and cosmetic demand for alleviating alopecia has driven research toward the discovery or synthesis of novel compounds that can promote hair growth by inducing HF stem cell activation or proliferation and initiating the anagen phase. Although several methods for evaluating the hair growth-promoting effects of candidate compounds are being used, most of these methods are difficult to use for large scale simultaneous screening of various compounds. Herein, we introduce a simple and reliable in vitro assay for the simultaneous screening of the hair growth-promoting effects of candidate compounds on a large scale. In this study, we first established a 3D co-culture system of human dermal papilla (hDP) cells and human outer root sheath (hORS) cells in an ultra-low attachment 96-well plate, where the two cell types constituted a polar elongated structure, named “two-cell assemblage (TCA).” We observed that the long axis length of the TCA gradually increased for 5 days, maintaining biological functional integrity as reflected by the increased expression levels of hair growth-associated genes after treatment with hair growth-promoting molecules. Interestingly, the elongation of the TCA was more prominent following treatment with the hair growth-promoting molecules (which occurred in a dose-dependent manner), compared to the control group (p < 0.05). Accordingly, we set the long axis length of the TCA as an endpoint of this assay, using a micro confocal high-content imaging system to measure the length, which can provide reproducible and reliable results in an adequate timescale. The advantages of this assay are: (i) it is physiologically and practically advantageous as it uses 3D cultured two-type human cells which are easily available; (ii) it is simple as it uses length as the only endpoint; and (iii) it is a high throughput system, which screens various compounds simultaneously. In conclusion, the “TCA” assay could serve as an easy and reliable method to validate the hair growth-promoting effect of a large volume of library molecules.

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species‐specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus‐induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus‐based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high‐level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research. [ABSTRACT FROM AUTHOR]

The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently published as an OECD Test Guideline for detecting chemicals acting on the thyroid axis. However, the OECD validation did not cover all mechanisms that can potentially be detected by the XETA. This study was therefore initiated to investigate and consolidate the applicability domain of the XETA regarding the following mechanisms: thyroid hormone receptor (THR) agonism, sodium-iodide symporter (NIS) inhibition, thyroperoxidase (TPO) inhibition, deiodinase (DIO) inhibition, glucocorticoid receptor (GR) agonism, and uridine 5′-diphospho-glucuronosyltransferase (UDPGT) induction. In total, 22 chemicals identified as thyroid-active or -inactive in Amphibian Metamorphosis Assays (AMAs) were tested using the XETA OECD Test Guideline. The comparison showed that both assays are highly concordant in identifying chemicals with mechanisms of action related to THR agonism, DIO inhibition, and GR agonism. They also consistently identified the UDPGT inducers as thyroid inactive. NIS inhibition, investigated using sodium perchlorate, was not detected in the XETA. TPO inhibition requires further mechanistic investigations as the reference chemicals tested resulted in opposing response directions in the XETA and AMA. This study contributes refining the applicability domain of the XETA, thereby helping to clarify the conditions where it can be used as an ethical alternative to the AMA. • The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently developed to screen for thyroid activity of chemicals. • XETA results were compared to Amphibian Metamorphosis Assay (AMA) outcomes for the detection of thyroid-active chemicals. • Results were concordant for all selected mechanisms, but the XETA did not detect inhibition of the sodium-iodide symporter. • Thyroperoxidase inhibition requires further mechanistic investigations due to equivocal responses in the XETA. • The XETA and the AMA consistently identified uridine 5′-diphospho-glucuronosyltransferase inducers as thyroid-inactive. [ABSTRACT FROM AUTHOR]

There is a need for rapidly screening thyroid hormone (TH) signaling disruptors in vivo considering the essential role of TH signaling in vertebrates. We aimed to establish a rapid in vivo screening assay using Xenopus laevis based on the T3-induced Xenopus metamorphosis assay we established previously, as well as the Xenopus Eleutheroembryonic Thyroid Assay (XETA). Stage 48 tadpoles were treated with a series of concentrations of T3 in 6-well plates for 24 h and the expression of six TH-response genes was analyzed for choosing a proper T3 concentration. Next, bisphenol A (BPA) and tetrabromobisphenol A (TBBPA), two known TH signaling disruptors, were tested for determining the most sensitive TH-response gene, followed by the detection of several suspected TH signaling disruptors. We determined 1 nM as the induction concentration of T3 and thibz expression as the sensitive endpoint for detecting TH signaling disruptors given its highest response to T3, BPA, and TBBPA. And we identified betamipron as a TH signaling agonist, and 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) as a TH signaling antagonist. Overall, we developed a multiwell-based assay for rapidly screening TH signaling disruptors using thibz expression as a sensitive endpoint in X. laevis.

Cyclin-dependent kinases (CDKs) constitute a family of 20 serine/threonine protein kinases that play pivotal roles in the regulation of numerous important molecular and cellular processes. CDKs have long been considered promising therapeutic targets in a variety of pathologies, and the recent therapeutic success of CDK4/6 inhibitors in breast cancers has renewed interest in their therapeutic potential. Small-molecule inhibitors have been identified for every human CDK, except for CDK10. The only recent discovery of an activating cyclin (CycM) for CDK10 enabled us to identify its first phosphorylation substrates and gain insights into its biological functions. Yet, our knowledge of this kinase remains incomplete, despite it being the only member of its family that causes severe human developmental syndromes, when mutated either on the cyclin or the CDK moiety. CDK10 small-molecule inhibitors would be useful in exploring the functions of this kinase and gauging its potential as a therapeutic target for some cancers. Here, we report the identification of an optimized peptide phosphorylation substrate of CDK10/CycM and the development of the first homogeneous, miniaturized CDK10/CycM in vitro kinase assay. We reveal the ability of known CDK inhibitors, among which clinically tested SNS-032, riviciclib, flavopiridol, dinaciclib, AZD4573 and AT7519, to potently inhibit CDK10/CycM. We also show that NVP-2, a strong, remarkably selective CDK9 inhibitor is an equally potent CDK10/CycM inhibitor. Finally, we validate this kinase assay for applications in high-throughput screening campaigns to discover new, original CDK10 inhibitors.

Autophagy is an evolutionary conserved stress survival pathway that has been shown to play an important role in the initiation, progression, and metastasis of multiple cancers; however, little progress has been made to date in translation of basic research to clinical application. This is partially due to an incomplete understanding of the role of autophagy in the different stages of cancer, and also to an incomplete assessment of potential drug targets in the autophagy pathway. While drug discovery efforts are on-going to target enzymes involved in the initiation phase of the autophagosome, e.g., unc51-like autophagy activating kinase (ULK)1/2, vacuolar protein sorting 34 (Vps34), and autophagy-related (ATG)7, we propose that the cysteine protease ATG4B is a bona fide drug target for the development of anti-cancer treatments. In this review, we highlight some of the recent advances in our understanding of the role of ATG4B in autophagy and its relevance to cancer, and perform a critical evaluation of ATG4B as a druggable cancer target. [ABSTRACT FROM AUTHOR]

The presence of reproductive endocrine‐disrupting compounds (REDCs) in the environment poses a potential threat to fish and wildlife, because exposures are capable of altering sexual development, reproductive success, and behavior. Fish‐based screening assays are often utilized to screen for the presence of REDCs in surface waters and to assess single chemicals for potential endocrine‐disrupting activity. In an effort to improve such screening assays, the goal of the present study was to determine whether the gonadosomatic index (GSI) of female fathead minnows (Pimephales promelas), as assessed via external characteristics, influences their response to REDC exposure. Specifically, we sought to determine whether low‐GSI females differed from high‐GSI females in their responses to the model anti‐estrogen fadrozole and the model androgen 17β‐trenbolone, and whether there was a preferable classification in the context of REDC screening. Low‐GSI females were more sensitive to fadrozole at the lower concentration of fadrozole (5 µg/L) and to the higher concentration of trenbolone (50 ng/L), whereas high‐GSI females were more sensitive at the lower concentration of trenbolone (5 ng/L). The differential response of low‐ and high‐GSI females to REDCs indicates that GSI influences exposure outcome, and should subsequently be taken into consideration in the implementation of screening assays, as failure to utilize fish of the appropriate reproductive status may skew the test results. Environ Toxicol Chem 2019;38:603–615. © 2019 SETAC [ABSTRACT FROM AUTHOR]

Numerous studies reported an association between GABA A R subunit genes and epilepsy, eating disorders, autism spectrum disorders, neurodevelopmental disorders, and bipolar disorders. This study was aimed to find some potential positive allosteric modulators and was performed by combining the in silico approach with further in vitro evaluation of its real activity. We started from the GABA A R-diazepam complexes and assembled a lipid embedded protein ensemble to refine it via molecular dynamics (MD) simulation. Then we focused on the interaction of α1β2γ2 with some Z-drugs (non-benzodiazepine compounds) using an Induced Fit Docking (IFD) into the relaxed binding site to generate a pharmacophore model. The pharmacophore model was validated with a reference set and applied to decrease the pre-filtered Enamine database before the main docking procedure. Finally, we succeeded in identifying a set of compounds, which met all features of the docking model. The aqueous solubility and stability of these compounds in mouse plasma were assessed. Then they were tested for the biological activity using the rat Purkinje neurons and CHO cells with heterologously expressed human α1β2γ2 GABA A receptors. Whole-cell patch clamp recordings were used to reveal the GABA induced currents. Our study represents a convenient and tunable model for the discovery of novel positive allosteric modulators of GABA A receptors. A High-throughput virtual screening of the largest available database of chemical compounds resulted in the selection of 23 compounds. Further electrophysiological tests allowed us to determine a set of 3 the most outstanding active compounds. Considering the structural features of leader compounds, the study can develop into the MedChem project soon., (© 2024 Wiley-VCH GmbH.)

Graphical abstract Highlights • A fluorescent assay for the screening of MBL inhibitors. • Compatible to B1, B2 and B3 subclass MBLs. • Highly sensitive. Abstract Reported herein is a fluorescence assay for the rapid screening of metallo-β-lactamase (MBL) inhibitors. This assay employs a fluorogenic carbapenem CPC-1 as substrate and is compatible with all MBLs, including B1, B2 and B3 subclass MBLs. The efficiency of this assay was demonstrated by the rapid inhibition screening of a number of molecules against B2 MBL CphA and 2,3-dimercaprol was identified as a potent CphA inhibitor. [ABSTRACT FROM AUTHOR]

The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 μM) and a broad linear detection range (5 to 250 μM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M). [ABSTRACT FROM AUTHOR]

Angiogenesis and its involved proteins, particularly Vascular Endothelial Growth Factor family (VEGFs) and VEGF receptors (VEGFRs), have been considered as a target of therapeutic interest for numerous inflammatory and vascular diseases. Acting on this biological process through interaction with VEGFs or VEGFRs has received considerable attention. Indeed, VEGFs and VEGFRs are currently targeted by drugs such as monoclonal antibodies. The feasibility of a therapeutic strategy based on blocking the VEGF/VEGFR interaction by using ligands “other-than-biologics” is also explored. To help to the discovery of new molecules, screening assays have been developed, particularly to evaluate the VEGFA/VEGFR1 interaction. Despite the therapeutic importance of VEGFB and PlGF (Placental Growth Factor), no assays have been developed to evaluate molecules against their interactions with VEGFR1. Here, we present new versatile colorimetric immunoassays to screen and evaluate the specific interaction of discovered molecules with different growth factors (VEGFA, VEGFB, PlGF) and receptors (VEGFR1, VEGFR2). These tests, based on competitive immunoassay format, will provide essential information on specificity and selectivity of molecules for their targets and will help to work on the pharmaco-modulation of molecules for targeting one specific interaction. [ABSTRACT FROM AUTHOR]

Background and Objectives: Anthocyanins are an important group of secondary metabolites, which are well-known and notablebecause of having high antioxidant activities, prevention of genetic mutation and cancers and also anti-aging properties. On the other hand, these compounds are also natural and widely used pigments in food and drug industries. The purpose of this research was to determine anthocyanin content, and cytotoxic activity in fruit peel of Solanum melongena cultivated in Kashan, and to compare the effect of extraction solvent. Methods: In this study, anthocyanin content of fruit peel of Solanum melongena with the common name of "egg plant" was determined using UV-Vis absorption method. To select the best extraction solvent, water, methanol, and ethanol were used to extract these valuable compounds. Also, the obtained extracts were investigated using brine shrimp lethality assay to determine cytotoxic activity and primary screening for the existence of anticancer effects. Results: The amount of extracted anthocyanins using water, methanol, and ethanol solvents, were 412, 354, and 293mg per 100g extract, and cytotoxic activity (LC 50 ) in these solvents were, respectively, 750, 525, and 1000µg/mL. Conclusion: In the present study, proving the existence of large amount of these compounds and their valuable properties, such as cytotoxic activity in different extracts of eggplant peel, which are discarded as garbage, shows the necessity of more attention to this type of valuable natural resources. Also, introducing “water” as the best solvent for extracting anthocyanins from eggplant peel, considering its low cost, non toxicity, and eco-friendliness , is one of the most important results of this research.

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases. [ABSTRACT FROM AUTHOR]

Many carbonyl species from either lipid peroxidation or glycoxidation are extremely reactive and can disrupt the function of proteins and enzymes. 4-hydroxynonenal and methylglyoxal are the most abundant and toxic lipid-derived reactive carbonyl species. The presence of these toxics leads to carbonyl stress and cause a significant amount of macromolecular damages in several diseases. Much evidence indicates trapping of reactive carbonyl intermediates may be a useful strategy for inhibiting or decreasing carbonyl stress-associated pathologies. There is no rapid and convenient analytical method available for the assessment of direct carbonyl scavenging capacity, and a very limited number of carbonyl scavengers have been identified to date, their therapeutic potential being highlighted only recently. In this context, we have developed a new and rapid sensitive fluorimetric method for the assessment of reactive carbonyl scavengers without involvement glycoxidation systems. Efficacy of various thiol- and non-thiol-carbonyl scavenger pharmacophores was tested both using this screening assay adapted to 96-well microplates and in cultured cells. The scavenging effects on the formation of Advanced Glycation End-product of Bovine Serum Albumin formed with methylglyoxal, 4-hydroxynonenal and glucose-glycated as molecular models were also examined. Low molecular mass thiols with an α-amino-β-mercaptoethane structure showed the highest degree of inhibitory activity toward both α,β-unsaturated aldehydes and dicarbonyls. Cysteine and cysteamine have the best scavenging ability toward methylglyoxal. WR-1065 which is currently approved for clinical use as a protective agent against radiation and renal toxicity was identified as the best inhibitor of 4-hydroxynonenal.

There is global demand for new in vitro testing tools for ecological risk assessment. The objective of the present study was to apply a set of cell-free neurochemical assays to screen many chemicals across many species in a relatively high-throughput manner. The platform assessed 7 receptors and enzymes that mediate neurotransmission of γ-aminobutyric acid, dopamine, glutamate, and acetylcholine. Each assay was optimized to work across 20 vertebrate species (5 fish, 5 birds, 7 mammalian wildlife, 3 biomedical species including humans). We tested the screening assay platform against 80 chemicals (23 pharmaceuticals and personal care products, 20 metal[loid]s, 22 polycyclic aromatic hydrocarbons and halogenated organic compounds, 15 pesticides). In total, 10 800 species-chemical-assay combinations were tested, and significant differences were found in 4041 cases. All 7 assays were significantly affected by at least one chemical in each species tested. Among the 80 chemicals tested, nearly all resulted in a significant impact on at least one species and one assay. The 5 most active chemicals were prochloraz, HgCl2, Sn, benzo[ a]pyrene, and vinclozolin. Clustering analyses revealed groupings according to chemicals, species, and chemical-assay combinations. The results show that cell-free assays can screen a large number of samples in a short period of time in a cost-effective manner in a range of animals not easily studied using traditional approaches. Strengths and limitations of this approach are discussed, as well as next steps. Environ Toxicol Chem 2017;36:3081-3090. © 2017 SETAC [ABSTRACT FROM AUTHOR]

Development of novel synthetic routes based on more eco-friendly processes, such as enzyme-based catalysis, are in great demand to produce biopolymers. Among them, enzymatic Ring Opening Polymerization (eROP) of lactones using lipases, such as Candida antarctica lipase B (CalB), offers new opportunities, although they have been mostly explored in wild-type form. In this study, we followed a structure-based engineering strategy to redesign CalB active site in order to improve catalytic efficiency of poly(ε-caprolactone) synthesis and size of polymer products. A library of 1410 single mutants was constructed by saturation mutagenesis that targeted 15 amino acid positions of the active site, identified by molecular modelling. Novel High-Throughput Screening (HTS) assays based either on the ring-opening of the lactone or the detection of poly(ε-caprolactone) production were developed and used to screen the library. This led to the identification of 8 improved mutants, among which mutant I285R showed nearly 3-fold increase in catalytic efficiency for the ε-caprolactone opening, first step of the reaction, and 30% increased poly(ε-caprolactone) molar mass compared to parental wild-type CalB enzyme in same reaction conditions. [ABSTRACT FROM AUTHOR]

Consumer products containing botanicals or natural substances (BNS) are often preferred because there is a perception that 'natural' is safe. As with any product ingredient, a thorough safety assessment must be conducted, including a determination of skin sensitization potential. A modification of the Peroxidase Peptide Reactivity Assay (PPRA) was explored for screening BNS (B-PPRA) for their reactivity to a model cysteine peptide. The PPRA incorporates a horseradish peroxidase‑hydrogen peroxide (+HRP/P) oxidation system for the activation of potential pre- and pro-haptens. BNS test materials contained <2% botanical constituent in either glycerin/water or propylene glycol/water. Stock solutions prepared in acetonitrile were diluted to 8 working concentrations. Direct reactivity was determined in reaction mixtures containing peptide and deferoxamine in potassium phosphate buffer. Enzyme-mediated reactivity determinations were performed with addition of +HRP/P. Initial studies demonstrated that results were reproducible and impact of carrier low. To determine the sensitivity of the assay, experiments were conducted with chamomile extract spiked with three sensitizers. Peptide depletion was observed in the +HRP/P reaction mixtures with isoeugenol spikes as low as 0.05%. The B-PPRA shows promise as a screening method for skin sensitization potential and could become part of a framework for the skin sensitization safety assessment of BNS. • Botanical natural substances (BNS) are preferred in consumer products as there is a perception that 'natural' is safe. • A safety assessment, including skin sensitization evaluation, must be conducted also for BNS containing products. • The Peroxidase Peptide Reactivity Assay (PPRA) was adapted for screening BNS semi quantitively for their reactivity. • The proof-of-concept studies with 6 BNS demonstrated reproducibility, low carrier impact and sufficient sensitivity. • A framework for skin sensitization safety assessment of BNS using B-PPRA will be developed. [ABSTRACT FROM AUTHOR]

The transportome of Saccharomyces cerevisiae comprises approximately 340 membrane-bound proteins, of which very few are well-characterized. Elucidating transporter proteins' function is essential not only for understanding central cellular processes in metabolite exchange with the external milieu but also for optimizing the production of value-added compounds in microbial cell factories. Here, we describe the application of 13 C-labeled stable isotopes and detection by targeted LC-MS/MS as a screening tool for identifying Saccharomyces cerevisiae metabolite transporters. We compare the transport assay's sensitivity, reproducibility, and accuracy in yeast transporter mutant cell lines and Xenopus oocytes. As proof of principle, we analyzed the transport profiles of five yeast amino acid transporters. We first cultured yeast transporter deletion or overexpression mutants on uniformly labeled 13 C-glucose and then screened their ability to facilitate the uptake or export of an unlabeled pool of amino acids. Individual transporters were further studied by heterologous expression in Xenopus oocytes, followed by an uptake assay with 13 C labeled yeast extract. Uptake assays in Xenopus oocytes showed higher reproducibility and accuracy. Although having lower accuracy, the results from S. cerevisiae indicated the system's potential for initial high-throughput screening for native metabolite transporters. We partially confirmed previously reported substrates for all five amino acid transporters. In addition, we propose broader substrate specificity for two of the transporter proteins. The method presented here demonstrates the application of a comprehensive screening platform for the knowledge expansion of the transporter-substrate relationship for native metabolites in S. cerevisiae., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stanchev, Møller-Hansen, Lojko, Rocha and Borodina.)