Investigating the impact of botulinum toxin type a on the migration of normal human dermal fibroblasts: An in vitro wound healing assay.

BACKGROUND: Botulinum toxin A (BoNT-A) is widely utilized in the management of hypertrophic and keloid scars. One proposed mechanism for scar prevention involves the inhibition of fibroblast migration in scars by BoNT-A. However, the data regarding the effect of BoNT-A on the migration of normal human dermal fibroblasts (NHDF) is limited. OBJECTIVES: The aim of this study was to investigate the inhibitory effect of different types and dilutions of BoNT-A on the migration of NHDF. METHODS: In vitro scratch wound assay, NHDF cells were cultured, incubated, and subjected to scratching using a sterile tip. Subsequently, the scratched NHDF monolayer was treated with different types of BoNT-A, including onabotulinumtoxinA (ONA), incobotulinumtoxinA (INCO), prabotulinumtoxinA (PRABO), or letibotulinumtoxinA (LETI), at varying concentrations of 10, 20, 25, 40, 50, and 100 units/milliliter (U/mL). Additionally, abobotulinumtoxinA (ABO) was administered at concentrations of 33, 50, 66, 71, 100, 150, 300, and 500 U/mL. Normal saline solution (NSS) served as a negative control. The extent of NHDF migration was evaluated by comparing each dilution of BoNT-A with the controls using high-content imaging at the 48-h time point. Furthermore, the viability of the of NHDF was assessed. RESULTS: The concentrations of 25, 40, and 50 U/mL of ONA (p < 0.001) and 25 U/mL of LETI (p < 0.05) demonstrated significantly inhibited NHDF migration in comparison to the control group. Conversely, all dilutions of PRABO, INCO, and ABO exhibited comparable NHDF migration to that of the control group. Regarding NHDF viability, no significant decrease was observed across any of the BoNT-A types and dilutions. CONCLUSION: Different types and dilutions of BoNT-A demonstrated variable inhibitory effects on NHDF migration in vitro. The selection of BoNT-A formulation may significantly impact the clinical outcome of scar prevention related to fibroblast migration.

An in vitro study of coagulation evaluation in obstetric hemorrhage for pregnancy-induced hypertension with coagulation and platelet function analyzer.

This study aimed to establish in vitro hemodilution and resupplementation assays for obstetric hemorrhage in pregnancy-induced hypertension (PIH) and to monitor the coagulation function dynamically using a coagulation and platelet function analyzer. Forty-seven singleton pregnant women were divided into normal (n = 24) and PIH (n = 23) groups. Peripheral blood samples were used to construct the assays, and the activated clotting time (ACT), clotting rate (CR), and platelet function index (PF) were measured. The results showed that the baseline ACT was higher in the PIH group (p < 0.01). Hemodilution assays showed decreased ACT and increased CR and PF, with ACT changes significantly lower in the PIH group (p < 0.05). CR changed most in both groups at lower dilution ratios (35% to 50%), while ACT changed most at a higher dilution ratio (75%). In the resupplementation assay, ACT exhibited the most significant response. The analyzer effectively detected differences between pregnant women with and without PIH. Thus, we need to pay more attention to the changes of ACT in the actual clinical application to assess the coagulation status of parturients.

RapidAIM 2.0: a high-throughput assay to study functional response of human gut microbiome to xenobiotics.

Aim: Our gut microbiome has its own functionalities which can be modulated by various xenobiotic and biotic components. The development and application of a high-throughput functional screening approach of individual gut microbiomes accelerates drug discovery and our understanding of microbiome-drug interactions. We previously developed the rapid assay of individual microbiome (RapidAIM), which combined an optimized culturing model with metaproteomics to study gut microbiome responses to xenobiotics. In this study, we aim to incorporate automation and multiplexing techniques into RapidAIM to develop a high-throughput protocol. Methods: To develop a 2.0 version of RapidAIM, we automated the protein analysis protocol, and introduced a tandem mass tag (TMT) multiplexing technique. To demonstrate the typical outcome of the protocol, we used RapidAIM 2.0 to evaluate the effect of prebiotic kestose on ex vivo individual human gut microbiomes biobanked with five different workflows. Results: We describe the protocol of RapidAIM 2.0 with extensive details on stool sample collection, biobanking, in vitro culturing and stimulation, sample processing, metaproteomics measurement, and data analysis. The analysis depth of 5,014 ± 142 protein groups per multiplexed sample was achieved. A test on five biobanking methods using RapidAIM 2.0 showed the minimal effect of sample processing on live microbiota functional responses to kestose. Conclusions: Depth and reproducibility of RapidAIM 2.0 are comparable to previous manual label-free metaproteomic analyses. In the meantime, the protocol realizes culturing and sample preparation of 320 samples in six days, opening the door to extensively understanding the effects of xenobiotic and biotic factors on our internal ecology.

Eco-friendly bio-larvicidal and antimicrobial activity of isolated bioactive compound from Kurthia gibsonii (Bacillales).

The targeted organisms include mosquito vectors, bacterial pathogens and non-targeted organisms. Preliminary mosquito larvicidal activity was conducted using cell-free supernatants (CFSs) from 11 gut bacteria. Among them, the bacterium SS11 exhibited promising results and was identified as Kurthia gibsonii based on its 16S rRNA sequence (1350 bp). The diethyl ether extract (DEE) of K. gibsonii demonstrated significant larvicidal effects, with LC50 values of 5.59 µL/mL and 8.59 µL/mL for 3rd instar larvae of Aedes aegypti and 2nd instar larvae of Anopheles stephensi, respectively. Analysis of the DEE using FT-IR, and GC-MS revealed the presence of 16 functional groups, and 7 bioactive compounds, respectively. A molecular docking study identified GC-MS compounds against odorant receptors from A. aegypti and odorant-binding proteins from A. stephensi was performed to assess the interaction and binding affinity. Overall, these findings suggest that the bioactive compounds 2, 4, 6-tribromoaniline from the DEE of K. gibsonii hold potential as an environmentally compatible alternative for biocontrol purposes, and compounds 9-tricosene and didecyl phthalate can be used for mosquito traps.

Design, synthesis, molecular docking, and molecular dynamic studies of novel quinazoline derivatives as phosphodiesterase 7 inhibitors.

Introduction: Phosphodiesterase 7 (PDE7) is a high-affinity cyclic AMP (cAMP)-specific PDE that is expressed in immune and proinflammatory cells. In this work, we explore the possibility that selective small molecule inhibitors of this enzyme family could provide a novel approach to alleviate the inflammation that is associated with many inflammatory diseases. Methods: A series of novel substituted 4-hydrazinoquinazoline derivatives and fused triazoloquinazolines were designed, synthesized, and evaluated in vitro for their PDE7A inhibition activities, in comparison with Theophylline, a non-selective PDE inhibitor, and BRL50481, a selective PDE7A inhibitor. This series of novel quinazoline derivatives were synthesized via multi-step reactions. The reaction sequence began with selective monohydrazinolysis of compounds 2a,b to give 3a,b. Schiff bases 4a-h were synthesized by the reaction of the quinazolylhydrazines 3a,b with various substituted aromatic aldehydes. The reaction of 4a-h with bromine in acetic acid, in turn, gave fused triazoloquinazolines 5a-h. These compounds were characterized by satisfied spectrum analyses mainly including 1HNMR, 13CNMR, and MS together with elemental analyses. Results and discussion: The results of in vitro PDE7A inhibition activity clearly indicated that compounds 4b, 4g, 5c, and 5f exhibited good potency. Molecular docking and molecular dynamic simulation studies further supported our findings and provided the basis of interaction in terms of conventional hydrogen bonds and π-π stacking patterns. The present results lay the groundwork for developing lead compounds with improved phosphodiesterase seven inhibitory activities.

Molecular and Clinical Features of Congenital Hypothyroidism Due to Multiple DUOX2 Variants.

Background: DUOX2 is one of the major causative genes of congenital hypothyroidism (CH). Still, the mutation spectrum and clinical outcomes of biallelic DUOX2 variants are not fully understood. This study aimed to elucidate the molecular features and long-term clinical manifestations of CH caused by multiple pathogenic DUOX2 variants. Methods: A total of 255 patients with CH were screened for rare variants of 11 known causative genes. DUOX2 variants were classified according to their protein structure and residual activity. In vitro assays were performed for several variants of unknown functions. Clinical analyses were conducted for patients with multiple pathogenic variants of DUOX2 but not of other genes. Results: We identified 24 pathogenic variants of DUOX2, together with two benign variants and seven variants of uncertain significance, in 63 patients. The pathogenic variants included three missense substitutions and one frameshift variant that have not yet been linked to CH. Twenty-one patients carried multiple pathogenic DUOX2 variants without any other pathogenic gene variants. Three of the 21 patients harbored homozygous variants. Family analysis, long-read amplicon sequencing, and haplotype phasing confirmed compound heterozygosity of the DUOX2 variants in 14 patients, whereas the allelic positions of the variants in the remaining four patients could not be determined. Of the 21 patients, 19 were treated with levothyroxine; their ages at drug withdrawal ranged from 9 months to 21.4 years. Three patients required retreatment after drug-free intervals of 6 months, 8 months, and 10 years. There were no differences in clinical severity among patients with DUOX2 amorphic/amorphic, amorphic/hypomorphic, and hypomorphic/hypomorphic variants. Conclusions: These results broaden the mutational spectrum of DUOX2. Furthermore, our data imply that patients with multiple pathogenic DUOX2 variants typically exhibit transient CH without significant genotype-phenotype correlations. Most importantly, this study demonstrated for the first time that these patients are at risk of developing recurrent hypothyroidism after a long drug-free interval.

A novel hepatocyte ketone production assay to help the selection of nutrients for the ketogenic diet treatment of epilepsy.

The classic ketogenic diet is an effective treatment option for drug-resistant epilepsy, but its high fat content challenges patient compliance. Optimizing liver ketone production guided by a method comparing substrates for their ketogenic potential may help to reduce the fat content of the diet without loss in ketosis induction. Here, we present a liver cell assay measuring the ß-hydroxybutyrate (ßHB) yield from fatty acid substrates. Even chain albumin-conjugated fatty acids comprising between 4 and 18 carbon atoms showed a sigmoidal concentration-ßHB response curve (CRC) whereas acetate and omega-3 PUFAs produced no CRC. While CRCs were not distinguished by their half-maximal effective concentration (EC50), they differed by maximum response, which related inversely to the carbon chain length and was highest for butyrate. The assay also suitably assessed the ßHB yield from fatty acid blends detecting shifts in maximum response from exchanging medium chain fatty acids for long chain fatty acids. The assay further detected a dual role for butyrate and hexanoic acid as ketogenic substrate at high concentration and ketogenic enhancer at low concentration, augmenting the ßHB yield from oleic acid and a fatty acid blend. The assay also found propionate to inhibit ketogenesis from oleic acid and a fatty acid blend at low physiological concentration. Although the in vitro assay shows promise as a tool to optimize the ketogenic yield of a fat blend, its predictive value requires human validation.

Kinetic analysis of T4 polynucleotide kinase via isothermal titration calorimetry.

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3'-monophosphate (3'-AMP) to adenosine-3',5'-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 µM, and the turnover number, kcat, was 8 s-1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3'-AMP were determined to be -25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.

In Silico and In Vitro multiple analysis approach for screening naturally derived ligands for red seabream aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a key ligand-dependent transcription factor that mediates the toxic effects of compounds such as dioxin. Recently, natural ligands of AHR, including flavonoids, have been attracting physiological and toxicological attention as they have been reported to regulate major biological functions such as inflammation and anti-cancer by reducing the toxic effects of dioxin. Additionally, it is known that natural AHR ligands can accumulate in wildlife tissues, such as fish. However, studies in fish have investigated only a few ligands in experimental fish species, and the AHR response of marine fish to natural AHR ligands of various other structures has not been thoroughly investigated. To explore various natural AHR ligands in marine fish, which make up the most fish, it is necessary to develop new screening methods that consider the specificity of marine fish. In this study, we investigated the response of natural ligands by constructing in vitro and in silico experimental systems using red seabream as a model species. We attempted to develop a new predictive model to screen potential ligands that can induce transcriptional activation of red seabream AHR1 and AHR2 (rsAHR1 and rsAHR2). This was achieved through multiple analyses using in silico/ in vitro data and Tox21 big data. First, we constructed an in vitro reporter gene assay of rsAHR1 and rsAHR2 and measured the response of 10 representatives natural AHR ligands in COS-7 cells. The results showed that FICZ, Genistein, Daidzein, I3C, DIM, Quercetin and Baicalin induced the transcriptional activity of rsAHR1 and rsAHR2, while Resveratrol and Retinol did not induce the transcriptional activity of rsAHR isoforms. Comparing the EC50 values of the respective compounds in rsAHR1 and rsAHR2, FICZ, Genistein, and Daidzein exhibited similar isoform responses, but I3C, Baicalin, DIM and Quercetin show the isoform-specific responses. These results suggest that natural AHR ligands have specific profiling and transcriptional activity for each rsAHR isoform. In silico analysis, we constructed homology models of the ligand binding domains (LBDs) of rsAHR1 and rsAHR2 and calculated the docking energies (U_dock values) of natural ligands with measured in vitro transcriptional activity and dioxins reported in previous studies. The results showed a significant correlation (R2=0.74(rsAHR1), R2=0.83(rsAHR2)) between docking energy and transcriptional activity (EC50) value, suggesting that the homology model of rsAHR1 and rsAHR2 can be utilized to predict the potential transactivation of ligands. To broaden the applicability of the homology model to diverse compound structures and validate the correlation with transcriptional activity, we conducted additional analyses utilizing Tox21 big data. We calculated the docking energy values for 1860 chemicals in both rsAHR1 and rsAHR2, which were tested for transcriptional activation in Tox21 data against human AHR. By comparing the U_dock energy values between 775 active compounds and 1085 inactive compounds, a significant difference (p<0.001) was observed between the U_dock energy values in the two groups, suggesting that the U_dock value can be applied to distinguish the activation of compounds. Furthermore, we observed a significant correlation (R2=0.45) between the AC50 of Tox21 database and U_dock values of human AHR model. In conclusion, we calculated equations to translate the results of an in silico prediction model for ligand screening of rsAHR1 and rsAHR2 transactivation. This ligand screening model can be a powerful tool to quantitatively estimate AHR transactivation of major marine agents to which red seabream may be exposed. The study introduces a new screening approach for potential natural AHR ligands in marine fish, based on homology model-docking energy values of rsAHR1 and rsAHR2, with implications for future agonist development and applications bridging in silico and in vitro data.

Protocol improvement and multisite validation of a digital soft agar colony formation assay for tumorigenic transformed cells intermingled in cell therapy products.

BACKGROUND AIMS: The administration of human cell-processed therapeutic products (hCTPs) is associated with a risk of tumorigenesis due to the transformed cellular contaminants. To mitigate this risk, these impurities should be detected using sensitive and validated assays. The digital soft agar colony formation (D-SAC) assay is an ultrasensitive in vitro test for detecting tumorigenic transformed cells in hCTPs. METHODS: In this study, we first evaluated the colony formation efficiency (CFE) precision of tumorigenic reference cells in positive control samples according to a previously reported D-SAC assay protocol (Protocol I) from multiple laboratories. However, the CFE varied widely among laboratories. Thus, we improved and optimized the test protocol as Protocol II to reduce variability in the CFE of tumorigenic reference cells. Subsequently, the improved protocol was validated at multiple sites. Human mesenchymal stromal cells (hMSCs) were used as model cells, and positive control samples were prepared by spiking them with HeLa cells. RESULTS: Based on the previously reported protocol, the CFE was estimated using an ultra-low concentration (0.0001%) of positive control samples in multiple plates. Next, we improved the protocol to reduce the CFE variability. Based on the CFE results, we estimated the sample size as the number of wells (Protocol II) and assessed the detectability of 0.0001% HeLa cells in hMSCs to validate the protocol at multiple sites. Using Protocol I yielded low CFEs (mean: 30%) and high variability between laboratories (reproducibility coefficient of variance [CV]: 72%). In contrast, Protocol II, which incorporated a relatively high concentration (0.002%) of HeLa cells in the positive control samples, resulted in higher CFE values (mean: 63%) and lower variability (reproducibility CV: 18%). Moreover, the sample sizes for testing were estimated as the number of wells per laboratory (314-570 wells) based on the laboratory-specific CFE (42-76%). Under these conditions, all laboratories achieved a detection limit of 0.0001% HeLa cells in hMSCs in a predetermined number of wells. Moreover, colony formation was not observed in the wells seeded with hMSCs alone. CONCLUSIONS: The D-SAC assay is a highly sensitive and robust test for detecting malignant cells as impurities in hCTPs. In addition, optimal assay conditions were established to test tumorigenic impurities in hCTPs with high sensitivity and an arbitrary false negative rate.

Sens-ocular model: Cell-based assay to evaluate eye stinging potential of chemicals and baby cosmetic formulations.

The TRPV1 receptor, which is known to contribute significantly to pain perception, has recently been identified as a useful tool for predicting eye stinging potential in cosmetics. In this study, HEK-293 cells with high TRPV1 expression were utilized to evaluate calcium influx related to receptor activation triggered by chemicals and cosmetic formulations. The cells were exposed to increasing concentrations of substances to cause or not some aggression to the eye, and TRPV1 activity was assessed by measuring intracellular FURA-2 AM fluorescence signal. To confirm TRPV1 channel activation, capsazepine, a capsaicin antagonist, was employed in addition to using capsaicin as a positive control. The study's results indicate that this novel model can identify compounds known to cause some aggression to the eye, such as stinging, considering a cut-off value of 60% of Ca2+ influx exposed to the lowest evaluated concentration (0.00032%). When applied to the cosmetic baby formulation, although the presented model exhibited higher sensitivity by classifying as stinging formulations that had previously undergone clinical testing and were deemed non-stinging, the assay could serve as a valuable in vitro tool for predicting human eye stinging sensation and can be used as a tier 1 in an integrated testing strategy.

Toxicity removal from contaminated water by constructed wetlands assessed using multiple biomarkers in human stem cell assays.

Constructed wetlands (CWs) have been developed rapidly as a sustainable water treatment technique. However, the capability of CWs for remediating the contaminated water based on toxicity assessment remains largely unknown. Four surface flow CWs and two integrated surface-subsurface flow CWs, from five cities in central and eastern region of China were evaluated, concerning the adverse effects of effluents and the toxicity reduction efficiency. Human bone marrow mesenchymal stem cells (hBMSCs) were employed as a human relevant in vitro model. The influent extractions caused cytotoxicity in a dose-dependent manner. The non-cytotoxic dilutions of the influents enhanced the genotoxicity marker γ-H2AX and reactive oxygen species levels. In addition, the influent repressed the osteogenic and neurogenic differentiation, and stimulated the adipogenic differentiation. Cytotoxicity of the contaminated water was reduced by 54 %-86 % after treatment with CWs. CWs were effective to remove part of the sub-lethal effects, with lower reduction than cytotoxicity. The integrated biomarker response (IBR) value of the effluents from the six CWs is lower than that of four secondary and one tertiary wastewater treatment plants. The IBR of the six CWs influents were in the range of 8.6-10.6, with a reduction of 15-50 % after the pollution restoration in CWs. The two integrated surface-subsurface flow CWs achieved higher IBR removal than the four surface flow CWs, possibly due to improved treatment effects by the combined systems. Cytotoxic and genotoxic effects of polar fractions in the CW effluents were stronger than the medium-polar and the non-polar fractions. Besides, PPARγ agonists present in the effluents played crucial roles and ERα agonists may make modest contributions. The present study enhances understanding of the role of CWs in achieving safe wastewater reclamation and provides evidence for further improving toxicity reduction in CWs performance.

Utilizing murine dendritic cell line DC2.4 to evaluate the immunogenicity of subunit vaccines in vitro.

Subunit vaccines hold substantial promise in controlling infectious diseases, due to their superior safety profile, specific immunogenicity, simplified manufacturing processes, and well-defined chemical compositions. One of the most important end-targets of vaccines is a subset of lymphocytes originating from the thymus, known as T cells, which possess the ability to mount an antigen-specific immune response. Furthermore, vaccines confer long-term immunity through the generation of memory T cell pools. Dendritic cells are essential for the activation of T cells and the induction of adaptive immunity, making them key for the in vitro evaluation of vaccine efficacy. Upon internalization by dendritic cells, vaccine-bearing antigens are processed, and suitable fragments are presented to T cells by major histocompatibility complex (MHC) molecules. In addition, DCs can secrete various cytokines to crosstalk with T cells to coordinate subsequent immune responses. Here, we generated an in vitro model using the immortalized murine dendritic cell line, DC2.4, to recapitulate the process of antigen uptake and DC maturation, measured as the elevation of CD40, MHC-II, CD80 and CD86 on the cell surface. The levels of key DC cytokines, tumor necrosis alpha (TNF-α) and interleukin-10 (IL-10) were measured to better define DC activation. This information served as a cost-effective and rapid proxy for assessing the antigen presentation efficacy of various vaccine formulations, demonstrating a strong correlation with previously published in vivo study outcomes. Hence, our assay enables the selection of the lead vaccine candidates based on DC activation capacity prior to in vivo animal studies.

A novel metabolite of Streptomyces coeruleorubidus exhibits antibacterial activity against Streptococcus agalactiae through modulation of physiological performance, inflammatory cytokines, apoptosis, and oxidative stress-correlated gene expressions in Nile tilapia (Oreochromis niloticus).

Using the unique structures found in natural materials to produce new antibacterial drugs is crucial. Actinobacteria is well-known for its ability to produce naturally occurring chemicals with a variety of structural features that can be used as weapons against infectious bacteria. In the present study, the Streptomyces coeruleorubidus metabolites were characterized and their efficacy in suppressing Streptococcus agalactiae growth was carried out both in vitro and in vivo. The metabolites of S. coeruleorubidus were purified and identified as octasiloxane-hexadecamethyl (OHM). In vivo antibacterial activity of OHM revealed an inhibitory minimum concentration value of 0.5 µg/ml against S. agalactiae and induced ultrastructural cell changes revealed by scanning electron microscope. The safe concentration of OHM was determined as 0.8 mg/L for Nile tilapia. Four in vivo treatments were treated with 0 and 0.8 mg/L OHM and with or without challenge by S. agalactiae (1 × 107 CFU/mL) named control, OHM, S. agalactiae, and S. agalactiae + OHM groups. The OHM treatment improved the survival of Nile tilapia by 33.33% than S. agalactiae challenge group. Waterborne OHM treatment significantly mitigated the deleterious effects of S. agalactiae on hematological, hepato-renal functions, stress indicators, and antioxidant balance. OHM significantly alleviated nitric oxide levels, complement 3, IgM, and lysozyme activity, downregulation of liver antioxidant genes expression in S. agalactiae group. Furthermore, the addition of OHM to challenged fish with S. agalactiae-significantly reversed dramatic negative regulation of inflammatory, apoptosis, and immune related gene expression (caspase-3, bax, pcna, tnf-α, ifn-γ, il-8 il-1ß, il-10, tgf-ß, and bcl-2 in the Nile tilapia spleen. Additionally, the damaged hepatic and splenic structure induced by bacterial infection was restored with OHM treatment. Finally, S. coeruleorubidus metabolites (mainly OHM) revealed in vitro and in vivo antibacterial activity and showed alleviated effects on the physiological status of S. agalactiae infected tilapia.

MIB Guides: Measuring the Immunoreactivity of Radioimmunoconjugates.

Immunoglobulins, both full-length antibodies and smaller antibody fragments, have long been regarded as effective platforms for diagnostic and therapeutic radiopharmaceuticals. The construction of radiolabeled immunoglobulins (i.e., radioimmunoconjugates) requires the manipulation of the biomolecule through the attachment of a radiohalogen or the bioconjugation of a chelator that is subsequently used to coordinate a radiometal. Both synthetic approaches have historically relied upon the stochastic modification of amino acids within the immunoglobulin, a process which poses a risk to the structural and functional integrity of the biomolecule itself. Not surprisingly, radioimmunoconjugates with impaired antigen binding capacity will inevitably exhibit suboptimal in vivo performance. As a result, the biological characterization of any newly synthesized radioimmunoconjugate must include an assessment of whether it has retained its ability to bind its antigen. Herein, we provide straightforward and concise protocols for three assays that can be used to determine the immunoreactivity of a radioimmunoconjugate: (1) a cell-based linear extrapolation assay; (2) a cell-based antigen saturation assay; and (3) a resin- or bead-based assay. In addition, we will provide a critical analysis of the relative merits of each assay, an examination of the inherent limitations of immunoreactivity assays in general, and a discussion of other approaches that may be used to interrogate the biological behavior of radioimmunoconjugates.

Utility of human cytochrome P450 inhibition data in the assessment of drug-induced liver injury.

1. Drug-induced liver injury (DILI) is a major cause of drug development discontinuation and drug withdrawal from the market, but there are no golden standard methods for DILI risk evaluation. Since we had found the association between DILI and CYP1A1 or CYP1B1 inhibition, we further evaluated the utility of cytochrome P450 (P450) inhibition assay data for DILI risk evaluation using decision tree analysis.2. The inhibitory activity of drugs with DILI concern (DILI drugs) and no DILI concern (no-DILI drugs) against 10 human P450s was assessed using recombinant enzymes and luminescent substrates. The drugs were also subjected to cytotoxicity assays and high-content analysis using HepG2 cells. Molecular descriptors were calculated by alvaDesc.3. Decision tree analysis was performed with the data obtained as variables with or without P450-inhibitory activity to discriminate between DILI drugs and no-DILI drugs. The accuracy was significantly higher when P450-inhibitory activity was included. After the decision tree discrimination, the drugs were further discriminated with the P450-inhibitory activity. The results demonstrated that many false-positive and false-negative drugs were correctly discriminated by using the P450 inhibition data.4. These results suggest that P450 inhibition assay data are useful for DILI risk evaluation.

Identification of new pharmacophore against SARS-CoV-2 spike protein by multi-fold computational and biochemical techniques.

COVID-19 appeared as a highly contagious disease after its outbreak in December 2019 by the virus, named SARS-CoV-2. The threat, which originated in Wuhan, China, swiftly became an international emergency. Among different genomic products, spike protein of virus plays a crucial role in the initiation of the infection by binding to the human lung cells, therefore, SARS-CoV-2's spike protein is a promising therapeutic target. Using a combination of a structure-based virtual screening and biochemical assay, this study seeks possible therapeutic candidates that specifically target the viral spike protein. A database of ~ 850 naturally derived compounds was screened against SARS-CoV-2 spike protein to find natural inhibitors. Using virtual screening and inhibitory experiments, we identified acetyl 11-keto-boswellic acid (AKBA) as a promising molecule for spike protein, which encouraged us to scan the rest of AKBA derivatives in our in-house database via 2D-similarity searching. Later 19 compounds with > 85% similarity with AKBA were selected and docked with receptor binding domain (RBD) of spike protein. Those hits declared significant interactions at the RBD interface, best possess and excellent drug-likeness and pharmacokinetics properties with high gastrointestinal absorption (GIA) without toxicity and allergenicity. Our in-silico observations were eventually validated by in vitro bioassay, interestingly, 10 compounds (A3, A4, C3, C6A, C6B, C6C, C6E, C6H, C6I, and C6J) displayed significant inhibitory ability with good percent inhibition (range: > 72-90). The compounds C3 (90.00%), C6E (91.00%), C6C (87.20%), and C6D (86.23%) demonstrated excellent anti-SARS CoV-2 spike protein activities. The docking interaction of high percent inhibition of inhibitor compounds C3 and C6E was confirmed by MD Simulation. In the molecular dynamics simulation, we observed the stable dynamics of spike protein inhibitor complexes and the influence of inhibitor binding on the protein's conformational arrangements. The binding free energy ΔGTOTAL of C3 (-38.0 ± 0.08 kcal/mol) and C6E (-41.98 ± 0.08 kcal/mol) respectively indicate a strong binding affinity to Spike protein active pocket. These findings demonstrate that these molecules particularly inhibit the function of spike protein and, therefore have the potential to be evaluated as drug candidates against SARS-CoV-2.

Chitosan-Calcium Aluminate as a Cell-homing Scaffold: Its Bioactivity Testing in a Microphysiological Dental Pulp Platform.

In vitro models of the dental pulp microenvironment have been proposed for the assessment of biomaterials, to minimise animal use in operative dentistry. In this study, a scaffold/3-D dental pulp cell culture interface was created in a microchip, under simulated dental pulp pressure, to evaluate the cell-homing potential of a chitosan (CH) scaffold functionalised with calcium aluminate (the 'CHAlCa scaffold'). This microphysiological platform was cultured at a pressure of 15 cm H2O for up to 14 days; cell viability, migration and odontoblastic differentiation were then assessed. The CHAlCa scaffold exhibited intense chemotactic potential, causing cells to migrate from the 3-D culture to its surface, followed by infiltration into the macroporous structure of the scaffold. By contrast, the cells in the presence of the non-functionalised chitosan scaffold showed low cell migration and no cell infiltration. CHAlCa scaffold bioactivity was confirmed in dentin sialophosphoprotein-positive migrating cells, and odontoblastic markers were upregulated in 3-D culture. Finally, in situ mineralised matrix deposition by the cells was confirmed in an Alizarin Red-based assay, in which the CHAlCa and CH scaffolds were adapted to fit within dentin discs. More intense deposition of matrix was observed with the CHAlCa scaffold, as compared to the CH scaffold. In summary, we present an in vitro platform that provides a simple and reproducible model for selecting and developing innovative biomaterials through the assessment of their cell-homing potential. By using this platform, it was shown that the combination of calcium aluminate and chitosan has potential as an inductive biomaterial that can mediate dentin tissue regeneration during cell-homing therapies.

Inhibition of Uridine 5'-diphospho-glucuronosyltransferases A10 and B7 by vitamins: insights from in silico and in vitro studies.

Uridine 5'-diphospho-glucuronosyltransferases (UGTs) have been considered as a family of enzymes responsible for the glucuronidation process, a crucial phase II detoxification reaction. Among the various UGT isoforms, UGTs A10 and B7 have garnered significant attention due to their broad substrate specificity and involvement in the metabolism of numerous compounds. Recent studies have suggested that certain vitamins may exert inhibitory effects on UGT activity, thereby influencing the metabolism of drugs, environmental toxins, and endogenous substances, ultimately impacting their biological activities. In the present study, the inhibition potential of vitamins (A, B1, B2, B3, B5, B6, B7, B9, D3, E, and C) on UGT1A10 and UGT2B7 was determined using in silico and in vitro approaches. A 3-dimensional model of UGT1A10 and UGT2B7 enzymes was built using Swiss Model, ITASSER, and ROSETTA and verified using Ramachandran plot and SAVES tools. Molecular docking studies revealed that vitamins interact with UGT1A10 and UGT2B7 enzymes by binding within the active site pocket and interacting with residues. Among all vitamins, the highest binding affinity predicted by molecular docking was - 8.61 kcal/mol with vitamin B1. The in vitro studies results demonstrated the inhibition of the glucuronidation activity of UGTs by vitamins A, B1, B2, B6, B9, C, D, and E, with IC50 values of 3.28 ± 1.07 µg/mL, 24.21 ± 1.11 µg/mL, 3.69 ± 1.02 µg/mL, 23.60 ± 1.08 µg/mL, 6.77 ± 1.08 µg/mL, 83.95 ± 1.09 µg/ml, 3.27 ± 1.13 µg/mL and 3.89 ± 1.12 µg/mL, respectively. These studies provided the valuable insights into the mechanisms underlying drug-vitamins interactions and have the potential to guide personalized medicine approaches, optimizing therapeutic outcomes, and ensuring patient safety. Indeed, further research in the area of UGT (UDP-glucuronosyltransferase) inhibition by vitamins is essential to fully understand the clinical relevance and implications of these interactions. UGTs play a crucial role in the metabolism and elimination of various drugs, toxins, and endogenous compounds in the body. Therefore, any factors that can modulate UGT activity, including vitamins, can have implications for drug metabolism, drug-drug interactions, and overall health. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00182-0.

In vitro Assay to Examine the Function of Tears on Corneal Epithelial Cells.

Tears contain numerous secreted factors, enzymes, and proteins that help in maintaining the homeostatic condition of the eye and also protect it from the external environment. However, alterations to these enzymes and/or proteins during pathologies such as mechanical injury and viral or fungal infections can disrupt the normal ocular homeostasis, further contributing to disease development. Several tear film components have a significant role in curbing disease progression and promoting corneal regeneration. Additionally, several factors related to disease progression are secreted into the tear film, thereby serving as a valuable reservoir of biomarkers. Tears are readily available and can be collected via non-invasive techniques or simply from contact lenses. Tears can thus serve as a valuable and easy source for studying disease-specific biomarkers. Significant advancements have been made in recent years in the field of tear film proteomics, lipidomics, and transcriptomics to allow a better understanding of how tears can be utilized to gain insight into the etiology of diseases. These advancements have enabled us to study the pathophysiology of various disease states using tear samples. However, the mechanisms by which tears help to maintain corneal homeostasis and how they are able to form the first line of defense against pathogens remain poorly understood and warrant detailed in vitro studies. Herein, we have developed an in vitro assay to characterize the functional importance of patient isolated tears and their components on corneal epithelial cells. This novel approach closely mimics real physiological conditions and could help the researchers gain insight into the underlying mechanisms of ocular pathologies and develop new treatments. Key features • This method provides a new technique for analyzing the effect of tear components on human corneal epithelial cells. • The components of the tears that are altered in response to diseases can be used as a biomarker for detecting ocular complications. • This procedure can be further employed as an in vitro model for assessing the efficacy of drugs and discover potential therapeutic interventions.