Dose-response Experiments Research Articles

Cytotoxic effects of ivermectin on Giardia lamblia: induction of apoptosis and cell cycle arrest

IntroductionGiardia lamblia is a flagellated protozoan parasite causing giardiasis, a common intestinal infection characterized by diarrhea, abdominal cramps, and nausea. Treatments employed to combat this parasitic infection have remained unchanged for the past 40 years, leading to the emergence of resistant strains and prompting the search for new therapeutic agents.MethodsThis study investigated the cytotoxic effects of ivermectin (IVM) on G. lamblia trophozoites. We conducted dose-response experiments to assess IVM-induced cytotoxicity. We utilized various biochemical and ultrastructural analyses to explore the underlying mechanisms of cell death, including reactive oxygen species (ROS) production, DNA fragmentation, cell cycle arrest, and apoptosis markers.ResultsOur findings demonstrate that IVM induces dose-dependent cytotoxicity and triggers cell death pathways. We found that IVM treatment generates elevated levels of reactive oxygen species (ROS), DNA fragmentation, and arrests of trophozoites in the cell cycle’s S phase. Additionally, ultrastructural analysis reveals morphological alterations consistent with apoptosis, such as cytoplasmic vacuolization, chromatin condensation, and tubulin distribution.DiscussionThe insights gained from this study may contribute to developing new therapeutic strategies against giardiasis, addressing the challenge posed by drug-resistant strains.

Multiple-resistance evolution to ACCase inhibitors and glyphosate in sourgrass (Digitaria insularis) is attributed to diverse polymorphisms in the herbicide target sites

Abstract Sourgrass [Digitaria insularis (L.) Mez ex Ekman] is considered the most troublesome weed in agronomic crops in South America. Overreliance on glyphosate has selected for resistant populations, although the resistance mechanisms remain unknown. Recently, populations were identified that exhibited multiple resistance to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetyl-CoA carboxylase (ACCase) inhibitors, posing a significant challenge due to the lack of alternative control options. This project aimed to identify the resistance patterns and levels to glyphosate and ACCase inhibitors of three suspected resistant populations (P1, P2, and P3), and elucidate the resistance mechanisms. We performed dose–response experiments with clethodim, fluazifop-P-butyl, glyphosate, and pinoxaden to identify the possibility of cross- and multiple resistance and to quantify the resistance levels. We sequenced the ACCase and EPSPS genes to test the hypothesis that target-site mutations were involved in the resistance mechanisms, given the resistance patterns observed. Our results indicated that two of the tested populations, P1 and P2, were multiple resistant to glyphosate and all ACCase-inhibitor classes, while P3 was resistant to glyphosate only. Resistance levels varied by herbicide, with resistance indices ranging from 2.7- to nearly 2,000-fold. We identified an amino acid substitution in ACCase at position 2078 (Asp-2078-Gly), homozygous for both P1 and P2, corroborating the resistance patterns observed. Interestingly, EPSPS sequencing identified multiple heterozygous DNA polymorphisms that resulted in amino acid substitutions at positions 106 (P1 and P2) or at both 102 and 106 (P3), indicating multiple evolutionary origins of glyphosate-resistance evolution. We show for the first time the genetic mechanisms of multiple resistance to glyphosate and ACCase in D. insularis, and provide a thorough discussion of the evolutionary and management implications of our work.

Antibiotic biocompatibility assay and anti-biofilm strategies for Pseudomonas aeruginosa infection in bioengineered artificial skin substitutes.

Bioengineered artificial skin substitutes (BASS) are an advanced therapy for treating extensively burned patients. Pseudomonas aeruginosa (P. aeruginosa) infections represent a major challenge in these patients as formation of biofilms impede wound healing and perpetuate a chronic inflammatory state. Here we assessed antibiotics (alone or in combination) with respect to cytotoxicity, as well as antimicrobial efficacy in P. aeruginosa biofilm formed on infection of BASS. Cell viability, structure and functionality were evaluated using microscopy and trans-epidermal water loss analyses, respectively. BASS were established and infected for 24 h to allow P. aeruginosa biofilm formation, after which two antimicrobial approaches, treatment and prevention, were tested. In the latter, antibiotics were added to BASS before infection. The antimicrobial effect was determined using real-time calorimetry. In dose-response experiments, 1.25 mg/mL amikacin, 0.02 mg/mL ciprofloxacin, 0.051 mg/mL colistin, 1 mg/mL meropenem and colistin in combination with either amikacin, ciprofloxacin and meropenem did not affect BASS' viability, structure and functionality. All antibiotics, except colistin, showed effective antimicrobial activity at these non-cytotoxic concentrations. For concentrations below the highest non-cytotoxic ones, successive treatments resulted in higher bacterial metabolic rates. Only the combinations managed to eradicate the infection with repeated treatments. With respect to prevention of infection, all antibiotics at the highest non-cytotoxic concentrations and the combinations were effective. This preventive capacity was maintained for at least 5 days. The findings highlight the potential for developing BASS with antimicrobial properties that can prevent infections during wound healing in burn patients.

Wild mustard (Sinapis arvensis L.) resistance to tribenuron methyl in wheat fields of Diyarbakır province, Türkiye

Chemical weed control is unavoidable in the fields where weeds cause problems in wheat production. However, resistance problems in weeds arise a few years later especially using herbicides with the same mode of action continuously. Frequent and inappropriate use of herbicides leads to various environmental, health and economic issues, as well as labour losses. Recently, growers complained that tribenuron-methyl (acetolactate synthase - ALS), commonly used to control wild mustard (Sinapis arvensis L.-SINAR), which is causing problems in the wheat fields of Diyarbakır province, is ineffective. To determine the extent of the problem, wild mustard seeds were collected from 56 suspected wheat fields in 16 districts of Diyarbakır. As a result of applications carried out with the method under controlled conditions, resistance was suspected in the population of 28 fields. The results of the dose-response experiments indicated that no resistance (RI

Taste characterization and molecular docking study of novel umami flavor peptides in Yanjin black bone Chicken meat

Five polypeptides with a potential umami taste were isolated and purified from Yanjin black bone chicken. However, the flavor characteristics and umami mechanism have not been clarified. The umami properties of these five peptides were investigated in this work using a range of analytical techniques, computer simulation, and sensory evaluation. HE-10 and TP-7 exhibited the strongest umami flavors. Furthermore, dose-response experiments showed that the umami peptides enhanced umami by generating peptide mineral chelates. Environmental scanning electron microscopy (ESEM) microstructural analyses supported this finding. The molecular docking results indicated that the five polypeptides bind to four critical amino acid residues, namely Glu217, Glu148, Asp216, and His145, of the T1R1/T1R3 receptor. The binding occurred through van der Waals, electrostatic interactions, hydrogen bonding, and hydrophobic interactions. The main surface forces implicated include aromatic interactions, hydrogen bonding, hydrophilicity, and solvent accessibility.

Lipid emulsion attenuates vasodilation by decreasing intracellular calcium and nitric oxide in vascular endothelial cells

Lipid emulsion (LE), a widely used parenteral nutrition, exhibits a well-documented ability to reverse the vasodilatory effects induced by acetylcholine in blood vessels. However, the specific mechanisms underlying this action are not yet fully understood. This study aimed to elucidate the mechanism by which LE reverses vasodilation in vitro through dose-response curve experiments, calcium imaging, and fluorescence assays. The results revealed a significant attenuation of acetylcholine (Ach)-induced vasodilation in rat thoracic aortic rings following LE exposure. In human aortic endothelial cells, pretreatment with LE significantly suppressed ATP-induced calcium elevation. This suppression persisted even after elimination of extracellular calcium with a calcium chelator. Moreover, LE pre-exposure reduced the intracellular calcium concentration ([Ca2+]i) elevation in endothelial cells following cyclopiazonic acid (CPA) treatment, suggesting enhanced endoplasmic reticulum (ER) calcium reuptake. Additionally, nitric oxide (NO) fluorescence assays showed a decrease in NO production upon ATP stimulation post-LE pretreatment of endothelial cells. Taken together, these results indicate that the reversal of vasodilation by LE may involve enhanced ER calcium uptake, leading to a reduction in intracellular calcium concentration and suppression of NO (key vasodilatory agent) synthesis.

Mechanistic modeling of cell viability assays with in silico lineage tracing.

Data from cell viability assays, which measure cumulative division and death events in a population and reflect substantial cellular heterogeneity, are widely available. However, interpreting such data with mechanistic computational models is hindered because direct model/data comparison is often muddled. We developed an algorithm that tracks simulated division and death events in mechanistically detailed single-cell lineages to enable such a model/data comparison and suggest causes of cell-cell drug response variability. Using our previously developed model of mammalian single-cell proliferation and death signaling, we simulated drug dose response experiments for four targeted anti-cancer drugs (alpelisib, neratinib, trametinib and palbociclib) and compared them to experimental data. Simulations are consistent with data for strong growth inhibition by trametinib (MEK inhibitor) and overall lack of efficacy for alpelisib (PI-3K inhibitor), but are inconsistent with data for palbociclib (CDK4/6 inhibitor) and neratinib (EGFR inhibitor). Model/data inconsistencies suggest (i) the importance of CDK4/6 for driving the cell cycle may be overestimated, and (ii) that the cellular balance between basal (tonic) and ligand-induced signaling is a critical determinant of receptor inhibitor response. Simulations show subpopulations of rapidly and slowly dividing cells in both control and drug-treated conditions. Variations in mother cells prior to drug treatment all impinging on ERK pathway activity are associated with the rapidly dividing phenotype and trametinib resistance. This work lays a foundation for the application of mechanistic modeling to large-scale cell viability assay datasets and better understanding determinants of cellular heterogeneity in drug response.

Syntheses and Applications of Coumarin-Derived Fluorescent Probes for Real-Time Monitoring of NAD(P)H Dynamics in Living Cells across Diverse Chemical Environments.

Fluorescent probes play a crucial role in elucidating cellular processes, with NAD(P)H sensing being pivotal in understanding cellular metabolism and redox biology. Here, the development and characterization of three fluorescent probes, A, B, and C, based on the coumarin platform for monitoring of NAD(P)H levels in living cells are described. Probes A and B incorporate a coumarin-cyanine hybrid structure with vinyl and thiophene connection bridges to 3-quinolinium acceptors, respectively, while probe C introduces a dicyano moiety for replacement of the lactone carbonyl group of probe A which increases the reaction rate of the probe with NAD(P)H. Initially, all probes exhibit subdued fluorescence due to intramolecular charge transfer (ICT) quenching. However, upon hydride transfer by NAD(P)H, fluorescence activation is triggered through enhanced ICT. Theoretical calculations confirm that the electronic absorption changes upon the addition of hydride to originate from the quinoline moiety instead of the coumarin section and end up in the middle section, illustrating how the addition of hydride affects the nature of this absorption. Control and dose-response experiments provide conclusive evidence of probe C's specificity and reliability in identifying intracellular NAD(P)H levels within HeLa cells. Furthermore, colocalization studies indicate probe C's selective targeting of mitochondria. Investigation into metabolic substrates reveals the influence of glucose, maltose, pyruvate, lactate, acesulfame potassium, and aspartame on NAD(P)H levels, shedding light on cellular responses to nutrient availability and artificial sweeteners. Additionally, we explore the consequence of oxaliplatin on cellular NAD(P)H levels, revealing complex interplays between DNA damage repair, metabolic reprogramming, and enzyme activities. In vivo studies utilizing starved fruit fly larvae underscore probe C's efficacy in monitoring NAD(P)H dynamics in response to external compounds. These findings highlight probe C's utility as a versatile tool for investigating NAD(P)H signaling pathways in biomedical research contexts, offering insights into cellular metabolism, stress responses, and disease mechanisms.

3D structure of acetolactate synthase explains why the Asp-376-Glu point mutation does not give the same resistance level to different imidazolinone herbicides

Resistance to ALS-inhibiting herbicides has dramatically increased worldwide due to the persisting evolution of target site mutations that reduce the affinity between the herbicide and the target. We evaluated the effect of the well-known ALS Asp-376-Glu target site mutation on different imidazolinone herbicides, including imazamox and imazethapyr. Greenhouse dose response experiments indicate that the Amaranthus retroflexus biotype carrying Asp-376-Glu was fully controlled by applying the field recommended dose of imazamox, whereas it displayed high level of resistance to imazethapyr. Likewise, Sorghum halepense, carrying Asp-376-Glu showed resistance to field recommended doses of imazethapyr but not of imazamox. Biochemical inhibition and kinetic characterization of the Asp-376-Glu mutant enzyme heterologously expressed using different plant sequence backbones, indicate that the Asp-376-Glu shows high level of insensitivity to imazethapyr but not to imazamox, corroborating the greenhouse results. Docking simulations revealed that imazamox can still inhibit the Asp-376-Glu mutant enzyme through a chalcogen interaction between the oxygen of the ligand and the sulfur atom of the ALS Met200, while imazethapyr does not create such interaction. These results explain the different sensitivity of the Asp-376-Glu mutation towards imidazolinone herbicides, thus providing novel information that can be exploited for defining stewardship guidelines to manage fields infested by weeds harboring the Asp-376-Glu mutation.

Diverse mechanisms confer bensulfuron-methyl resistance in Schoenoplectiella juncoides (Roxb.) lye

The effectiveness of bensulfuron-methyl in controlling Schoenoplectiella juncoides (Roxb.) Lye has significantly decreased in rice fields in China. Hence, a bensulfuron-methyl-resistant S. juncoides population (W15) was collected from Dandong City, Liaoning Province, China, to investigate the underlying resistance mechanisms. Whole-plant dose-response experiments and ALS activity assay confirmed that W15 has evolved high-level resistance to bensulfuron-methyl compared with the susceptible S. juncoides population (W4). Molecular analysis revealed a Pro-197-Ser mutation in ALS1, while there was no significant difference in the relative ALS gene expression between W15 and W4. LC-MS/MS analysis showed W15 metabolized bensulfuron-methyl more rapidly than W4. Furthermore, bensulfuron-methyl resistance in W15 was significantly alleviated by malathion and 4-chloro-7-nitrobenzoxadiazole (NBD-Cl). Glutathione S-transferase activity was higher in W15 than in W4. Meanwhile, W15 displayed cross-resistance to halosulfuron-methyl and multi-resistance to MCPA-Na. In summary, these findings demonstrated for the first time that both target- and non-target-site resistance are relevant in the resistance of S. juncoides to bensulfuron-methyl.

Myokine secretion during moderate-intensity physical activity: Dose-response of interleukin 6 to walking duration.

During vigorous physical activity, contracting muscles secrete a variety of immunomodulatory and metabolic factors called myokines that perform a variety of functions. Foremost among these is interleukin 6 (IL-6), which increases fatty acid mobilization and stimulates anti-inflammatory cytokine release. Despite being well characterized in vigorous exercise contexts, whether IL-6 is secreted during moderate-intensity activities such as walking is unclear. This is especially pertinent as regular walking is one of the oldest and most common forms of physical activity and comes with a variety of health benefits, which may be coordinated in part by IL-6 signaling. To test the hypothesis that IL-6 release occurs during evolutionarily normal physical activity contexts like long distance walking, we performed a dose-response experiment to test the effect of walking duration on IL-6 secretion. Thirteen participants completed four moderate-intensity walking trials (55% HRmax) of varying durations (30 min, 1 h, 2 h, and 3 h) in a randomized order with intervening washout periods of at least 1 week. Using a linear mixed effects model, we found that IL-6 levels modestly increased during only the 2 hand 3 h walking trials. These results suggest that small frequent increases in IL-6 signaling may be an important mechanism underlying the health benefits of regular walking. Furthermore, this finding raises the possibility that IL-6 secretion is an adaptation to fuel physical activity, especially during periods of negative energy balance.

Effects of sodium lactate injection on meat quality and lactate content in broiler chickens: emphasis on injection method and dosage

This study aims to develop an experimental model of high lactate levels in broilers to mimic the condition of birds under stress or diseases and evaluate its consequent effects on meat quality. The injection sites and dosage effects were compared separately in 2 experiments. Experiment 1 includes 3 injection sites: intraperitoneal injection, intramuscular injection, and subcutaneous injection. Experiment 2 was a dosage experiment based on the results of Experiment 1: sodium lactate intraperitoneal injection group with 1.5, 3, 6 mM concentration. The results showed that injecting sodium lactate intraperitoneally, intramuscularly, or subcutaneously all significantly decreased body weight and breast muscle weight while elevating lactic acid levels in both the blood and breast muscle of broilers. Moreover, all 3 injection methods caused a significant reduction in pH24h and an increase in the shear force value of breast muscle. In addition, dose-response experiments of intraperitoneal injection showed that a concentration of 3 mM and 6 mM were significantly decreased body weight and breast muscle weight in broiler chickens, accompanied by a notable increase in breast muscle lactate content. Compared to the control group, intraperitoneal injections of 1.5 mM, 3 mM, and 6 mM sodium lactate treatments significantly reduced the yellowness values of the breast muscle. As the dose of sodium lactate increased, the shear force value of the breast meat exhibited linear and quadratic increments, while the drip loss decreased linearly. Intraperitoneal injection of 3 mM sodium lactate also significantly reduced the pH24h of broiler breast muscle. In addition, an increased dose of lactate injections up-regulated the glycolytic pathway responsible for endogenous lactate production in the breast muscle by upregulating the expression of phosphofructokinase, pyruvate kinase and lactate dehydrogenase A. In conclusion, intraperitoneal injection of sodium lactate at 3 mM directly increased breast muscle lactate levels, providing a valuable method for establishing a high-level lactate model in poultry.

Addition of GM-CSF during in vitro oocyte maturation improves embryo development and implantation and birth rate in mice.

The present study determined whether adding granulocyte macrophage colony stimulating factor (GM-CSF) during in vitro oocyte maturation (IVM) could improve oocyte developmental competence by examining embryo development and implantation and birth rates following embryo transfer in mice. In an initial dose response experiment, we demonstrated that the addition of 2 and 10 ng/mL of GM-CSF during IVM increased cumulus expansion (P<0.05) but did not affect fertilisation rate compared with the control group. The addition of 10 ng/mL increased blastocyst rate (17.0%; P<0.05) and tended to increase the number of good quality blastocysts present at 96 h of culture (+19.4%; P=0.06) and increased blastocyst inner cell mass (+25.2%; P<0.001), trophectoderm (+29.9%; P<0.01), and total cell numbers (+28.6%; P<0.05). GM-CSF also reduced the incidence of DNA damage in blastocysts in the 10 ng/mL group (-16.2%) compared with the control group. These improvements translated into increases in implantation rate (+21.0%; P<0.05) and birth rate (+17.0%; P<0.05) following the transfer of vitrified blastocysts.GM-CSF treatment did not alter any fetal and placental parameters. Together these results suggest that the addition of GM-CSF during IVM may improve livestock in vitro embryo production and human IVM.

Pharmacological and behavioral investigation of putative self-medicative plants in Budongo chimpanzee diets.

Wild chimpanzees consume a variety of plants to meet their dietary needs and maintain wellbeing. While some plants have obvious value, others are nutritionally poor and/or contain bioactive toxins which make ingestion costly. In some cases, these nutrient-poor resources are speculated to be medicinal, thought to help individuals combat illness. In this study, we observed two habituated chimpanzee communities living in the Budongo Forest, Uganda, and collected 17 botanical samples associated with putative self-medication behaviors (e.g., bark feeding, dead wood eating, and pith-stripping) or events (e.g., when consumer had elevated parasite load, abnormal urinalysis, or injury). In total, we selected plant parts from 13 species (nine trees and four herbaceous plants). Three extracts of different polarities were produced from each sample using n-hexane, ethyl acetate, and methanol/water (9/1, v/v) and introduced to antibacterial and anti-inflammatory in vitro models. Extracts were evaluated for growth inhibition against a panel of multidrug-resistant clinical isolates of bacteria, including ESKAPE strains and cyclooxygenase-2 (COX-2) inhibition activity. Pharmacological results suggest that Budongo chimpanzees consume several species with potent medicinal properties. In the antibacterial library screen, 45 out of 53 extracts (88%) exhibited ≥40% inhibition at a concentration of 256 μg/mL. Of these active extracts, 41 (91%) showed activity at ≤256μg/mL in subsequent dose-response antibacterial experiments. The strongest antibacterial activity was achieved by the n-hexane extract of Alstonia boonei dead wood against Staphylococcus aureus (IC50: 16 μg/mL; MIC: 32 μg/mL) and Enterococcus faecium (IC50: 16 μg/mL; MIC: >256 μg/mL) and by the methanol-water extract of Khaya anthotheca bark and resin against E. faecium (IC50: 16 μg/mL; MIC: 32 μg/mL) and pathogenic Escherichia coli (IC50: 16 μg/mL; MIC: 256 μg/mL). We observed ingestion of both these species by highly parasitized individuals. K. anthotheca bark and resin were also targeted by individuals with indicators of infection and injuries. All plant species negatively affected growth of E. coli. In the anti-inflammatory COX-2 inhibition library screen, 17 out of 51 tested extracts (33%) showed ≥50% COX-2 inhibition at a concentration of 5 μg/mL. Several extracts also exhibited anti-inflammatory effects in COX-2 dose-response experiments. The K. anthotheca bark and resin methanol-water extract showed the most potent effects (IC50: 0.55 μg/mL), followed by the fern Christella parasitica methanol-water extract (IC50: 0.81 μg/mL). This fern species was consumed by an injured individual, a feeding behavior documented only once before in this population. These results, integrated with associated observations from eight months of behavioral data, provide further evidence for the presence of self-medicative resources in wild chimpanzee diets. This study addresses the challenge of distinguishing preventative medicinal food consumption from therapeutic self-medication by integrating pharmacological, observational, and health monitoring data-an essential interdisciplinary approach for advancing the field of zoopharmacognosy.

Quantifying and normalizing the combined phytotoxicity of mixed-valence chromium in soil integrated by toxicity equivalence

When Cr(VI) and Cr(III) coexist, the reasonable assessment of the combined toxicity of chromium in soil and its ecological risk is still not well resolved. In the present study, exogenous mixed concentration combinations were set up to determine the interaction and combined toxicity of Cr(VI) and Cr(III), which were quantified as measured total and resin extractable forms for dose-response experiments with barley root elongation. The concept of toxicity equivalence "α" (the ratio of toxicity intensity coefficient between Cr(VI) and Cr(III), which can be expressed as the relative toxic strength of Cr(VI) to Cr(III)) was proposed for the toxicity assessment of mixed-valence chromium in soil. The results showed that the dose-response relationship was determined more precisely by the extended independent action model (e-IA) than traditional models (e.g., concentration addition model), and the mutual antagonism for resin extractable form (Resin-Cr) was stronger than the measured total form (T-Cr). The values of toxicity equivalence (α) between coexisting Cr(VI) and Cr(III) as Resin-Cr and T-Cr were 0.74 and 160, respectively, which indicated Resin-Cr(III) had relatively stronger toxicity than Resin-Cr(VI), while T-Cr(III) was much less than T-Cr(VI). The α values between Cr(VI) and Cr(III) decreased with their more active forms (decreased to about 0.5% of the original), even as total concentration and activity in solutions, making a dialectical view of the toxicity of both in different forms necessary. Finally, the log-logistic models were developed, enabling mixed-valence Cr toxicity to be assessed from a unilateral perspective using the Cr(III) equivalence concentration (Cr(III)-eq). This work provided innovative ideas for ecological threshold studies for mixed-valence metals in soils.

Comparative assessment of microplastics and microalgae as vectors of mercury and chlorpyrifos in the copepod Acartia tonsa

Microplastics (MPs) raise concerns not only as pollutants themselves, but also due to their ability to act as vectors of pollutants adsorbed from seawater, transferring them to marine organisms. However, the relevance of MPs as carriers of pollutants compared to microalgae needs further exploration. This study compared the role of MPs (2–10 μm non-oxidized and 10–15 μm oxidized high-density polyethylene) and natural organic particles (Rhodomonas lens microalgae, MA) as carriers of mercury (Hg, 2.3 μg Hg/L) and chlorpyrifos (CPF, 1.0 μg CPF/L) to adult Acartia tonsa copepods, after 24–48 h exposure. Dose-response experiments were first performed with adult female copepods exposed to oxidized MPs (0.25–4.0 mg/L), waterborne Hg (0.01–10.0 μg/L) and Ox MPs + Hg (0.25–4.0 mg oxidized MPs/L + 0.50–8.0 μg Hg/L) for 48 h, to complement previous studies that focused on the pesticide CPF. Effects were evaluated with four replicates for physiological and reproductive responses (6 females/replicate), biochemical techniques (40 individuals/replicate) and Hg/CPF bioaccumulation measurements (1000 individuals/replicate). Copepods accumulated Hg/CPF similarly from dissolved pollutants (6204 ± 2265 ng Hg/g and 1251 ± 646 ng CPF/g) and loaded MPs (3125 ± 1389 ng Hg/g and 1156 ± 266 ng CPF/g), but significantly less from loaded MA (21 ± 8 ng Hg/g and 173 ± 80 ng CPF/g). After 24–48 h, copepods exposed to MPs + Hg/CPF showed generally greater biological effects than those exposed to dissolved Hg/CPF or to MA + Hg/CPF, although differences were not statistically significant. MA + CPF had significantly lower AChE inhibition (1073.4 nmol min−1 mg−1) and MA + Hg lower GRx induction (48.8 nmol min−1 mg−1) compared to MPs + Hg/CPF and dissolved Hg/CPF (182.8–236.4 nmol min−1 mg−1 of AChE and 74.1–101.7 nmol min−1 mg−1 of GRx). Principal component analysis suggested different modes of action for Hg and CPF.

Effectiveness of integrating mowing and systemic herbicides applied with a weed wiper for Sporobolus indicus var. pyramidalis management in Florida

Abstract Giant smutgrass [Sporobolus indicus (L.) R. Br. var. pyramidalis (P. Beauv.) Veldkamp] is an invasive species in grasslands, and herbicide application has been the most efficient management method to suppress this weed. Experiments were conducted in 2017 and 2018 to determine the effects of wiping glyphosate and hexazinone on S. indicus var. pyramidalis. A dose–response experiment using a handheld weed wiper was established with 20 treatments comprising two herbicides (glyphosate and hexazinone), uni- and bidirectional wiping methods, and 5 herbicide concentrations (6.25% v/v, 12.5% v/v, 25.0% v/v, 50.0% v/v, and 100% v/v basis). Data were collected 30 and 60 d after treatment (DAT). An ATV-mounted roto-type weed-wiper experiment was established in a strip-plot arrangement, with mowing as the horizontal strip, the wiping method (unidirectional vs. bidirectional) randomized as the vertical strip with three dosages of each herbicide for a total of 12 wiping treatments. Data were collected at 35 and 90 DAT. The percent plant mortality was calculated using differences in pre- and posttreatment plant counts. ANOVA and log-logistic linear regression were used to analyze the data. The dose–response experiment showed that S. indicus var. pyramidalis mortality increased with herbicide concentration, and mortality was greater with the bidirectional wiping method compared with the unidirectional method. Treatments wiped bidirectionally with glyphosate at 70% v/v, hexazinone at 30% v/v, and hexazinone at 60% v/v resulted in S. indicus var. pyramidalis mortality ranging from 75% to 98% by 90 DAT across all locations. The ATV-mounted weed-wiper experiment showed that mowing before herbicide application with weed wipers decreased the efficacy of both herbicides. Overall, both experiments indicate that S. indicus var. pyramidalis should be wiped bidirectionally using either glyphosate (70% v/v) or hexazinone (at least 30% v/v) to obtain satisfactory control. Further work should be conducted to determine whether seasonality impacts the response of S. indicus var. pyramidalis to mowing and the application of these herbicides.

Identification of the Sex Pheromone of the Asparagus Moth, Parahypopta Caestrum (Lepidoptera, Cossidae)

Chemical, electrophysiological, and field trapping experiments were carried out to identify the female-produced sex pheromone of the asparagus moth, Parahypopta caestrum, a very serious pests of asparagus cultivations in southern Europe. Gas chromatography coupled with mass spectrometry and electroantennogram detection (GC-MS-EAD) analysis of hexane and solid-phase microextraction (SPME) extracts of sex pheromone glands of calling females consistently detected four compounds eliciting EAG responses in male moth antennae. According to their GC retention times, mass spectra, and comparative EAG analyses with reference standards, these EAD-active compounds were identified as (Z)-9-tetradecenol (Z9-14:OH), (Z)-5-tetradecenyl acetate (Z5-14:Ac), (Z)-7-tetradecenyl acetate (Z7-14:Ac), and (Z)-9-tetradecenyl acetate (Z9-14:Ac), respectively. In the SPME extracts from the head-space of individual abdominal tips, Z9-14:Ac, Z5-14:Ac, Z7-14:Ac, and Z9:14 OH were detected in the ratio of 82:9:5:4. In EAG dose-response experiments, Z9-14:Ac was the strongest antennal stimulant at different doses tested. In field trapping experiments, Z9-14:Ac, Z7-14:Ac, and Z5-14:Ac proven to be essential for male attraction and a their 85:5:10 blend loaded onto green rubber septum dispensers was significantly more effective than single-, two-, and any other three-component blend of these compounds. The addition of Z9-14:OH to the optimal blend resulted in a significant reduction of male catches. The attractive blend here identified allowed for an effective and accurate monitoring of P. caestrum flight activity in southern Italy.

Abstract PR007: Identifying targeted therapies for muscle invasive bladder cancer via STAG2 expression

Abstract High stromal antigen 2 (STAG2) protein expression is significantly associated with shorter overall and progression free survival of muscle invasive bladder cancer (MIBC) patients. Unfortunately, STAG2 itself is difficult to target as it functions as a transcriptional regulator and has no intrinsic enzymatic activity. Therefore, it becomes essential to identify downstream therapeutic vulnerabilities based on STAG2 expression. In this study, we accomplish this by employing a drug screen in isogenic STAG2 wild-type (WT) and knock-out (KO) MIBC cell lines, analyzing drug sensitivity of DepMap bladder cancer cell lines, and performing in vitro dose-response experiments based on our drug screen findings. To delineate changes in drug sensitivity due specifically to altered STAG2 expression, we used CRISPR-Cas9 to knock out STAG2 expression in T24 MIBC cells and generated two clonal STAG2 KO cell lines to compare to control. Then, we performed a drug screen with 312 drugs targeting oncogenic signaling pathways. We treated cells with drugs at 534 nM and read viability after 72 hours. To expand our drug screen findings to a larger group of cell lines and drugs, we further queried the DepMap database which includes drug sensitivity data for 4,868 drugs and approximately 2,000 cancer cell lines. We selected drug screened bladder cancer cell lines from DepMap and grouped them into ‘STAG2-high’ or ‘STAG2-low’ cohorts based on STAG2 mRNA expression. We then compared these two groups to identify if either group was more sensitive to drugs in the DepMap database. From these drug screen and DepMap analyses, we identified TAK-733 (MEKi), Berzosertib (ATRi), Prexasertib (CHKi), PI-103 (PI3Ki), and Rigosertib (PLKi) as candidate drugs that are differentially effective based on STAG2 expression level. We performed validation studies with these candidate drugs by treating matched STAG2 WT and KO T24 cells with each drug at concentrations ranging from 0.001 to 100 µM, then computationally modeled EC50 values in R. Cells with STAG2 KO were more sensitive to Berzosertib (T24 Control EC50: 1.78 µM, T24 KO EC50s: 1.03, 0.77 µM) compared to cells with WT STAG2 expression. Interestingly, cells with WT STAG2 expression were more sensitive to PI-103 (T24 Control EC50: 0.33 µM, T24 KO EC50s: 0.97, 1.41 µM) compared to cells with STAG2 KO. This suggests that STAG2 expressing cells may have a unique dependency on PI3K signaling, which has previously been reported in sarcoma cell lines. These findings lay the foundation for further investigation of Berzosertib as a targeted therapy for MIBC patients with mutated or low STAG2, and PI-103 for MIBC patients with high STAG2 who currently suffer worse clinical outcomes. Citation Format: Sarah R. Athans, Henry Withers, Zara Kazmierczak, Katerina Gurova, Anna Woloszynska. Identifying targeted therapies for muscle invasive bladder cancer via STAG2 expression [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR007.

Preimplantation development of in vitro-produced bovine embryos treated with hydroxychloroquine

Hydroxychloroquine (HCQ) is a safe antimalarial drug but its overdosage or inappropriate use, such as during the pandemic, may cause adverse effects once this drug is considered a potent inhibitor of autophagy. Information about HCQ's effects on the reproductive field, including gametes and initial embryos, is limited. In this study, we evaluated the effect of HCQ (1, 6, 12, and 24 μM) on pre-implantation embryo development, autophagy, and apoptosis of bovine embryos produced in vitro. A dose-response experiment showed a reduction (p < 0.05) in cleavage only at the highest concentration. Blastocyst rate was gradually reduced (p < 0.05) with the increase of HCQ dosage starting at 6 μM, with no embryo formation occurring at 24 μM. Further analysis showed that embryos treated with 12 μM of HCQ had a higher (p < 0.05) accumulation of acidic autophagic vesicles on Days 5 and 7 of development and a higher (p < 0.01) apoptotic index on Day 7. To our knowledge, this is the first study to evaluate the effects of HCQ on embryo pre-implantation development in mammals. The results contribute with more information related to the study of autophagy in embryology as well as add some discussion on HCQ toxicology and its effects on reproductive cells.