Faster ciguatoxin extraction methods for toxicity screening.

Ciguatera poisoning (CP) is a severe global public health problem caused by the consumption of seafood products contaminated with ciguatoxins (CTXs). The growing demand for seafood products requires high-throughput testing for CTX-susceptible seafood, however complex extraction and slow cleanup methods inhibit this goal. Herein, several methods for extracting CTXs from fish tissue were established and compared; these methods are sensitive, specific, and valid while achieving higher sample extraction throughput than currently established protocols. The trial fish material was generated from multiple species, with different physical conditions (wet and freeze-dried tissue), and naturally contaminated with various CTXs (i.e., CTX-1B, CTX-3C, and C-CTX-1), thus ensuring these methods are robust and broadly applicable. The extraction methods used were based on mechanical maceration with acetone or methanol or enzymatic digestion followed by acetone and ethyl acetate extraction. Crude extracts were investigated for CTX-like toxicity using an in vitro mouse neuroblastoma (N2a) cell-based assay (CBA). Among the three methods, there was no significant difference in toxin estimates (p = 0.219, two-way ANOVA), indicating their interchangeability. For speed (> 16 samples/day), accuracy (100%), and CTX analog retention confirmation by liquid chromatography-tandem mass spectrometry (LC‒MS/MS), the preferred extraction methods were both methanol and enzyme-based. All extraction methods post hoc confirmation of CTX analogs successfully met international seafood market-based CTX contaminant guidance. These methods can drastically increase global CTX screening capabilities and subsequently relieve sample processing bottlenecks, inhibiting environmental and human health-based CTX analysis.

Assessment of receptor-mediated activity (AhR and ERα), mutagenicity, and teratogenicity of metal shredder wastes in Wallonia, Belgium.

In this study, hazardous wastes including fluff, dust, and scrubbing sludge were sampled in 2019 from two metal shredding facilities located in Wallonia, Belgium. To assess the extent of the contamination, a global approach combining chemical and biological techniques was used, to better reflect the risks to health and the environment. The samples investigated induced significant in vitro aryl hydrocarbon receptor (AhR) agonistic bioactivities and estrogenic receptor (ERα) (ant)agonistic bioactivities in the respective CALUX (chemical activated luciferase gene expression) bioassays. The mutagenicity of the samples was investigated with the bacterial reverse gene mutation test using the Salmonella typhimurium TA98 and TA100 strains. Except for the sludge sample (site 3), all samples induced a mutagenic response in the TA98 strain (± S9 metabolic fraction) whereas in the TA100 strain (+ S9 metabolic fraction), only the sludge sample (site 2) showed a clear mutagenic effect. The in vivo toxicity/teratogenicity of the shredder wastes was further evaluated with zebrafish embryos. Except for the dust sample (site 2), all samples were found to be teratogenic as they returned teratogenic indexes (TIs) > 1. The high levels of contamination, the mutagenicity, and the teratogenicity of these shredder wastes raise significant concerns about their potential negative impacts on both human health and environment.

Application of Microbiological Screening Tests in Assessment of Environmental Exposure to Antibiotics: Preliminary Studies.

Contact of aquatic microbiocenoses with antibiotics present in the environment can cause the former to develop resistance to antimicrobial drugs. Therefore, the search for methods to detect antibiotics and drug-resistant microorganisms in the environment is important. The presented paper proposes a simple procedure to assess environmental exposure to antibiotics and the presence of non-susceptible microorganisms. Medium solutions with selected antibiotics and a microbial growth indicator were applied to test plates, and were inoculated with water samples from various ecosystems. After incubation, the susceptibility of the microorganisms to antibiotics was determined and presented in chronic microbial toxic concentration (MTC) values. It was confirmed that the presented procedure enables the assessment of the antibiotic susceptibility and adaptation potential of unselected microorganisms from different aquatic ecosystems. However, the MTC values depend on the inoculum volume, the density and seasonal activity of the microorganisms, the method of inoculum preparation, and the incubation time of the test plate. The described procedure may be practically applied as a screening test to identify the presence of drug-resistant microorganisms. Additionally, it may also be suitable as a method to assess environmental exposure to antibiotics. However, prior standardisation is required before implementing this procedure in quantitative studies.

Aligning Post-Column ESI-MS, MALDI-MS, and Coagulation Bioassay Data of Naja spp., Ophiophagus hannah, and Pseudonaja textillis Venoms Chromatographically to Assess MALDI-MS and ESI-MS Complementarity with Correlation of Bioactive Toxins to Mass Spectrometric Data.

Snakebite is a serious health issue in tropical and subtropical areas of the world and results in various pathologies, such as hemotoxicity, neurotoxicity, and local swelling, blistering, and tissue necrosis around the bite site. These pathologies may ultimately lead to permanent morbidity and may even be fatal. Understanding the chemical and biological properties of individual snake venom toxins is of great importance when developing a newer generation of safer and more effective snakebite treatments. Two main approaches to ionizing toxins prior to mass spectrometry (MS) analysis are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). In the present study, we investigated the use of both ESI-MS and MALDI-MS as complementary techniques for toxin characterization in venom research. We applied nanofractionation analytics to separate crude elapid venoms using reversed-phase liquid chromatography (RPLC) and high-resolution fractionation of the eluting toxins into 384-well plates, followed by online LC-ESI-MS measurements. To acquire clear comparisons between the two ionization approaches, offline MALDI-MS measurements were performed on the nanofractionated toxins. For comparison to the LC-ESI-MS data, we created so-called MALDI-MS chromatograms of each toxin. We also applied plasma coagulation assaying on 384-well plates with nanofractionated toxins to demonstrate parallel biochemical profiling within the workflow. The plotting of post-column acquired MALDI-MS data as so-called plotted MALDI-MS chromatograms to directly align the MALDI-MS data with ESI-MS extracted ion chromatograms allows the efficient correlation of intact mass toxin results from the two MS-based soft ionization approaches with coagulation bioassay chromatograms. This facilitates the efficient correlation of chromatographic bioassay peaks with the MS data. The correlated toxin masses from ESI-MS and/or MALDI-MS were all around 6-8 or 13-14 kDa, with one mass around 20 kDa. Between 24 and 67% of the toxins were observed with good intensity from both ionization methods, depending on the venom analyzed. All Naja venoms analyzed presented anticoagulation activity, whereas pro-coagulation was only observed for the Pseudonaja textillis venom. The data of MALDI-MS can provide complementary identification and characterization power for toxin research on elapid venoms next to ESI-MS.

Utility of Cry1Ja for Transgenic Insect Control.

Insect control traits are a key component of improving the efficacy of insect pest management and maximizing crop yields for growers. Insect traits based on proteins expressed by the bacteria Bacillus thuringiensis (Bt) have proven to be very effective tools in achieving this goal. Unfortunately, the adaptability of insects has led to resistance to certain proteins in current commercial products. Therefore, new insecticidal traits representing a different mode of action (MoA) than those currently in use are needed. Cry1Ja has good insecticidal activity against various lepidopteran species, and it provides robust protection against insect feeding with in planta expression. For Bt proteins, different MoAs are determined by their binding sites in the insect midgut. In this study, competitive binding assays are performed using brush border membrane vesicles (BBMVs) from Helicoverpa zea, Spodoptera frugiperda, and Chrysodeixis includens to evaluate the MoA of Cry1Ja relative to representatives of the various Bt proteins that are expressed in current commercial products for lepidopteran insect protection. This study highlights differences in the shared Cry protein binding sites in three insect species, Cry1Ja bioactivity against Cry1Fa resistant FAW, and in planta efficacy against target pests. These data illustrate the potential of Cry1Ja for new insect trait development.

Non-constant mean relative potency for antibody-dependent cellular cytotoxicity assays.

Bioassays are regulated, analytical methods used to ensure proper activity (potency) of biological products at release and during long-term storage. Potency is commonly reported on a relative basis by comparing and calibrating a concentration-response curve from the test material to that of a reference standard material. The relative potency approach depends on an assumption that the two concentration-response curves exhibit similar (equivalent) shapes, except for a potency shift. In certain circumstances, however, biological factors preclude the similarity assumption, and the traditional approach becomes unworkable. The antibody-mediated cytotoxicity assay is one example where the similarity assumption does not always hold. Other examples also arise in the fields of toxicology and pharmacology. In this work, we present a non-constant mean relative potency approach which averages the relative potency across a common range of the concentration-response curves. The proposed method captures the changing nature of the relative potency into a summary statistic that can be reported for batch calibration and quality control purposes. We provide inferential methods for this statistic and summarize the results of a simulation comparing these methods across a number of non-constant relative potency scenarios and assay conditions.

Chemosensory protein 22 in Riptortus pedestris is involved in the recognition of three soybean volatiles.

Riptortus pedestris (Hemiptera: Alydidae), a common agricultural pest, is the major causative agent of "soybean staygreen." However, the interactions between chemosensory proteins (CSPs) in R. pedestris and host plant volatiles have yet to be comprehensively studied. In this study, we performed real-time fluorescence quantitative polymerase chain reaction (PCR) to analyze the antennal expression of RpedCSP22 and subsequently analyzed the interactions between 21 soybean volatiles, five aggregation pheromones, and RpedCSP22 protein in vitro using a protein expression system, molecular docking, site-directed mutagenesis, and fluorescence competitive binding experiments. The RpedCSP22 protein showed binding affinity to three soybean volatiles (benzaldehyde, 4-ethylbenzaldehyde, and 1-octene-3-ol), with optimal binding observed under neutral pH conditions, and lost binding ability after site-directed mutagenesis. In subsequent RNA interference (RNAi) studies, gene silencing was more than 90 %, and in silenced insects, electroantennographic responses were reduced by more than 75 % compared to non-silenced insects. Moreover, Y-tube olfactory behavioral assessments revealed that the attraction of R. pedestris to the three soybean volatiles was significantly attenuated. These findings suggest that RpedCSP22 plays an important role in the recognition of host plant volatiles by R. pedestris andprovides a theoretical basis for the development of novel inhibitors targeting pest behavior.

Ecotoxicological effects of suspended sediments on marine microalgae using flow cytometry and pulse-amplitude modulation (PAM) fluorometry.

Microalgal bioassays were conducted to evaluate the ecotoxicological effects of suspended sediments (SS) collected from coastal environments. Growth inhibition was assessed for six microalgal species, and multiple endpoints were measured using flow cytometry (FCM) and pulse-amplitude modulation (PAM) fluorometry for three species (Dunaliella tertiolecta, Isochrysis galbana, and Phaeodactylum tricornutum). Among these, the EC50 for growth inhibition of D. tertiolecta (6700 mg L-1) was notably lower compared to the other species, and among several endpoints, esterase activity was the most inhibited. Species-specific responses to SS exposure were identified, with D. tertiolecta exhibiting greater susceptibility across most endpoints. Meanwhile, measurements of Fo', Fm', and Y(NPQ) in P. tricornutum using PAM fluorometry revealed greater sensitivity. Based on the results of this study and review, the tentative predicted no-effect concentration was calculated as 12.1 mg L-1. Overall, this study provides novel insights into SS ecotoxicity, establishing a crucial baseline for future investigations.

Isolation and genetic characterization of Toxoplasma gondii from chickens from public markets in Pernambuco State, Brazil.

This study aimed to evaluate the presence and viability of Toxoplasma gondii in chickens intended for human consumption in the Pernambuco State, Brazil. Blood and tissue samples were collected from 25 chickens sold in markets in Recife, Pernambuco. Samples were evaluated by indirect immunofluorescence assay (IFA) to detect antibodies to T. gondii. Pools of brain and heart of seropositive chickens were subjected to bioassay in two Swiss Webster mice, which were evaluated for 45 days then tested by IFA to detect seroconversion. The mice were euthanized, and their brains were evaluated for cysts. Peritoneal lavage was also conducted in mice that exhibited clinical signs. Brains containing cysts or peritoneal lavage with tachyzoites were inoculated into MA-104 cells. Brains of mice inoculated with the same tissue were pooled and analysed by ITS1-PCR. We obtained a frequency of antibodies to T. gondii of 68.00% (17/25) in chickens, and a seroconversion rate of 70.58% (24/34) in mice. Detection of Toxoplasma ITS1 DNA confirmed an isolation rate of 41.1% (7/17). Three isolates were characterized by mnPCR-RFLP as genotypes ToxoDB#36 and ToxoDB#114. We highlight the occurrence of ToxoDB#36 in chickens in Pernambuco State and the parasites' viability in chickens intended for human consumption.

Niche construction and niche choice by aphids infesting wheat ears.

The niche of aphids is largely defined by their consumption of plant phloem sap and its composition, including nutrients and specialized metabolites. Niche construction is the change of the environment by organisms, which may influence the fitness of these organisms and their offspring. To better understand interactions between plants and aphids, it is necessary to investigate whether aphids modify the chemical composition of the phloem sap of their host plants and whether conspecifics are affected by previous infestation. In the current study, ears of wheat (Triticum aestivum) plants were infested with clonal lineages of the English grain aphid (Sitobion avenae) or were left uninfested. The metabolic composition of ear phloem sap exudates was analyzed through amino acid profiling and metabolic fingerprinting. Aphids of the clonal lineages were either put on previously aphid-infested or on uninfested ears and their colony sizes followed over time. Furthermore, it was investigated whether aphids choose one treatment group over another. Sitobion avenae infestation affected the relative concentrations of some metabolites in the phloem exudates of the ears. Compared to uninfested plants, the relative concentration of asparagine was higher after aphid infestation. Colonies grew significantly larger on previously aphid-infested ears, which the aphids also clearly chose in the choice experiment. The pronounced positive effect of previous infestation on aphid colonies indicates niche construction, while the choice of these constructed niches reveals niche choice by S. avenae on wheat. The interplay between these different niche realization processes highlights the complexity of interactions between aphids and their hosts.

Methodological advances in formulation and assay of herbal resources-based topical drug delivery systems.

Medicinal plants have been utilized for centuries as a source of healing compounds, which consist of thousands of known bioactive molecules with therapeutic potentials. This article aims to explore and emphasize the significance of medicinal plants and bioactive compounds in the development of topical pharmaceutical formulations. The journey from the extraction of phytochemicals to the development of topical pharmaceutical formulations is described with the aid of scientific evidence selected from PubMed, Google Scholar, ScienceDirect, and Web of Science. Articles published in English during 2018-2023 period were considered and selected randomly. The review discusses the extraction process of medicinal plants, solvent selection, and green synthesis of metal nanoparticles. Subsequently, various biological activities of plant extracts are elaborated especially focusing on antimicrobial, antioxidant, anti-inflammatory, and sun protection activities, along with the corresponding in vitro assays commonly employed for the evaluation. The article presents the process of compound isolation through bioactivity-guided fractionation and also the toxicity evaluation of isolated fractions. Finally, the formulation of medicinal plant extracts into topical pharmaceuticals is addressed, emphasizing the stability evaluation procedures necessary for ensuring product quality and efficacy.

Quantitative analysis and characterization of floral volatiles, and the role of active compounds on the behavior of Heortia vitessoides.

This study explores the role of floral volatile organic compounds (FVOCs) in insect behavior, focusing on Aquilaria sinensis (AS), a valuable tropical plant threatened by Heortia vitessoides Moore. Despite H. vitessoides' attraction to AS and non-host plants like Elaeocarpus decipiens (ED) and Dalbergia odorifera (DO), little is known about their chemical interactions. FVOCs from these plants were analyzed at 9:00 and 18:00 using GC×GC-QTOF-MS and HS-SPME. The results showed that ED exhibiting the highest concentration (92.340 ng/mg), followed by DO (75.167 ng/mg) and AS (64.450 ng/mg). Through GC-EAD and EAG, a total of 11 FVOC compounds with electrophysiological activates were identified. These compounds, except linalool, showed dose-dependent responses. Y-Tube bioassays confirmed phenylethyl alcohol or the mixture of EAD-active compounds produced positive chemotactic responses in both males and females. FVOCs have the potential to be used as a natural and sustainable alternative to chemical insecticides in pest control.

A bioassay that yields quantifiable symptoms of cucurbit yellow vine disease caused by Serratia marcescens.

Cucurbit yellow vine disease (CYVD), which is caused by the gram-negative bacterium Serratia marcescens and transmitted by squash bugs (Anasa tristis DeGeer), is a devastating disease of cucurbit crops that is emerging rapidly in the eastern half of the U.S. The lack of a robust pathogenicity assay for CYVD in the laboratory has hampered functional tests using genomic sequences to investigate the biology of this phytopathogen. In this study we developed and validated a bioassay that yielded consistent and quantifiable CYVD symptoms on squash in the lab. We compared inoculation by wounding with a multipronged floral pin frog to inoculation by injection in which a needle was moved in and out of the stem multiple times in each of multiple piercings to mimic the feeding behavior of squash bugs. We found that inoculation by needle injection of ≥108 CFU/ml of S. marcescens into the stem of squash (Cucurbita pepo) plants at the cotyledon growth stage reproducibly induced CYVD symptoms, whereas injecting 106 or 107 CFU/ml did not. Additionally, we found that S. marcescens induced symptoms on all of the squash cultivars tested, and induced symptoms that have not been previously reported, including stem elongation and leaf cupping. In short, through our injection approach of mimicking the natural process of S. marcescens transmission by squash bug feeding, we obtained robust and quantifiable CYVD symptoms. This laboratory bioassay provides a crucial tool for investigating the biology and pathology of this emerging pathogen and for plant breeding screens aimed at combatting CYVD.

Isolation and antimicrobial activity of secondary metabolites of pothos tener wall.

The Pothos genus is extensively utilised in traditional medicine in China and India. An underexplored species of Pothos tener Wall was identified in Sulawesi, Indonesia. Antimicrobial activity was assessed using microdilutions and streak plates against Staphylococcus aureus, Eschericia coli, Aeromonas hydrophila, Aspergillus niger, and Candida albicans. Significant effectiveness was observed in the methanol extract, as indicated by the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values for three different extracts (methanol, ethyl acetate, and n-hexane) of P. tener. The isolates obtained were structurally analysed using Ultraviolet (UV)-spectroscopy, Fourier-transform Infra Red-Spectroscopy (FT-IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance (NMR), and antimicrobial testing after undergoing fractionation and subfractionation. The isolate obtained was stigmasterol with moderate antimicrobial activity against A. niger and A. hydrophila, with MIC equivalent to MBC of 500 µg/ml. The first report of stigmasterol from P. tener has potent antimicrobial properties, bolstering empirical data in this field.

Insights into the whole genome sequence of Bacillus thuringiensis NBAIR BtPl, a strain toxic to the melon fruit fly, Zeugodacus cucurbitae.

Bacillus thuringiensis is the most widely used biopesticide, targets a diversity of insect pests belonging to several orders. However, information regarding the B. thuringiensis strains and toxins targeting Zeugodacus cucurbitae is very limited. Therefore, in the present study, we isolated and identified five indigenous B. thuringiensisstrains toxic to larvae of Z. cucurbitae. However, of five strains NBAIR BtPl displayed the highest mortality (LC50 = 37.3 µg/mL) than reference strain B. thuringiensis var. israelensis (4Q1) (LC50 = 45.41 µg/mL). Therefore, the NBAIR BtPl was considered for whole genome sequencing to identify the cry genes present in it. Whole genome sequencing of our strain revealed genome size of 6.87 Mb with 34.95% GC content. Homology search through the BLAST algorithm revealed that NBAIR BtPl is 99.8% similar to B. thuringiensis serovar tolworthi, and gene prediction through Prokka revealed 7406 genes, 7168 proteins, 5 rRNAs, and 66 tRNAs. BtToxin_Digger analysis of NBAIR BtPl genome revealed four cry gene families: cry1, cry2, cry8Aa1, and cry70Aa1. When tested for the presence of these four cry genes in other indigenous strains, results showed that cry70Aa1 was absent. Thus, the study provided a basis for predicting cry70Aa1 be the possible reason for toxicity. In this study apart from novel genes, we also identified other virulent genes encoding zwittermicin, chitinase, fengycin, and bacillibactin. Thus, the current study aids in predicting potential toxin-encoding genes responsible for toxicity to Z. cucurbitae and thus paves the way for the development of B. thuringiensis-based formulations and transgenic crops for management of dipteran pests.

Bioactivity of silverleaf nightshade (Solanum elaeagnifolium Cav.) berries parts against Galleria mellonella and Erwinia carotovora and LC-MS chemical profile of its potential extract.

Natural products received much attention as an environmentally beneficial solution for pest management. Therefore, the extracts of invasive silverleaf nightshade (Solanum elaeagnifolium Cav.) weeds using their berries parts (seeds, peels and mucilage) supported by bioassay-guided fractionation were tested against both the greater wax moth (Galleria mellonella) and Erwinia carotovora pv. carotovora causes of the blackleg of potatoes. The seeds and peels of S. elaeagnifolium were successively extracted by maceration using dichloromethane (DCM), ethyl acetate (EtOAc), and ethanol (EtOH), respectively. While, its mucilage was extracted using EtOAc. The successive EtOH extract of the plant seeds had promising inhibition efficacy and the best minimal inhibition concentration (MIC) of 50 µg/ml against E. Carotovora amongst other extracts (DCM and EtOAc of the plant berries parts). Depending on dose response activity, EtOH extract had G. mellonella larval mortality and pupal duration rates (LC50; 198.30 and LC95; 1294.73 µg/ml), respectively. Additionally, this EtOH extract of seeds was fractionated using preparative TLC to three characteristic bands. The insecticidal and bacterial activities of these isolated bands (SEA, SEB, and SEC) were evaluated at a dose of 100 µg/ml, causing mortality by 48.48, 62.63 and 92.93% (G. mellonella larvae) and inhibition by 15.22, 0.00 and 31.66 mm (E. carotovora), respectively. Moreover, the separated major three bands were tentatively identified using LC-ESI-MS analysis revealing the presence of two phenolic acids; chlorogenic acid (SEA) and dicaffeoyl quinic acid (SEB) in addition to one steroidal saponin (SEC) annotated as borassoside E or yamoscin. Finally, the plant seeds' successive EtOH extract as well as its active constituents, exhibited potential broad-spectrum activity and the ability to participate in future pest management initiatives. A field study is also recommended to validate its bio-efficacy against selected pests and to develop its formulations.

Investigation of pharmaceutical bioaccumulation in Daphnia sp. living in a wastewater treatment plant.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used pharmaceuticals. Their presence in natural waters is due to the low removal efficiency in conventional wastewater treatment plants (WWTPs). Interestingly, certain zooplankton species can survive the mixture of pollution and abnormal water conditions in WWTPs. In our study, for the first time, we tested the in-situ bioaccumulation of NSAIDs and their metabolites in Daphnia pulex, which were obtained in high numbers in one WWTP during the summer. It was found that diclofenac (DCF) and 4-hydroxy DCF were present in the studied clarifiers and ponds. Among these chemicals, only DCF was detected in daphnia. The bioaccumulation factor of DCF in daphnia was below 36 L kg-1ww and was lower than those obtained under experimental conditions for Daphnia magna. The tested daphnia adapted to chronic exposure to mixtures of drugs in µg L-1 level and could be implemented in biobased WWTPs. According to our data, there is a need to supplement the risk assessment of anthropogenic pollutants with in-situ cases to demonstrate the adaptation possibilities of wild-living organisms.

Air-Drying Time Affects Mortality of Pyrethroid-Susceptible Aedes aegypti Exposed to Transfluthrin-Treated Filter Papers.

Increasing temperature can enhance the geographical spread and behavior of disease vector mosquitoes, exposing vulnerable populations to Aedes-borne viruses and infections. To address this risk, cost-effective and sustained intervention vector control tools are required, such as volatile pyrethroid spatial repellents. This study used a high-throughput screening system toxicity bioassay to determine the discriminating concentrations of transfluthrin-treated filter papers with variable air-drying times exposed to pyrethroid-susceptible Aedes aegypti mosquitoes. At the highest transfluthrin concentration (0.01706%), a significant reduction in mosquito mortality was observed in filter papers air-dried for 24 h compared to those air-dried for 1 h (odds ratio = 0.390, p < 0.001, 95% confidence interval: 0.23-0.66). Conversely, no significant difference in mortality was found between filter papers air-dried for 1 h and those air-dried for 12 h (odds ratio = 0.646, p = 0.107, 95% confidence interval: 0.38-1.10). The discriminating concentration was 2.8-fold higher for transfluthrin-treated filter papers air-dried for 24 h than it was for papers air-dried for 1 h, and it increased 5-fold from 1 h to 336 h of air-drying. These results show that the optimal air-drying period of transfluthrin-treated filter paper is critical, as higher discriminating concentration values may lead to underestimations of insecticide resistance. The instability of transfluthrin-treated papers necessitates the use of the World Health Organization (WHO) bottle bioassay, which is the preferred method for determining mosquito susceptibility to volatile insecticides.

Development of a Gold Nanoparticle-Based Sensor for Authentication of Organic Milk Based on Differential Levels of miRNA.

Dairy production systems significantly impact environmental sustainability, animal welfare, and human health. Intensive farming maximizes output through high-input practices, raising concerns about environmental degradation, animal welfare, and health risks from antibiotic residues. Conversely, organic farming emphasizes sustainable practices, animal welfare, and minimal synthetic inputs, potentially enhancing biodiversity, soil health, and milk quality. MicroRNAs (miRNAs), non-coding RNAs regulating gene expression, are promising biomarkers due to their response to various conditions. In this study, miRNAs bta-miR-103 and bta-miR-155, which are abundant in milk from pasture-fed cows, were selected. Additionally, bta-miR-215, which is abundant in milk fat from intensive systems, was also studied, in order to differentiate dairy production systems. A novel, cost-effective gold nanoparticle (AuNP)-based sensor was developed for miRNA detection, leveraging the unique plasmonic properties of AuNPs for visual detection. The method involves functionalizing AuNPs with complementary RNA probes and detecting miRNA-induced aggregation through colorimetric changes. This rapid, results in 30 min, and sensitive, visual limit of detection of 200 nM, assay requires minimal instrumentation and can be easily interpreted, offering significant advantages for field implementation in characterizing dairy production systems. This study demonstrates the successful application of this sensor in detecting miRNAs in 350 nM miRNA spiked raw milk, highlighting its potential for in situ dairy industry applications.

Development of bioassay platforms for biopharmaceuticals using Jurkat-CAR cells by AICD.

The assessment of bioactivity for therapeutic antibody release assay poses challenges, particularly when targeting immune checkpoints. An in vitro bioassay platform was developed using the chimeric antigen receptor on Jurkat cells (Jurkat-CAR) to analyze antibodies targeting immune checkpoints, such as CD47/SIRPα, VEGF/VEGFR1, PD-1/PD-L1, and CD70/CD27. For CD47/SIRPα, the platform involved a Jurkat-CAR cell line expressing the chimeric SIRPα receptor (CarSIRPα). CarSIRPα was created by sequentially fusing the SIRPα extracellular region with the CD8α hinge region, the transmembrane (TM) and intracellular (IC) domains of CD28, and the intracellular signaling domain of CD3ζ. The resulting Jurkat-CarSIRPα cells can undergo "activation-induced cell death (AICD)" upon incubation with purified or cellular CD47, as evidenced by the upregulation of CD69, IL-2, and IFN-γ. Similar results also appeared in Jurkat CarVEGFR1, Jurkat CarPD1 and Jurkat CARCD27 cells. These cells are perfectly utilized for the bioactivity analysis of therapeutic antibody. Our study indicates that the established in vitro assay platform based on Jurkat-CAR has been confirmed repeatedly and has shown robust reproducibility; thus, this platform can be used for screening or for release assays of given antibody drugs targeting immune checkpoints.