Dose-response Experiments Research Articles

Enhanced tolerance to narcosis in starved Daphnia magna neonates

Abstract Guidelines for ecotoxicity testing with Daphnia magna specify particular feeding protocols during the exposure, yet standardization for preexposure feeding remains ambiguous despite its recognized significance in affecting organismal metabolic capacity and tolerance. This ambiguity may contribute to disparate responses and heightened uncertainty in determining the effect concentrations of test chemicals, particularly those inducing metabolic effects through narcosis. Here, we address this gap through a three-step dose-response experiment with neonates of D. magna subjected to two alternative feeding regimes in the preexposure phase: starved and moderately fed during the first 24 hr after birth. Following this treatment, the daphnids were exposed to narcosis-inducing substances (polycyclic aromatic hydrocarbons; PAHs) for 72 hr before being transferred to clean media with algal food ad libitum for a 48 hr recovery phase. Daphnid survivorship, individual protein content, and body size at the end of each experiment phase—pre-exposure, postexposure and postrecovery—were compared between the treatments. Significant treatment effects were observed, including lower and less variable protein content in the starved daphnids entering the PAH exposure phase, yet higher survivorship and greater recovery potential in these daphnids compared with the fed individuals. Our findings underscore the importance of early-life food access and advocate for mandatory reporting of pre-exposure feeding regimes, particularly when testing substances acting via nonpolar narcosis.

Azoxystrobin hides the respiratory failure of low dose sulfoxaflor in bumble bees

Pollinators are exposed to multiple pesticides during their lifetime. Various pesticides are used in agriculture and thus not all mixtures have been tested against each other and little is known about them. In this article, we investigate the impact of sulfoxaflor, a novel sulfoximine insecticide, and azoxystrobin, a widely used strobilurin fungicide, on bumble bee Bombus terrestris worker survival and physiological functions. The dosages used in this experiment are selected from dose response experiments based on LD50 data. Due to variable interactive effects on survival, our findings reveal distinct effects on bumble bee metabolic rate and respiratory patterns induced by sulfoxaflor in combination with azoxystrobin, shedding light on previously unexplored aspects of its physiological impact. Notably, we observed noteworthy differences between oral and contact treatments, emphasizing the importance of considering distinct application methods when evaluating pesticide effects and interactions. Specifically, our results indicate that azoxystrobin can mitigate the impact of sulfoxaflor, suggesting dose-dependent antagonistic interaction between these pesticides in contact exposure. In oral exposure, however, Amistar tended to potentiate the sulfoxaflor effect. This study contributes valuable insights into the multifaceted dynamics of pesticide exposure and interactions, paving the way for a more nuanced understanding of their implications on pollinator health.

Pyroxsulam Resistance in Apera spica-venti: An Emerging Challenge in Crop Protection.

Apera spica-venti, a prevalent weed in Czech winter wheat fields, has developed resistance to ALS-inhibiting herbicides due to their frequent use. This study reports a biotype of A. spica-venti resistant to pyroxsulam, with cross and multiple resistance to iodosulfuron, propoxycarbazone, pinoxaden, and chlortoluron. Dose-response experiments revealed high resistance of both R1 and R2 biotypes to pyroxsulam, with resistance factors (RF) of 6.69 and 141.65, respectively. Pre-treatment with malathion reduced RF by 2.40× and 1.25× in R1 and R2, indicating the potential involvement of cytochrome P450 (CytP450). NBD-Cl pre-treatment decreased RF only in R2, suggesting possible GST involvement. Gene analysis revealed no mutations (at previously reported sites) or overexpression in the acetolactate synthase (ALS) gene. However, a significant difference in ALS enzyme activity between resistant and susceptible biotypes points to target-site resistance mechanisms. Studies with 14C-labeled pyroxsulam showed that reduced absorption and translocation were not likely resistance mechanisms. In summary, herbicide resistance in A. spica-venti appears to result from multiple mechanisms. Possible causes include target-site resistance from an unidentified ALS mutation (within coding or regulatory regions). Enhanced herbicide metabolism via CytP450s and GSTs is also a contributing factor. Further experimental validation is needed to confirm these mechanisms and fully understand the resistance. This evolution underscores the adaptive capacity of weed populations under herbicide pressure, emphasizing the need for alternative control strategies.

Senescent myoblasts exhibit an altered exometabolome that is linked to senescence-associated secretory phenotype (SASP) signalling.

Cellular senescence has been implicated in the aging-related dysfunction of satellite cells, the resident muscle stem cell population primarily responsible for the repair of muscle fibres. Despite being in a state of permanent cell cycle arrest, these cells remain metabolically active and release an abundance of factors that can have detrimental effects on the cellular microenvironment. This phenomenon is known as the senescence-associated secretory phenotype (SASP), and its metabolic profile is poorly characterized in senescent muscle. In the present investigation, we examined the intracellular and extracellular metabolome of C2C12 myoblasts using a bleomycin-mediated model of DNA damage-induced senescence. We also evaluated the relationship between the senescent metabolic phenotype and SASP signalling through molecular and network-based analyses. Senescent myoblasts exhibited a significantly altered extracellular metabolome (i.e. exometabolome), including increased secretion of several aging-associated metabolites. Four of these metabolites - trimethylamine-N-oxide (TMAO), xanthine, choline, and oleic acid - were selected for individual dose-response experiments to determine if they could drive the senescence phenotype. While most of the tested metabolites did not independently alter senescence markers, oleic acid treatment of healthy myoblasts significantly upregulated the SASP genes Ccl2, Cxcl12, and Il33 (p<0.05). A gene-metabolite interaction network further revealed that oleic acid was one of the most interconnected metabolites to key senescence-associated genes. Notably, oleic acid interacted with several prominent SASP genes, suggesting a potential epigenetic effect between this monounsaturated fatty acid and SASP regulation. In summary, the exometabolome, particularly oleic acid, is implicated in SASP signalling within senescent myoblasts.

Droplet-Based Microfluidic Photobioreactor as a Growth Optimization Tool for Cyanobacteria and Microalgae

Microalgae and cyanobacteria are photosynthetic microorganisms with significant biotechnological potential for the production of bioactive compounds, making them a promising resource for diverse industrial applications. This study presents the development and validation of a modular, droplet-based microfluidic photobioreactor (µPBR) designed for high-throughput screening and cultivation under controlled light conditions. The µPBR, based on polytetrafluoroethylene (PTFE) tubing and a 4-channel LED illumination system, enables precise modulation of light intensity, wavelength, and photoperiod, facilitating dose–response experiments. Synechococcus elongatus UTEX 2973 and Chlorella vulgaris were used to demonstrate the system’s capacity to support photosynthetic growth under various conditions. The results indicate that continuous illumination, particularly under blue and mixed blue-red light, promotes higher autofluorescence and chlorophyll a content in cyanobacteria Synechococcus elongatus UTEX2973, while Chlorella vulgaris achieved optimal growth under a 16:8 light-dark cycle with moderate light intensity. This µPBR offers not only a flexible, scalable platform for optimizing growth parameters but also allows for the investigation of highly resolved dose response screenings of environmental stressors such as salinity. The presented findings highlight its potential for advancing microalgal biotechnology research and applications.

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

Giardia 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. This 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. Our 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. The 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.

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.

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

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.