A large scale bacterial attraction assay: A new quantitative bacterial migration assay suitable for genetic screens.

Bacteria use various motility mechanisms to explore their environments. Chemotaxis is the ability of a motile bacterial cell to direct its movement in response to chemical gradients. A number of methods have been developed and widely used to study chemotactic responses to chemoeffectors including capillary, agar plug, microscopic slide, and microfluidic assays. While valuable, these assays are primarily designed to monitor rapid chemotactic responses to chemoeffectors on a small scale, which poses challenges in collecting large quantities of attracted bacteria. Consequently, these setups are not ideal for experiments like forward genetic screens. To overcome this limitation, we developed the Large Scale Bacterial Attraction assay (LSBA), which relies on the use of a Nalgene™ Reusable Filter Unit and other materials commonly found in laboratories. We validate the LSBA by investigating chemoeffector kinetics in the setup and by using chemoattractants to quantify the chemotactic response of wild-type, and motility impaired strains of the plant pathogenic bacterium Xanthomonas campestris pv. campestris and the environmental bacterium Shewanella oneidensis. We show that the LSBA establishes a long lasting chemoeffector gradient, that the setup can be used to quantify bacterial migration over time and that the LSBA offers the possibility to collect high numbers of attracted bacteria, making it suitable for genetic screens.

Application of ecotoxicological tools to evaluate the quality status of mangroves under restoration in the Yucatán Peninsula, Mexico.

Ecotoxicological tools, namely biomarkers and bioassays, may provide insights on the ecological quality status of mangroves under restoration. We investigated how 1) physicochemical parameters and water bioassays using Artemia franciscana; and 2) quantification of sublethal (osmoregulatory capacity, biochemical, and oxidative stress) and individual biomarkers (density, length-weight relationship [LWR], parasitic prevalence) in the sentinel fiddler crab Minuca rapax, can improve restoration indicators in mangroves from the Yucatán Peninsula, Southern Gulf of Mexico. We showed that water quality was improved with restoration, but still presented toxicity. Regarding sublethal biomarkers, M rapax from restored areas lower osmotic regulatory capacity, higher oxidative stress, and showed lipid peroxidation. As to the individual biomarkers, the density, LWR, and the prevalence of parasites in M. rapax was higher in restored areas. The use of bioassays/biomarkers were useful as early warning indicators to better assess the health of mangroves under restoration.

Bioassay-Guided and DeepSAT-Driven Precise Mining of Monoterpenoid Coumarin Derivatives with Antifeedant Effects from the Leaves of Ailanthus altissima.

Demand for the exploration of botanical pesticides continues to increase due to the detrimental effects of synthetic chemicals on human health and the environment and the development of resistance by pests. Under the guidance of a bioactivity-guided approach and HSQC-based DeepSAT, 16 coumarin derivatives were discovered from the leaves of Ailanthus altissima (Mill.) Swingle, including seven undescribed monoterpenoid coumarins, three undescribed monoterpenoid phenylpropanoids, and two new coumarin derivatives. The structure and configurations of these compounds were established and validated via extensive spectroscopic analysis, acetonide analysis, and quantum chemical calculations. Biologically, 5 exhibited significant antifeedant activity toward the Plutella xylostella. Moreover, tyrosinase being closely related to the growth and development of larva, the inhibitory potentials of 5 against tyrosinase was evaluated in vitro and in silico. The bioactivity evaluation results highlight the prospect of 5 as a novel category of botanical insecticide.

Identifying suitable methods for evaluating the sterilizing effects of pyriproxyfen on adult malaria vectors: a comparison of the oviposition and ovary dissection methods.

BACKGROUND: Nets containing pyriproxyfen, an insect growth regulator that sterilizes adult mosquitoes, have become available for malaria control. Suitable methods for investigating vector susceptibility to pyriproxyfen and evaluating its efficacy on nets need to be identified. The sterilizing effects of pyriproxyfen on adult malaria vectors can be assessed by measuring oviposition or by dissecting mosquito ovaries to determine damage by pyriproxyfen (ovary dissection). METHOD: Laboratory bioassays were performed to compare the oviposition and ovary dissection methods for monitoring susceptibility to pyriproxyfen in wild malaria vectors using WHO bottle bioassays and for evaluating its efficacy on nets in cone bioassays. Blood-fed mosquitoes of susceptible and pyrethroid-resistant strains of Anopheles gambiae sensu lato were exposed to pyriproxyfen-treated bottles (100 µg and 200 µg) and to unwashed and washed pieces of a pyriproxyfen long-lasting net in cone bioassays. Survivors were assessed for the sterilizing effects of pyriproxyfen using both methods. The methods were compared in terms of their reliability, sensitivity, specificity, resources (cost and time) required and perceived difficulties by trained laboratory technicians. RESULTS: The total number of An. gambiae s.l. mosquitoes assessed for the sterilizing effects of pyriproxyfen were 1745 for the oviposition method and 1698 for the ovary dissection method. Fertility rates of control unexposed mosquitoes were significantly higher with ovary dissection compared to oviposition in both bottle bioassays (99-100% vs. 34-59%, P < 0.05) and cone bioassays (99-100% vs. 18-33%, P < 0.001). Oviposition rates of control unexposed mosquitoes were lower with wild pyrethroid-resistant An. gambiae s.l. Cové, compared to the laboratory-maintained reference susceptible An gambiae sensu stricto Kisumu (18-34% vs. 58-76%, P < 0.05). Sterilization rates of the Kisumu strain in bottle bioassays with the pyriproxyfen diagnostic dose (100 µg) were suboptimal with the oviposition method (90%) but showed full susceptibility with ovary dissection (99%). Wild pyrethroid-resistant Cové mosquitoes were fully susceptible to pyriproxyfen in bottle bioassays using ovary dissection (> 99%), but not with the oviposition method (69%). Both methods showed similar levels of sensitivity (89-98% vs. 89-100%). Specificity was substantially higher with ovary dissection compared to the oviposition method in both bottle bioassays (99-100% vs. 34-48%) and cone tests (100% vs.18-76%). Ovary dissection was also more sensitive for detecting the residual activity of pyriproxyfen in a washed net compared to oviposition. The oviposition method though cheaper, was less reliable and more time-consuming. Laboratory technicians preferred ovary dissection mostly due to its reliability. CONCLUSION: The ovary dissection method was more accurate, more reliable and more efficient compared to the oviposition method for evaluating the sterilizing effects of pyriproxyfen on adult malaria vectors in susceptibility bioassays and for evaluating the efficacy of pyriproxyfen-treated nets.

Exploiting Cell-Based Assays to Accelerate Drug Development for G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are relevant targets for health and disease as they regulate various aspects of metabolism, proliferation, differentiation, and immune pathways. They are implicated in several disease areas, including cancer, diabetes, cardiovascular diseases, and mental disorders. It is worth noting that about a third of all marketed drugs target GPCRs, making them prime pharmacological targets for drug discovery. Numerous functional assays have been developed to assess GPCR activity and GPCR signaling in living cells. Here, we review the current literature of genetically encoded cell-based assays to measure GPCR activation and downstream signaling at different hierarchical levels of signaling, from the receptor to transcription, via transducers, effectors, and second messengers. Singleplex assay formats provide one data point per experimental condition. Typical examples are bioluminescence resonance energy transfer (BRET) assays and protease cleavage assays (e.g., Tango or split TEV). By contrast, multiplex assay formats allow for the parallel measurement of multiple receptors and pathways and typically use molecular barcodes as transcriptional reporters in barcoded assays. This enables the efficient identification of desired on-target and on-pathway effects as well as detrimental off-target and off-pathway effects. Multiplex assays are anticipated to accelerate drug discovery for GPCRs as they provide a comprehensive and broad identification of compound effects.

The use of bioluminescent enzyme bioassay for the analysis of human saliva: Advantages and disadvantages.

The purpose of the work was to find optimal conditions for bioluminescent enzymatic analysis of saliva (based on the use of NADH:FMN oxidoreductase + luciferase) and then to determine the biological effect of using bioluminescence assay of saliva to study the physiological state of the body under normal and pathological conditions. The saliva of snowboarders and students were studied in the "rest-training" model. The saliva of patients diagnosed with acute pharyngitis was examined in the "sick-healthy" model. Bioluminescence assay was performed with a lyophilized and immobilized bi-enzyme system using cuvette, plate, and portable luminometers. The concentrations of secretory immunoglobulin A (sIgA) and cortisol were determined by enzyme immunoassay, and the total protein content was measured by spectrophotometric method. The activity of the bioluminescent system enzymes increased as the amount and volume of saliva in the sample was decreased. The cuvette and plate luminometers were sensitive to changes in the luminescence intensity in saliva assay. Luminescence intensity correlated with the concentrations of sIgA and cortisol. The integrated bioluminescent index for saliva was reduced in the "rest-training" model and increased in the "sick-healthy" model. Thus, the non-invasive bioluminescent saliva analysis may be a promising tool for assessing the health of the population.

An Au bipyramids@CuZn MOF core-shell nanozyme enables universal SERS and a colorimetric dual-model bioassay.

In this study, a new type of gold nano-bipyramids@CuZn bimetallic organic framework (AuNBPs@CuZn MOF) nanozyme with high peroxidase (POD)-like activity and surface enhanced Raman scattering (SERS) activity was constructed with a special core-shell structure, which can catalyze the oxidation of TMB (colourless and Raman-inactive) into ox-TMB (blue and Raman-active). An AuNBPs@CuZn MOF-enabling universal SERS and colorimetric dual-model bioassay was thus developed for biomolecules with excellent performance, and has promising application prospects in the biosensing fields.

Toxoplasma gondii infection in European brown hares (Lepus europaeus) in Brazil.

Toxoplasmosis is a worldwide zoonosis that affects warm-blooded animals, including humans. Wild animals can act as intermediate hosts of this pathogen; thus, this study aims to detect Toxoplasma gondii infection in invasive European brown hares in Brazil. For this, 72 wild European brown hares were captured from July 2020 to June 2022 in three Brazilian states: São Paulo, Paraná, and Rio Grande do Sul. The diagnostic of Toxoplasma gondii infection was performed by bioassay in mouse, histopathology in Hematoxylin-Eosin-stained tissue sections (brain, liver, lungs, kidneys, and small intestine), serology by IFAT, and molecular techniques by conventional PCR and qPCR. The combined prevalence of the different diagnostic methods was 51.4% (37/72, CI= 40.1 - 62.6 %), and there was no statistical difference between sexes, age range, or geographical region of the hosts. Mouse bioassay was the technique that detected more positive hares. To our knowledge, this is the first confirmation of Toxoplasma gondii infection in invasive European brown hares in Brazil. These animals act as reservoirs and potential infection source for carnivores and other wild and domestic animals, including humans, thus contributing to perpetuate the disease cycle in São Paulo, Paraná, and Rio Grande do Sul States. Research such as the present study is necessary to raise awareness about the role of animals in the disease cycle.

Biocontrol potential of six Heterorhabditis bacteriophora strains isolated in the Azores Archipelago.

Entomopathogenic nematodes (EPNs) are closely associated with Popillia japonica and potentially used as their biological control agents, although field results proved inconsistent and evoked a continual pursuit of native EPNs more adapted to the environment. Therefore, we surveyed the Azorean Archipelago to isolate new strains of Heterorhabditis bacteriophora and to evaluate their virulence against the model organism Galleria mellonella under laboratory conditions. Six strains were obtained from pasture and coastal environments and both nematode and symbiont bacteria were molecularly identified. The bioassays revealed that Az172, Az186, and Az171 presented high virulence across the determination of a lethal dose (LD50) and short exposure time experiments with a comparable performance to Az29. After 72 hours, these virulent strains presented a mean determination of a lethal dose of 11 infective juveniles cm-2, a lethal time (LT50) of 34 hours, and achieved 40% mortality after an initial exposure time of only 60 minutes. Az170 exhibited an intermediate performance, whereas Az179 and Az180 were classified as low virulent strains. However, both strains presented the highest reproductive potential with means of 1700 infective juveniles/mg of larvae. The bioassays of the native EPNs obtained revealed that these strains hold the potential to be used in biological control initiatives targeting P. japonica because of their high virulence and locally adapted to environmental conditions.

Novel polymorphisms in the octopamine receptor gene of amitraz resistant population of Rhipicephalus sanguineus sensu lato, in south India.

The brown dog tick or Rhipicephalus sanguineus sensu lato is an ixodid tick, responsible for the dissemination of pathogens that cause canine infectious diseases besides inflicting the direct effects of tick bite. The hot humid climate of Kerala, a south Indian state, is favorable for propagation of tick vectors and acaricides are the main stay of tick control. Though the resistance against synthetic pyrethroids is reported among these species, the status of amitraz resistance in R. sanguineus s. l. in the country is uncertain due to the lack of molecular characterisation data and scarce literature reports. Hence the present study was focused on the phenotypic detection and preliminary genotypic characterisation of amitraz resistance in the R. sanguineus s. l. A modified larval packet test (LPT) on a susceptible isolate was performed to determine the discriminating dose (DD). Further LPT-DD on 35 tick isolates was carried out to detect amitraz resistance robustly, along with that full dose response bioassays on the resistant isolates were performed. The results indicated that amitraz resistance is prevalent with 49 per cent of the samples being resistant. Amplification of exon 3 of octopamine receptor gene from both the susceptible and resistant larval isolates was carried out. Amplicons of ten pooled amitraz susceptible and ten pooled amitraz resistant representative samples were sequenced and analysed, unveiling a total of three novel non-synonymous mutations in the partial coding region at positions V32A, N41D and V58I in phenotypically resistant larval DNA samples. In silico analysis by homology modelling and molecular docking of the mutated and unmutated receptors showed that these mutations had reduced the binding affinity to amitraz. However, lack of mutations in the octopamine receptor gene in three of the pooled low order resistant R. sanguineus s. l. larval samples could be suggestive of other mechanisms associated with amitraz resistance in the region. Hence, further association studies should be carried out to confirm the association of these mutations with target insensitivity in R. sanguineus s. l. ticks, along with exploring the status of metabolic resistance and other mechanisms of resistance.

Versatile Platinum Nanoparticles-Decorated Phage Nanozyme Integrating Recognition, Bacteriolysis, and Catalysis Capabilities for On-Site Detection of Foodborne Pathogenic Strains Vitality Based on Bioluminescence/Pressure Dual-Mode Bioassay.

Sensitive and on-site discrimination of live and dead foodborne pathogenic strains remains a significant challenge due to the lack of appropriate assay and signal probes. In this work, a versatile platinum nanoparticle-decorated phage nanozyme (P2@PtNPs) that integrated recognition, bacteriolysis, and catalysis was designed to establish the bioluminescence/pressure dual-mode bioassay for on-site determination of the vitality of foodborne pathogenic strains. Benefiting from the bacterial strain-level specificity of phage, the target Salmonella typhimurium (S.T) was specially captured to form sandwich complexes with P2@PtNPs on another phage-modified glass microbead (GM@P1). As the other part of the P2@PtNPs nanozyme, the introduced PtNPs could not only catalyze the decomposition of hydrogen peroxide to generate a significant oxygen pressure signal but also produce hydroxyl radicals around the target bacteria to enhance the bacteriolysis of phage and adenosine triphosphate release. It significantly improved the bioluminescence signal. The two signals corresponded to the total and live target bacteria counts, so the dead target could be easily calculated from the difference between the total and live target bacteria counts. Meanwhile, the vitality of S.T was realized according to the ratio of live and total S.T. Under optimal conditions, the application range of this proposed bioassay for bacterial vitality was 102-107 CFU/mL, with a limit of detections for total and live S.T of 30 CFU/mL and 40 CFU/mL, respectively. This work provides an innovative and versatile nanozyme signal probe for the on-site determination of bacterial vitality for food safety.

Bioassay-guided isolation of anti-inflammatory and antinociceptive metabolites among three Moroccan Juniperus leaves extract supported with in vitro enzyme inhibitory assays.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbs of the genus Juniperus (family Cupressaceae) have been commonly used in ancestral folk medicine known as "Al'Araar" for treatment of rheumatism, diabetes, inflammation, pain, and fever. Bioassay-guided isolation of bioactives from medicinal plants is recognized as a potential approach for the discovery of novel drug candidates. In particular, non-addictive painkillers are of special interest among herbal phytochemicals. AIM OF THE STUDY: The current study aimed to assess the safety of J. thurifera, J. phoenicea, and J. oxycedrus aqueous extracts in oral treatments; validating the traditionally reported anti-inflammatory and analgesic effects. Further phytochemical investigations, especially for the most bioactive species, may lead to isolation of bioactive metabolites responsible for such bioactivities supported with in vitro enzyme inhibition assays. MATERIALS AND METHODS: Firstly, the acute toxicity study was investigated following the OECD Guidelines. Then, the antinociceptive, and anti-inflammatory bioactivities were evaluated based on chemical and mechanical trauma assays and investigated their underlying mechanisms. The most active J. thurifera n-butanol fraction was subjected to chromatographic studies for isolating the major anti-inflammatory metabolites. Moreover, several enzymatic inhibition assays (e.g., 5-lipoxygenase, protease, elastase, collagenase, and tyrosinase) were assessed for the crude extracts and isolated compounds. RESULTS: The results showed that acute oral administration of the extracts (300-500 mg/kg, p. o.) inhibited both mechanically and chemically triggered inflammatory edema in mice (up to 70% in case of J. thurifera) with a dose-dependent antinociceptive (tail flick) and anti-inflammatory pain (formalin assay) activities. This effect was partially mediated by naloxone inhibition of the opioid receptor (2 mg/kg, i. p.). In addition, 3-methoxy gallic acid (1), quercetin (2), kaempferol (3), and ellagic acid (4) were successfully identified being involved most likely in J. thurifera extract bioactivities. Nevertheless, quercetin was found to be the most potent against 5-LOX, tyrosinase, and protease with IC50 of 1.52 ± 0.01, 192.90 ± 6.20, and 399 ± 9.05 µM, respectively. CONCLUSION: J. thurifera extract with its major metabolites are prospective drug candidates for inflammatory pain supported with inhibition of inflammatory enzymes. Interestingly, antagonism of opioid and non-opioid receptors is potentially involved.

Bioassay predictive values for chemical health risks in drinking water.

Bioanalytical tools can be used for assessment of the chemical quality of drinking water and its sources. For water managers it is important to know the probability that a bioassay response above an established health-based 'effect-based trigger value' (EBT) indeed implies a harmful chemical (mixture) concentration. This study presents and applies a framework, based on Bayes' theorem, to derive such risk probabilities for bioassay responses. These were evaluated under varying (in silico) chemical mixture concentrations relevant to drinking water (sources), with toxicity data for six in vitro assays from the ToxCast database. For single chemicals and in silico mixtures, the negative predictive value (NPV) was 100 % for all assays. For water managers, this means that when a bioassay response is below the EBT, a chemical risk is reliably absent, and no further action is required. The positive predictive value (PPV) increased with increasing chemical concentrations (2 µg/L) up to 40-80 %, depending on the assay. For in silico mixtures of increasing numbers of chemicals, the PPV did not increase until higher sum concentrations (>2-10 µg/L). Hence, the ability to accurately signal a harmful chemical (mixture) using bioassays will be lowest for highly diverse, low-concentration chemical mixtures. For water managers, this means in practice that further investigations after an EBT exceedance will, in many cases, not reveal chemicals at harmful concentrations. A solution offered is to increase the trigger value for positive responses to achieve a higher PPV and maintain the EBT for negative responses to ensure an optimal NPV.

Identification of novel GSPT1 degraders by virtual screening and bioassay.

GSPT1 plays crucial physiological functions, such as terminating protein translation, overexpressed in various tumors. It is a promising anti-tumor target, but is also considered as an "undruggable" protein. Recent studies have found that a class of small molecules can degrade GSPT1 through the "molecular glue" mechanism with strong antitumor activity, which is expected to become a new therapy for hematological malignancies. Currently available GSPT1 degraders are mostly derived from the scaffold of immunomodulatory imide drug (IMiD), thus more active compounds with novel structure remain to be found. In this work, using computer-assisted multi-round virtual screening and bioassay, we identified a non-IMiD acylhydrazone compound, AN5782, which can reduce the protein level of GPST1 and obviously inhibit the proliferation of tumor cells. Some analogs were obtained by a substructure search of AN5782. The structure-activity relationship analysis revealed possible interactions between these compounds and CRBN-GSPT1. Further biological mechanistic studies showed that AN5777 decreased GSPT1 remarkably through the ubiquitin-proteasome system, and its effective cytotoxicity was CRBN- and GSPT1-dependent. Furthermore, AN5777 displayed good antiproliferative activities against U937 and OCI-AML-2 cells, and dose-dependently induced G1 phase arrest and apoptosis. The structure found in this work could be good start for antitumor drug development.

Pathogens of the oak processionary moth Thaumetopoea processionea: Developing a user-friendly bioassay system and metagenome analyses for microorganisms.

The oak processionary moth (OPM) Thaumetopoea processionea is a pest of oak trees and poses health risks to humans due to the urticating setae of later instar larvae. For this reason, it is difficult to rear OPM under laboratory conditions, carry out bioassays or examine larvae for pathogens. Biological control targets the early larval instars and is based primarily on commercial preparations of Bacillus thuringiensis ssp. kurstaki (Btk). To test the entomopathogenic potential of other spore-forming bacteria, a user-friendly bioassay system was developed that (i) applies bacterial spore suspensions by oak bud dipping, (ii) targets first instar larvae through feeding exposure and (iii) takes into account their group-feeding behavior. A negligible mortality in the untreated control proved the functionality of the newly established bioassay system. Whereas the commercial Btk HD-1 strain was used as a bioassay standard and confirmed as being highly efficient, a Bacillus wiedmannii strain was ineffective in killing OPM larvae. Larvae, which died during the infection experiment, were further subjected to Nanopore sequencing for a metagenomic approach for entomopathogen detection. It further corroborated that B.wiedmannii was not able to infect and establish in OPM, but identified potential insect pathogenic species from the genera Serratia and Pseudomonas.

Application of in vitro bioassays to monitor pharmaceuticals in water: A synthesis of chronological analysis, mode of action, and practical insights.

Pharmaceutical compounds in wastewater have emerged as a significant concern for the aquatic environment. The use of in vitro bioassays represents a sustainable and cost-effective approach for assessing the potential toxicological risks of these biologically active compounds in wastewater and aligns with ethical considerations in research. It facilitates high-throughput analysis, captures mixture effects, integrates impacts of both known and unknown chemicals, and reduces reliance on animal testing. The core aim of the current review was to explore the practical application of in vitro bioassays in evaluating the environmental impacts of pharmaceuticals in wastewater. This comprehensive review strives to achieve several key objectives. First, it provides a summary categorisation of pharmaceuticals based on their mode of action, providing a structured framework for understanding their ecological significance. Second, a chronological analysis of pharmaceutical research aims to document their prevalence and trends over time, shedding light on evolving environmental challenges. Third, the review critically analyses existing bioassay applications in wastewater, while also examining bioassay coverage of representative compounds within major pharmaceutical classes. Finally, it explores the potential for developing innovative bioassays tailored for water quality monitoring of pharmaceuticals, paving the way for more robust environmental monitoring and risk assessment. Overall, adopting effect-based methods for pharmaceutical monitoring in water holds significant promise. It encompasses a broad spectrum of biological impacts, promotes standardized protocols, and supports a bioassay test battery approach indicative of different endpoints, thereby enhancing the effectiveness of environmental risk assessment.

Chemical effects on neural network activity: Comparison of acute versus network formation exposure in microelectrode array assays.

New approach methodologies (NAMs) can address information gaps on potential neurotoxicity or developmental neurotoxicity hazard for data-poor chemicals. Two assays have been previously developed using microelectrode arrays (MEA), a technology which measures neural activity. The MEA acute network function assay (AcN) uses dissociated rat cortical cells cultured at postnatal day 0 and evaluates network activity during a 40-minute chemical exposure on day in vitro (DIV)13 or 15. In contrast, the MEA network formation assay (NFA) uses a developmental exposure paradigm spanning DIV0 through DIV12. Measures of network activity over time at DIV5, 7, 9, and 12 in the NFA are reduced to an estimated area under the curve to facilitate concentration-response evaluation. Here, we evaluated the hypothesis that chemicals with effects in the AcN also perturb the NFA by examining quantitative and qualitative concordance between assays. Out of 243 chemicals screened in both assays, we observed 70.3% concordance between the AcN and NFA after eliminating activity inferred to be cytotoxic (selective activity), with the majority of discordance explained by chemicals that altered selective activity in the AcN but not NFA. The NFA detected more active chemicals when evaluating activity associated with cytotoxicity. Median potency values were lower in the NFA compared to the AcN, but within-chemical potency values were not uniformly lower in the NFA than the AcN. Lastly, the AcN and NFA captured unique bioactivity fingerprints; the AcN was more informative for identifying chemicals with a shared mode of action, while the NFA provided information relevant to developmental exposure. Taken together, this analysis provides a rationale for using both approaches for chemical evaluation with consideration of the context of use, such as screening/ prioritization, hazard identification, or to address questions regarding biological mechanism or function.

Concept of a six-fold multiplex planar bioassay to distinguish endocrine agonist, antagonist, cytotoxic and false-positive responses.

To analyze a complex sample for endocrine activity, different tests must be performed to clarify androgen/estrogen agonism, antagonism, cytotoxicity, anti-cytotoxicity, and corresponding false-positive reactions. This means a large amount of work. Therefore, a six-fold planar multiplex bioassay concept was developed to evaluate up to the mentioned six endpoints or mechanisms simultaneously in the same sample analysis. Separation of active constituents from interfering matrix via high-performance thin-layer chromatography and effect differentiation via four vertical stripes (of agonists and end-products of the respective enzyme-substrate reaction) applied along each separated sample track were key to success. First, duplex endocrine bioassay versions were established. For the androgen/anti-androgen bioassay applied via piezoelectric spraying, the mean limit of biological detection of bisphenol A was 14 ng/band and its mean half maximal inhibitory concentration IC50 was 116 ng/band. Applied to trace analysis of six migrate samples from food packaging materials, 19 compound zones with agonistic or antagonistic estrogen/androgen activities were detected, with up to seven active compound zones within one migrate. For the first time, the S9 metabolism of endocrine effective compounds was studied on the same surface and revealed partial deactivation. Coupled to high-resolution mass spectrometry, molecular formulas were tentatively assigned to compounds, known to be present in packaging materials or endocrine active or previously unknown. Finally, the detection of cytotoxicity/anti-cytotoxicity and false-positives was integrated into the duplex androgen/anti-androgen bioassay. The resulting six-fold multiplex planar bioassay was evaluated with positive control standards and successfully applied to one migrate sample. The streamlined stripe concept for multiplex planar bioassays made it possible to assign different mechanisms to individual active compounds in a complex sample. The concept is generic and can be transferred to other assays.

Development of a reliable cell-based reporter gene assay to measure the bioactivity of anti-HER2 therapeutic antibodies.

Human epidermal growth factor receptor 2 (HER2) is a key player in the pathogenesis and progression of breast cancer and is currently a primary target for breast cancer immunotherapy. Bioactivity determination is necessary to guarantee the safety and efficacy of therapeutic antibodies targeting HER2. Nevertheless, currently available bioassays for measuring the bioactivity of anti-HER2 mAbs are either not representative or have high variability. Here, we established a reliable reporter gene assay (RGA) based on T47D-SRE-Luc cell line that expresses endogenous HER2 and luciferase controlled by serum response element (SRE) to measure the bioactivity of anti-HER2 antibodies. Neuregulin-1 (NRG-1) can lead to the heterodimerization of HER2 on the cell membrane and induce the expression of downstream SRE-controlled luciferase, while pertuzumab can dose-dependently reverse the reaction, resulting in a good dose-response curve reflecting the activity of the antibody. After optimizing the relevant assay parameters, the established RGA was fully validated based on ICH-Q2 (R1), which demonstrated that the method had excellent specificity, accuracy, precision, linearity, and stability. In summary, this robust and innovative bioactivity determination assay can be applied in the development and screening, release control, biosimilar assessment and stability studies of anti-HER2 mAbs.

Repellent activity of essential oils to the Lone Star tick, Amblyomma americanum.

BACKGROUND: The Lone Star tick, Amblyomma americanum is important to human health because of a variety of pathogenic organisms transmitted to humans during feeding events, which underscores the need to identify novel approaches to prevent tick bites. Thus, the goal of this study was to test natural and synthetic molecules for repellent activity against ticks in spatial, contact and human fingertip bioassays. METHODS: The efficacy of essential oils and naturally derived compounds as repellents to Am. americanum nymphs was compared in three different bioassays: contact, spatial and fingertip repellent bioassays. RESULTS: Concentration response curves after contact exposure to 1R-trans-chrysanthemic acid (TCA) indicated a 5.6 µg/cm2 concentration required to repel 50% of ticks (RC50), which was five- and sevenfold more active than DEET and nootkatone, respectively. For contact repellency, the rank order of repellency at 50 µg/cm2 for natural oils was clove > geranium > oregano > cedarwood > thyme > amyris > patchouli > citronella > juniper berry > peppermint > cassia. For spatial bioassays, TCA was approximately twofold more active than DEET and nootkatone at 50 µg/cm2 but was not significantly different at 10 µg/cm2. In spatial assays, thyme and cassia were the most active compounds tested with 100% and 80% ticks repelled within 15 min of exposure respectively and was approximately twofold more effective than DEET at the same concentration. To translate these non-host assays to efficacy when used on the human host, we quantified repellency using a finger-climbing assay. TCA, nootkatone and DEET were equally effective in the fingertip assay, and patchouli oil was the only natural oil that significantly repelled ticks. CONCLUSIONS: The differences in repellent potency based on the assay type suggests that the ability to discover active tick repellents suitable for development may be more complicated than with other arthropod species; furthermore, the field delivery mechanism must be considered early in development to ensure translation to field efficacy. TCA, which is naturally derived, is a promising candidate for a tick repellent that has comparable repellency to commercialized tick repellents.