Toxicity Assays Research Articles

A pumpless liver-on-a-chip for drug hepatotoxicity analysis.

This study presents the development and validation of an innovative microfluidic liver-on-a-chip device utilizing gravity-driven perfusion for the evaluation of drug hepatotoxicity. This research involved the construction of a hydrogel-based coculture chip that integrates liver parenchymal and stellate cells within a tri-channel configuration. The assembly and operation of the liver-on-a-chip and its accompanying custom rocker were straightforward. The cells in the chip maintained high viability and continuously synthesized liver albumin over extended culture durations. Acetaminophen (APAP), a hepatic injury-inducing drug, was utilized as a positive control in hepatic toxicity assays on the chip. The liver chip exhibited hepatotoxic responses comparable to those observed in 2D models. Furthermore, in this study we evaluated the effects of two plant-derived natural compounds, aristolochic acid I (AA) and its analog aristolactam AII (AL), in both 2D cell models and the liver-on-a-chip system. AA, known for its hepatorenal toxicity, was observed to cause hepatotoxicity in both the 2D models and on the chip. The flow cytometry and mRNA sequencing results confirmed the propensity of these compounds to induce liver cell apoptosis. Notably, AL, previously considered nontoxic, provoked a significant decrease in the hepatic functionality marker albumin exclusively in the liver chip but not in 2D models, indicating the liver chip's enhanced sensitivity to toxic substances. In summary, this pumpless liver-on-a-chip is a simple yet powerful tool for drug hepatotoxicity studies.

A First Report on the Isolation and Characterization of a Highly Potential Indigenous Mosquitocidal Bacterium (Bacillus thuringiensis Subspecies Israelensis VCRC B647) from Red Soil, India.

To control mosquito vectors causing human diseases, bacterial biopesticides are currently in use. Indeed, the recent development of resistance to these bacterial agents has impeded its applications. Under these circumstances, the search for novel bacterial agents with mosquitocidal activity is unavoidable. In this study, a novel mosquitocidal bacterium was isolated from red soils of agricultural field. The objective of this study was to isolate and identify new mosquitocidal bacteria from the natural environment. Soil samples were collected during 2021-2022 from Tirupathur district of Tamil Nadu, South India. The samples were bioprocessed for culturing the bacterial colony in a suitable culture medium (Nutrient Yeast Salt Medium), and after 72 h, the bacterial cell mass was removed and lyophilized. Bioassays (mosquito toxicity assays) were carried out to screen the bacterial colonies for mosquitocidal effect. The potential colony was further analyzed, and identified for its application in mosquito control. The new isolate screened from red soil was identified as Bacillus thuringiensis subspecies israelensis (VCRC B647) as per the ilvD gene sequence analysis. The strain was found to be potentially effective in controlling mosquito larvae, and further biochemical analyses, bacterial growth, biomass, and protein content were investigated. The new isolate did not show any toxic effect on nontarget aquatic organisms. It is significant to depict that the mosquitocidal action of this new isolate (Bti) is highly significant than the reference strain of Bti-H14. It is concluded that this is the first report that an indigenous strain of Bti VCRC B647 is very effective in mosquito control.

Lysinibacillus fusiformis: A novel mosquitocidal bacterium isolated from Western Ghats, Kerala, India

An extensive field survey was carried out from December 2021 to January 2022 for the collection of soil samples in the Western Ghat region of the Wayanad district of Kerala, India, to isolate potent novel mosquitocidal bacteria which could be used as efficient formulations to control mosquito vectors. Several bacterial colonies were isolated and screened, for mosquitocidal activity. Toxicity assay showed that only one bacterium isolated from forest loamy soil had promising larvicidal activity against Culex quinquefasciatus the lymphatic filariasis vector (LC50: 0.03 mg L-1 & LC90: 0.07 mg L-1) and moderate activity was shown against the dengue vector, Aedes aegypti (LC50: 1.03 mg L-1 & LC90: 1.8 mg L-1). The bacterium was identified as Lysnibacillus fusiformis by a phylogenetic tree constructed using 16S rRNA genome sequencing. This is the first report that L. fusiformis isolated from the forest loamy soil of the Western Ghats, Kerala, which showed proficient mosquitocidal activity against disease-transmitting mosquito vectors.

Exploring the Efficacy of Essential Oils in Laboratory Conditions for Controlling Mediterranean Fruit Fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

The Mediterranean fruit fly (Medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), poses a significant threat to agriculture worldwide. This study examines the potential insecticidal effects of essential oils from Mentha arvensis and Cinnamomum zeylanicum on controlling C. capitata under laboratory conditions. Even at low concentrations, toxicity assays indicated that both essential oils significantly increased the mortality of adult Medflies. The concentration-dependent effect of these oils on C. capitata mortality is demonstrated, with Mentha arvensis achieving 100% mortality within 48 hours at 1% concentration and Cinnamomum zeylanicum exhibiting rapid efficacy, reaching a low LC50 value after only 1 hour of application. The concentration and application time of essential oils were found to have a significant impact on their effectivness. This study highlights the potential of essential oils for controlling C. capitata populations. Essential oils offer a sustainable and eco-friendly alternative for managing C. capitata but further studies are necessary for their successful incorporation into integrated pest management programs.

Antibiofilm activity of lawsone against polymicrobial enterohemorrhagic Escherichia coli O157:H7 and Candida albicans by suppression of curli production and hyphal growth

Most bacteria and fungi form biofilms that attach to living or abiotic surfaces. These biofilms diminish the efficacy of antimicrobial agents and contribute to chronic infections. Furthermore, multispecies biofilms composed of bacteria and fungi are often found at chronic infection sites. In this study, lawsone (2‑hydroxy-1,4-naphthoquinone) and its parent 1,4-naphthoquinone were studied for antimicrobial and antibiofilm activities against single-species and multispecies biofilms of enterohemorrhagic Escherichia coli O157:H7 (EHEC) and Candida albicans. Biofilm formation assays, biofilm eradication assays, antimicrobial assays, live cell imaging microscopy, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), extracellular polymeric substances and indole production, cell surface hydrophilicity assay, cell motility, cell aggregation, hyphal growth, dual species biofilm formation, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and toxicity assays on plant seed germination and nematode model were utilized to investigate how lawsone affect biofilm development. Sub-inhibitory concentrations of lawsone (35µg/ml) significantly inhibited single-and multispecies biofilm development. Lawsone reduced the production of curli and indole, and the swarming motility of EHEC, efficiently inhibited C. albicans cell aggregation and hyphal formation, and increased the cell surface hydrophilicity of C. albicans. Transcriptomic analysis showed that lawsone suppressed the expression of the curli-related genes csgA and csgB in EHEC, and the expression of several hypha- and biofilm-related genes (ALS3, ECE1, HWP1, and UME6) in C. albicans. In addition, lawsone up to 100µg/ml was nontoxic to the nematode Caenorhabditis elegans and to the seed growth of Brassica rapa and Triticum aestivum. These results show that lawsone inhibits dual biofilm development and suggest that it might be useful for controlling bacterial or fungal infections and multispecies biofilms.

Acute Toxicity Assays with Adult Coral Fragments: A Method for Standardization.

Coral reefs are globally declining due to various anthropogenic stressors. Amongst those, chemical pollutants, such as pesticides from agricultural runoff, sewage or an overabundance of personal care products in coastal waters due to intense tourism, may be considered as a local stressor for reef-building corals. The extent to which such chemicals exhibit toxic effects towards corals at environmentally relevant concentrations is currently controversially discussed and existing studies are often based on varying and sometimes deficient test methods. To address this uncertainty, we adapted available methods into a reliable and comprehensive acute coral toxicity test method for the reef-building coral Montipora digitata. The toxicities of the four substances benzophenone-3 (BP-3), Diuron (DCMU), copper (Cu2+ as CuCl2, positive control) and dimethylformamide (DMF, solvent) were assessed in a 96 h semi-static test design. Endpoints such as maximum quantum yield, bleaching, tissue loss and mortality were evaluated with respect to their suitability for regulatory purposes. Overall, the endpoints bleaching and mortality yielded sensitive and robust results for the four tested substances. As the test method follows the principles of internationally standardized testing methods (ISO, OECD), it can be considered suitable for further validation and standardization. Once validated, a standardized test method will help to obtain reproducible toxicity results useful for marine hazard and risk assessment and regulatory decision making.

Toxicity Evaluation and Controlled-Release of Curcumin-Loaded Amphiphilic Poly-N-vinylpyrrolidone Nanoparticles: In Vitro and In Vivo Models.

Curcumin attracts huge attention because of its biological properties: it is antiproliferative, antioxidant, anti-inflammatory, immunomodulatory and so on. However, its usage has been limited by poor water solubility and low bioavailability. Herein, to solve these problems, we developed curcumin-loaded nanoparticles based on end-capped amphiphilic poly(N-vinylpyrrolidone). Nanoparticles were obtained using the solvent evaporation method and were characterized by dynamic and electrophoretic light scattering, transmission electron (TEM) and atomic force (AFM) microscopy. The average particle size was 200 nm, and the ζ-potential was -4 mV. Curcumin-release studies showed that nanoparticles are stable in aqueous solutions. An in vitro release study showed prolonged action in gastric, intestinal and colonic fluids, consistently, and in PBS. In vitro studies on epidermoid carcinoma and human embryonic kidney cells showed that the cells absorbed more curcumin in nanoparticles compared to free curcumin. Nanoparticles are safe for healthy cells and show high cytotoxicity for glioblastoma cells in cytotoxicity studies in vitro. The median lethal dose was determined in an acute toxicity assay on zebrafish and was 23 μM. Overall, the curcumin-loaded nanoparticles seem promising for cancer treatment.

OXIDOSQUALENE CYCLASE 1 and 2 influence triterpene biosynthesis and defense in Nicotiana attenuata.

Triterpenes are a class of bioactive compounds with diverse biological functions, playing pivotal roles in plant defense against biotic stressors. Oxidosqualene cyclases (OSCs) serve as gatekeepers in the biosynthesis of triterpenes. In this study, we utilized a Nicotiana benthamiana heterologous expression system to characterize NaOSC1 from Nicotiana attenuata as a multifunctional enzyme capable of synthesizing lupeol, dammarenediol II, 3-alpha,20-lupanediol, and 7 other triterpene scaffolds. We also demonstrated that NaOSC2 is, in contrast, a selective enzyme, producing only the β-amyrin scaffold. Through virus-induced gene silencing and in vitro toxicity assays, we elucidated the roles of NaOSC1 and NaOSC2 in the defense of N. attenuata against Manduca sexta larvae. Metabolomic and feature-based molecular network analyses of leaves with silenced NaOSC1 and NaOSC2 unveiled 3 potential triterpene glycoside metabolite clusters. Interestingly, features identified as triterpenes within these clusters displayed a significant negative correlation with larval mass. Our study highlights the pivotal roles of NaOSC1 and NaOSC2 from N. attenuata in the initial steps of triterpene biosynthesis, subsequently influencing defense against M. sexta through the modulation of downstream triterpene glycoside compounds.

Application of the white-rot fungus Trametes sp. (C3) laccase in the removal of acetaminophen from aqueous solutions

Effluents containing drugs originating from hospitals, veterinary clinics, residences, and pharmaceutical industries, pass through the sewage treatment system and are discharged into the aquatic environment, causing water contamination. The methods used in sewage treatment are ineffective for these types of micropollutants. Laccase, an enzyme produced by white-rot fungi, has been used to successfully remove drugs in waters. The objective of this work was to evaluate the removal of acetaminophen from aqueous solutions by the fungus Trametes sp. (C3). Ethanol showed efficiency in inducing the production of the laccase enzyme and in vitro removal tests the amount of drug decreased by 76 %. The use of the 1-hydroxybenzotriazole (HBT) as mediator caused a lower removal rate than the assay without its presence, indicating that it acted as an inhibitor of degradation. In in vivo tests, there was a 79 % removal rate of acetaminophen on the fourth day of culture. Toxicity assays showed that after the action of the laccase there was toxicity to the larval stage of Artemia salina.

Lactobacillus paracasei R3 Alleviates Tumor Progression in Mice with Colorectal Cancer.

Lactobacillus paracasei (L. paracasei), a common probiotic lactobacillus, has important functions in the food industry and human health. However, different strains of L. paracasei inevitably show differences in activity and colonization resistance, leading to differentiation in their functions, as well as their physical or chemical properties. The purpose of this study was to evaluate the characteristics of L. paracasei R3 (L.p R3) isolated from healthy human feces and determine whether the criteria for edible probiotics is met. The hemolysis type, biofilm-forming ability, antibiotic susceptibility, toxicity, and effective activity of L.p R3 were determined by establishing its probiotic activity traits in vitro and in vivo. The results showed that L.p R3 had a moderate biofilm formation ability, was sensitive to 11 antibiotics, was resistant to eight antibiotics, and was not hemolytic. The culture characteristics, morphology, and biochemical responses of the strain were consistent with the seed batch characteristics. In toxicity assays, L.p R3-fed mice showed no abnormalities in body weight, growth, or various organs. Additionally, L.p R3 was found to be effective in the prevention and treatment of colorectal cancer. In conclusion, our results revealed that L.p R3 has potential value as an edible probiotic without toxic side effects and alleviated the tumor progression of colorectal cancer in mice.

Selenium‐doped albumin nanoparticles enhance tamoxifen‐induced anticancer effects in 4T‐1 mouse breast cancer cells

In this study, we aimed to improve the delivery and efficacy of the well‐known anticancer drug, tamoxifen (TAM), through its encapsulation in bovine serum albumin nanoparticles (BSA NPs) and their hybridization with selenium nanoparticles (Se NPs). The developed nanostructures were characterized by Fourier transform infrared (FT‐IR) spectroscopy, ultraviolet–visible (UV–Vis) spectroscopy, energy‐dispersive X‐ray spectroscopy (EDS) analysis, dynamic light scattering (DLS), and field emission scanning electron microscopy (FESEM) imaging. The impact of nanostructures on 4T‐1 cancer cells was assessed through MTT and flow cytometry assays to measure cytotoxicity and apoptosis rates, respectively. FT‐IR and UV–Vis spectroscopy confirmed the successful encapsulation of TAM within BSA NPs. EDS analysis confirmed the Se integration into BSA‐TAM@Se NPs, signifying successful hybridization. Size and zeta potential measurements revealed the average hydrodynamic size and surface charge of the nanostructures. BSA NPs, BSA@Se NPs, and Se NPs showed the sizes ranging from 153.37 ± 1.02 to 229.63 ± 1.36 nm, with zeta potential values from −10.12 ± 0.26 to −28.45 ± 0.40 mV. Hemocompatibility studies demonstrated minimal hemolytic activity of the nanostructures, indicating their biocompatibility for intravenous administration. Cellular toxicity assays showed higher cytotoxicity of BSA‐TAM@Se NPs compared to other formulations (****p < 0.0001). The increased cell death in 4T‐1 breast cancer cells highlights the enhanced therapeutic potential of BSA‐TAM@Se NPs. Apoptosis analysis revealed a significant increase in apoptosis rate in 4T‐1 cells treated with BSA‐TAM@Se NPs compared to other formulations (****p < 0.0001), emphasizing their ability to induce programmed cell death. These findings support the rational importance of BSA NPs as drug nanocarriers and their hybridization with Se NPs to improve therapeutic effects. Finally, we suggest that the developed BSA‐TAM@Se NPs can be used as a potential therapeutic approach in combination with other tools such as X‐ray irradiation in future studies.

Review of machine learning and deep learning models for toxicity prediction.

The ever-increasing number of chemicals has raised public concerns due to their adverse effects on human health and the environment. To protect public health and the environment, it is critical to assess the toxicity of these chemicals. Traditional in vitro and in vivo toxicity assays are complicated, costly, and time-consuming and may face ethical issues. These constraints raise the need for alternative methods for assessing the toxicity of chemicals. Recently, due to the advancement of machine learning algorithms and the increase in computational power, many toxicity prediction models have been developed using various machine learning and deep learning algorithms such as support vector machine, random forest, k-nearest neighbors, ensemble learning, and deep neural network. This review summarizes the machine learning- and deep learning-based toxicity prediction models developed in recent years. Support vector machine and random forest are the most popular machine learning algorithms, and hepatotoxicity, cardiotoxicity, and carcinogenicity are the frequently modeled toxicity endpoints in predictive toxicology. It is known that datasets impact model performance. The quality of datasets used in the development of toxicity prediction models using machine learning and deep learning is vital to the performance of the developed models. The different toxicity assignments for the same chemicals among different datasets of the same type of toxicity have been observed, indicating benchmarking datasets is needed for developing reliable toxicity prediction models using machine learning and deep learning algorithms. This review provides insights into current machine learning models in predictive toxicology, which are expected to promote the development and application of toxicity prediction models in the future.

Prediction of Spheroid Cell Death Using Fluorescence Staining and Convolutional Neural Networks.

Three-dimensional (3D) cell culture is emerging for drug design and drug screening. Skin toxicity is one of the most important assays for determining the toxicity of a compound before being used in skin application. Much work has been done to find an alternative assay without animal experiments. 3D cell culture is one of the methods that provides clinically relevant models with superior clinical translation compared to that of 2D cell culture. In this study, we developed a spheroid toxicity assay using keratinocyte HaCaT cells with propidium iodide and calcein AM. We also applied the transfer learning-containing convolutional neural network (CNN) to further determine spheroid cell death with fluorescence labeling. Our result shows that the morphologies of the spheroid are the key features in determining the apoptosis cell death of the HaCaT spheroid. Our CNN model provided good statistical measurement in terms of accuracy, precision, and recall in both validation and external test data sets. One can predict keratinocyte spheroid cell death if that spheroid image contains the fluorescence signals from propidium iodide and calcein AM. The CNN model can be accessed in the web application at https://qsarlabs.com/#spheroiddeath.

In Vitro Investigation of Gelatin/Polycaprolactone Nanofibers in Modulating Human Gingival Mesenchymal Stromal Cells.

The aesthetic constancy and functional stability of periodontium largely depend on the presence of healthy mucogingival tissue. Soft tissue management is crucial to the success of periodontal surgery. Recently, synthetic substitute materials have been proposed to be used for soft tissue augmentation, but the tissue compatibility of these materials needs to be further investigated. This study aims to assess the in vitro responses of human gingival mesenchymal stromal cells (hG-MSCs) cultured on a Gelatin/Polycaprolactone prototype (GPP) and volume-stable collagen matrix (VSCM). hG-MSCs were cultured onto the GPP, VSCM, or plastic for 3, 7, and 14 days. The proliferation and/or viability were measured by cell counting kit-8 assay and resazurin-based toxicity assay. Cell morphology and adhesion were evaluated by microscopy. The gene expression of collagen type I, alpha1 (COL1A1), α-smooth muscle actin (α-SMA), fibroblast growth factor (FGF-2), vascular endothelial growth factor A (VEGF-A), transforming growth factor beta-1 (TGF-β1), focal adhesion kinase (FAK), integrin beta-1 (ITG-β1), and interleukin 8 (IL-8) was investigated by RT-qPCR. The levels of VEGF-A, TGF-β1, and IL-8 proteins in conditioned media were tested by ELISA. GPP improved both cell proliferation and viability compared to VSCM. The cells grown on GPP exhibited a distinct morphology and attachment performance. COL1A1, α-SMA, VEGF-A, FGF-2, and FAK were positively modulated in hG-MSCs on GPP at different investigation times. GPP increased the gene expression of TGF-β1 but had no effect on protein production. The level of ITG-β1 had no significant changes in cells seeded on GPP at 7 days. At 3 days, notable differences in VEGF-A, TGF-β1, and α-SMA expression levels were observed between cells seeded on GPP and those on VSCM. Meanwhile, GPP showed higher COL1A1 expression compared to VSCM after 14 days, whereas VSCM demonstrated a more significant upregulation in the production of IL-8. Taken together, our data suggest that GPP electrospun nanofibers have great potential as substitutes for soft tissue regeneration in successful periodontal surgery.

Rapid development of increased neonicotinoid tolerance in non-target freshwater amphipods

The comprehensive assessment of the long-term impacts of constant exposure to pollutants on wildlife populations remains a relatively unexplored area of ecological risk assessment. Empirical evidence to suggest that multigenerational exposure affects the susceptibility of organisms is scarce, and the underlying mechanisms in the natural environment have yet to be fully understood. In this study, we first examined the arthropod candidate species, Gammarus roeselii that – unlike closely related species – commonly occurs in many contaminated river systems of Central Europe. This makes it a suitable study organism to investigate the development of tolerances and phenotypic adaptations along pollution gradients. In a 96-h acute toxicity assay with the neonicotinoid thiacloprid, we indeed observed a successive increase in tolerance in populations coming from contaminated regions. This was accompanied by a certain phenotypic change, with increased investment into reproduction. To address the question of whether these changes are plastic or emerged from longer lasting evolutionary processes, we conducted a multigeneration experiment in the second part of our study. Here, we used closely-related Hyalella azteca and pre-exposed them for multiple generations to sublethal concentrations of thiacloprid in a semi-static design (one week renewal of media containing 0.1 or 1.0 µg/L thiacloprid). The pre-exposed individuals were then used in acute toxicity assays to see how quickly such adaptive responses can develop. Over only two generations, the tolerance to the neonicotinoid almost doubled, suggesting developmental plasticity as a plausible mechanism for the rapid adaptive response to strong selection factors such as neonicotinoid insecticides. It remains to be discovered whether the plasticity of rapidly developed tolerance is species-specific and explains why closely related species – which may not have comparable adaptive response capabilities – disappear in polluted habitats. Overall, our findings highlight the neglected role of developmental plasticity during short- and long-term exposure of natural populations to pollution. Moreover, our results show that even pollutant levels seven times lower than concentrations found in the study region have a clear impact on the developmental trajectories of non-target species.

Apolipoprotein E Receptor 2 is associated with Alzheimer’s disease pathogenesis

AbstractBackgroundGenetic variations to apolipoprotein E (apoE) underlie the strongest risk for Alzheimer´s disease (AD) after aging but it is not yet known how apoE confers risk for AD. While most research is focusing on dysregulations associated with the presence of the risk allele of apoE, several aspects of apoE biology remain poorly understood. In particular, the physiological relevance of apoE interaction with members of the low‐density lipoprotein (LDL) receptor gene family is not well understood.MethodFerroptosis was induced in the N27 neuronal cell model of ferroptosis using multiple agents (erastin, RSL3, iron, cysteine depletion). Genetic variants of apolipoprotein E receptor 2 (apoER2) were generated via site‐directed mutagenesis and expressed in N27 neurons. The severity of ferroptosis was measured by several toxicity assays (MTT, LDH) and lipid peroxide probe (C11BODIPY). The association between apoER2 polymorphism and cognition were investigated in two longitudinal studies: the Alzheimer’s Disease Neuroimaging Initiative (ADNI) and the Australian Imaging Biomarkers and Lifestyle Study of Ageing (AIBL).ResultWe recently reported that apoE is a potent inhibitor of ferroptosis in neuronal cells in a mechanism that involves a receptor‐mediated activation of the PI3K/AKT signaling pathway. In this study, we identified genetic variants of apoER2 (also known as LRP8, a member of the LDL receptor family) that were associated with slower cognitive decline in clinical cohorts of AD. In preliminary work, we show that one apoER2 variant was associated with an increased protection from ferroptosis in an apoE‐dependent process.ConclusionWe present evidence for a biological role of apoER2 and its genetic variation on pathologies associated with Alzheimer’s disease, which may help develop new diagnostic and therapeutic approaches towards the disease.

Gp350-Targeted CAR-T Therapy in EBV-Positive NK/T-Cell Lymphoma: Pre-Clinical Development of gp350CAR-T

Introduction: Epstein-Barr virus (EBV) positive NK/T cell lymphoma is a subtype of non-Hodgkin's lymphoma strongly associated with EBV infection. EBV infection has been reported in 85-100% of cases. It has been suggested to be central in pathogenesis. The incidence of EBV+ NK/T cell lymphoma is increasing worldwide, particularly in Asia and Latin America, and it has a poor prognosis with few effective treatment options. Therefore, novel approaches for treating EBV-associated NK/T cell lymphoma are urgently needed. Developing chimeric antigen receptor (CAR)-T cells therapy targeting EBV specific protein in the context of this EBV-positive aggressive lymphoma will provide a novel therapeutic strategy for patients with limited treatment options. Targeting EBV lytic protein for control EBV in vivo replication and tumorigenesis have been explored lately. The gp350 protein is a major glycoprotein on the surface of EBV during its lytic phase, after fusion between virus and host cell membrane, gp350 is left on the host cell surface. The distinct surface distribution pattern of gp350 in both freshly infected host cells and the lytic phase host cells renders it an attractive target for immunotherapy. Method and Result: We developed EBV specific CAR-T cells targeting gp350. We utilized single-chain variable fragments (scFvs) derived from four novel, gp350-binding, neutralizing monoclonal antibodies (mAbs) (12C10, 7D11, 6A12, 7A8) to generate the CAR constructs. We demonstrated that these scFvs, when fused to a second-generation CAR backbone consisting of 4-1BB and CD3ζ signal domains, are efficiently expressed on the surface of transduced T cells. These gp350 CAR-T cells exhibit specific activation and cytotoxic effects in vitro. Of note, the clone 6A12 scFv of CAR displayed the most potent cytotoxicity effects in gp350 positive cancer coculture toxicity assays. To optimize the CAR structure further, we designed various linker and transmembrane domains based on the clone 6A12 scFv, including CD8 linker, IgG4 hinge, IgG1 Fc linker, and CD28 linker and transmembrane domains. Our cytotoxicity assay screening indicated that the CD8 linker and transmembrane domain resulted in the most advanced cytotoxicity and target specificity (Figure A, B). To assess gp350 CAR-T efficacy in vivo, we established a T-cell leukaemia animal model in NOD immune-deficient mice through intravenous injection of Jurkat-gp350-luc cells. On post xenograft day 7, the Jurkat-gp350-luc xenografted mice were separated into three groups and each group with 5 mice (except PBS group with 2 mice). CAR-T and T cell were infused by tail vein injection with mock T 2x10^6/mouse, 6A12CAR-T 2x10^6/mouse and PBS control. The tumor growth in mice was monitored by in vivo luminescence with IVIS imaging system. Upon engraftment, Jurkat-gp350-luc cells established disseminated leukaemia expanding preferentially in the spine, femur, head, and pelvis, with few cells in peripheral blood. Mice receiving control mock T cells or without T cell PBS control developed rapid disease progression and were euthanized by day 35 after Jurkat-gp350-luc engraftment. In contrast, mice receiving gp350 CART cells were significantly protected from rapid progression (Figure C, D). Conclusion: In pre-clinical development, we designed, constructed, and screened gp350 CAR-T cells to demonstrate its efficacy in vitro and in vivo against and kill gp350 positive leukaemia cells and prevent formation and development leukaemia in xenograft mice model.

Susceptibility of northern corn rootworm (Diabrotica barberi) populations to Cry3Bb1 and Gpp34/Tpp35Ab1 proteins in seedling and diet overlay toxicity assays.

The northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae) is a major pest of maize in the United States Corn Belt. Recently, resistance to Bacillus thuringiensis (Bt) maize was reported in North Dakota and increased use of Bt maize hybrids could facilitate resistance evolution in other maize-producing states. In this study, susceptibility to Bt proteins was evaluated in wild D. barberi populations from 8 fields collected in 5 different states (Minnesota, Missouri, Nebraska, Iowa, and North Dakota). Field populations were compared to a susceptible D. barberi colony in seedling and diet toxicity assays conducted with 3 concentrations of Cry3Bb1 (0.4, 4.0, and 40.0 µg/cm2) and Gpp34/Tpp35Ab1 (previously called Cry34/35Ab1; 1.4, 14.0, and 140.0 µg/cm2). The 2019 population from Meeker Co., Minnesota (MN-2019), exhibited the lowest mortality to Cry3Bb1 and also had nominally lowest mortality to Gpp34/Tpp35Ab1 at the highest concentrations tested in diet toxicity assays. Percent second instar was also highest for larvae of the Minnesota population surviving Cry3Bb1. In seedling assays, MN and IA-2018 populations exhibited the highest proportion survival and dry weight to both proteins expressed in corn. No significant differences in mortality, percent second instar, and dry weight were observed at the highest concentration for both proteins among the populations collected in in 2020. Most D. barberi populations were still highly susceptible to Cry3Bb1 and Gpp34/Tpp35Ab1 proteins based on diet and seedling assays, but resistance appears to be developing in some D. barberi populations. Now that methods are available, resistance monitoring may also be needed for D. barberi in some regions.

Toxicity and Repellency Efficacy of Benzyl Alcohol and Benzyl Benzoate as Eco-Friendly Choices to Control the Red Flour Beetle Tribolium castaneum (Herbst. 1797)

Tribolium castaneum is a damaging pest of stored grains, causing significant losses and secreting lethal quinones, which render the grains unfit for human consumption. Chemical insecticides are the most commonly used approach for control; however, they create insecticide resistance and affect the health of humans, animals, and the environment. As a result, it is critical to find an environmentally friendly pest-management strategy. In this study, two naturally occurring chemicals, benzyl alcohol (BA) and benzoyl benzoate (BB), were investigated for insecticidal activity against T. castaneum using different assays (impregnated-paper, contact toxicity, fumigant, and repellency assays). The results showed that BA had a significant insecticidal effect, with the LC50 achieved at a lower concentration in the direct-contact toxicity test (1.77%) than in the impregnated-paper assay (2.63%). BB showed significant effects in the direct-contact toxicity test, with an LC50 of 3.114%, and a lower toxicity in the impregnated-paper assay, with an LC50 of 11.75%. Furthermore, BA exhibited significant fumigant toxicity against T. castaneum, with an LC50 of 6.72 µL/L, whereas BB exhibited modest fumigant toxicity, with an LC50 of 464 µL/L. Additionally, at different concentrations (0.18, 0.09, 0.045, and 0.0225 µL/cm2), BA and BB both showed a notable and potent repelling effect. BA and BB significantly inhibited acetylcholinesterase, reduced glutathione (GSH), and increased malondialdehyde (MDA) in treated T. castaneum. This is the first report of BA insecticidal activity against the red flour beetle. Also, the outcomes of various assays demonstrated that the application of BA induces a potent bio-insecticidal effect. BA may be a promising eco-friendly alternative to control T. castaneum due to its safety and authorization by the EFSA (European Food Safety Authority).

In vitro and in vivo antimicrobial effects of domiphen combined with itraconazole against Aspergillus fumigatus

Aspergillus fumigatus, a prevalent saprophytic fungus in the atmosphere, is known to rapidly induce severe invasive aspergillosis (IA) upon inhalation of its conidia by humans or animals. The mortality rate associated with IA exceeds 50%. The misuse of antifungal agents has contributed to the emergence of numerous highly pathogenic drug-resistant strains of A. fumigatus. Our study found that the combination of domiphen and itraconazole had sound synergistic antimicrobial effects against wild-type and itraconazole-resistant A. fumigatus in vivo and in vitro through MIC, FIC, plate inoculation, growth curve experiments, and Galleria mellonella infection model. Drug cytotoxicity and pharmacological tests for acute toxicity assays demonstrated that both itraconazole and domiphen showed minimal cytotoxicity and good biocompatibility. The transcriptome sequencing experiment demonstrated that domiphen exerted a suppressive effect on the expression of various genes, including those involved in drug efflux, redox regulation, and cellular membrane and cell wall remodeling. The present investigation explores the synergistic antimicrobial mechanisms of domiphen and itraconazole, encompassing three key aspects: (i) domiphen inhibited the efflux of itraconazole by reducing the expression of drug efflux-related genes, (ii) the combination has good ability to disrupt the cell membrane and cell wall, (iii) the combination also can remove biofilm more effectively. In summary, the utilization of domiphen as a synergist of itraconazole exhibited disruptive effects on the biofilm, cell wall, and cell membrane of A. fumigatus. This subsequently led to a modified distribution of itraconazole within the fungal organism, ultimately resulting in enhanced antifungal efficacy. The results of this study may provide a new therapeutic strategy for the treatment of IA caused by drug-resistant A. fumigatus.