Normalization of data for viability and relative cell function curves.

Application of Benchmark Concentration (BMC) Analysis on Zebrafish Data: A New Perspective for Quantifying Toxicity in Alternative Animal Models.

Many toxicological test methods, including assays of cell viability and function, require an evaluation of concentration-response data. This often involves curve fitting, and the resulting mathematical functions are then used to determine the concentration at which a certain deviation from the control value occurs (e.g. a decrease of cell viability by 15%). Such a threshold is called the benchmark response (BMR). For a toxicological test, it is often of interest to determine the concentration of test compound at which a pre-defined BMR of e.g. 10, 25 or 50% is reached. The concentration at which the modelled curve crosses the BMR is called the benchmark concentration (BMC). We present a user-friendly, web-based tool (BMCeasy), designed for operators without programming skills and profound statistical background, to determine BMCs and their confidence intervals. BMCeasy allows simultaneous analysis of viability plus a functional test endpoint, and it yields absolute BMCs with confidence intervals for any BMR. Besides an explanation of the algorithm underlying BMCeasy, this article also gives multiple examples of data outputs. BMCeasy was used within the EU-ToxRisk project for preparing data packages that were submitted to regulatory authorities, demonstrating the real-life applicability of the tool.

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts.

Acute and short-term toxicity tests are foundational to toxicology research. These tests offer preliminary insights into the fundamental toxicity characteristics of the chemicals under evaluation and provide essential data for chronic toxicity assessments. Fluoride is a common chemical in aquatic environments; however, the findings of toxicological data, such as LC50 for aquatic organisms, often exhibit inconsistency. Consequently, this study employed zebrafish as a model organism during their early life stages to assess the acute and short-term toxicity of fluoride exposure. Bayesian model averaging was utilized to calculate the LC50/EC50 values and establish baseline concentrations. The results indicated a dose–response relationship between water fluoride concentration and harmful outcomes. The 20 mg/L group was identified as the lowest observed adverse effect level (LOAEL) for the majority of toxicity indicators and warrants special attention. Based on the BBMD model averages, the LC50 of fluoride for 1 to 5 days post-fertilization (dpf) zebrafish was 147.00, 80.80, 61.25, 56.50, and 37.50 mg/L, while the EC50 of cumulative malformation rate for 5 dpf zebrafish was 59.75 mg/L. As the benchmark response (BMR) increased, both the benchmark concentrations (BMCs) and benchmark dose levels (BMDLs) also increased. The research aims to provide essential data for the development of environmental water guidelines and to mitigate ecological risks associated with fluoride in aquatic ecosystems.

A Bayesian benchmark concentration analysis for urinary fluoride and intelligence in adults in Guizhou, China

The effects of ethanol on striatal dopamine and frontal cortical D-[3H]aspartate efflux oscillate with repeated treatment: Relevance to individual differences in drug responsiveness

Evidence from human studies suggests that high expression of brain mineralocorticoid receptors (MR) may promote resilience against negative consequences of stress exposure, including childhood trauma. We examined, in mice, whether brain MR overexpression can alleviate the effects of chronic early life stress (ELS) on contextual memory formation under low and high stress conditions, and neurogenesis and synaptic function of dentate gyrus granular cells. Male mice were exposed to ELS by housing the dam with limited nesting and bedding material from postnatal day (PND) 2 to 9. We investigated the moderating role of MRs by using forebrain-specific transgenic MR overexpression (MR-tg) mice. Low-stress contextual (i.e., object relocation) memory formation was hampered by ELS in wildtype but not MR-tg mice. Anxiety like behavior and high-stress contextual (i.e., fear) memory formation were unaffected by ELS and/or MR expression level. At the cellular level, an interaction effect was observed between ELS and MR overexpression on the number of doublecortin-positive cells, with a significant difference between the wildtype ELS and MR-tg ELS groups. No interaction was found regarding Ki-67 and BrdU staining. A significant interaction between ELS and MR expression was further observed with regard to mEPSCs and mIPSC frequency. The ratio of evoked EPSC/IPSC or NMDA/AMPA responses was unaffected. Overall, these results suggest that ELS affects contextual memory formation under low stress conditions as well as neurogenesis and synaptic transmission in dentate granule cells, an effect that can be alleviated by MR-overexpression.

The benchmark dose (BMD) concept is increasingly utilized to analyze quantitative dose-response relationships in genetic toxicology. This methodology requires the user (i.e. the toxicologist) to a priori define a small increase over controls that is "acceptable" to be induced by a genotoxic test substance. The increase is called benchmark response (BMR) or critical effect size (CES), depending on the software used. To render the metrics calculated from the data of animals treated with the test substance applicable for risk assessment, the BMR or CES must represent biologically relevant changes of parameters measured in in vivo genotoxicity assays such as the Micronucleus, Comet, Transgenic rodent or Pig-a assay. Current recommendations for CES in genotoxicology are arbitrary (10% increase over mean vehicle controls) or based on limited, usually 5-6, data points (i.e. the standard deviation of the concurrent vehicle control group). We have, therefore, analyzed historical vehicle control data of standard in vivo genotoxicity test systems with statistical methods. Based on this evaluation, we illustrate limitations of the currently recommended CES values and propose a pragmatic approach that may contribute to better defining endpoint-specific CES values for BMD software like PROAST.

The world market for bovine embryos has increased in the past few years. However, sanitary problems such as foot and mouth disease in Brazil, vesicular stomatitis in South America, and bovine spongiform encephalopathy (BSE) in North America and Europe have increased concerns regarding the risk of introducing exotic diseases and/or more virulent serotypes of endemic diseases by embryo transfer. Many countries are trying to develop and/or improve new techniques for infectious disease detection, with the scientific basis to support the import and export of animal germplasm. Therefore, the epidemiology of the diseases and the interaction between pathogens and cumulus–oocyte complexes (COCs), embryos, and semen must be investigated. Despite the many studies that have been carried out to evaluate the possibility of transmission of infectious agents by the embryo, few data are available regarding COC susceptibility (Tsuboi et al. 1992 J. Vet. Med. Sci. 54, 1179–1181). The aim of this study was to evaluate the presence of bovine herpes virus serotype 1 (BHV-1) in COCs and follicular fluid (FF) collected from naturally infected animals in a low stress condition. Blood samples of non-lactating Gyr breed (Bos indicus) cows were collected and evaluated for BHV-1 antibodies by the serum neutralization microplate test, performed as described in the Manual for Standards for Diagnostic Tests and Vaccines (OIE, 1992). The cows were diagnosed as serologically positive (n = 38) or serologically negative (n = 8), and kept under grazing in Brachiaria decumbens pasture with mineral supplementation. The cows considered as positive showed titers greater than 1/4. COCs and follicular fluid (FF) were obtained by ovum pick-up (OPU) using sterile and disposable materials for each animal. Virus detection was performed by the PCR technique. PCR sensitivity was made using COCs and FF recovered from eight BHV-1 serologically negative animals. These samples were either artificially infected on plates with 106.5 TCID in 50 µL of IBR Colorado 1 reference serotype (ATCC, VR-864) or used as a negative control. The PCR analitical sensitivity was 100.5 TCID. The presence of BHV-1 in COCs and FF was not detected in any of the animals, despite the high sensitivity of the PCR technique. In the present in vivo model, results show that COCs collected from serologically BHV-1 positive cows presenting no clinical signs of the illness and managed in a low stress condition could be used as donors for in vitro fertilization procedures with minimal sanitary risks. Also, the absence of the virus in COCs and FF cannot be used as a predictor of BHV-1 infection status in bovine herds.

NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His(44), Met(90), His(97), and His(127), and CuB, a second degenerate octahedral geometry with the addition of Glu(46). The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.

Retrospective analyses of large medical databases: what do they tell us?

Dynorphin/Kappa Opioid Receptor Signaling in Preclinical Models of Alcohol, Drug, and Food Addiction.

Stress exposure is associated with psychiatric conditions, such as depression, anxiety, and post-traumatic stress disorder (PTSD). It is also a vulnerability factor to developing or reinstating substance use disorder. Stress causes several changes in the neuro-immune-endocrine axis, potentially resulting in prolonged dysfunction and diseases. Changes in several transmitters, including serotonin, dopamine, glutamate, gamma-aminobutyric acid (GABA), glucocorticoids, and cytokines, are associated with psychiatric disorders or behavioral alterations in preclinical studies. Complex and interacting mechanisms make it very difficult to understand the physiopathology of psychiatry conditions; therefore, studying regulatory mechanisms that impact these alterations is a good approach. In the last decades, the impact of stress on biology through epigenetic markers, which directly impact gene expression, is under intense investigation; these mechanisms are associated with behavioral alterations in animal models after stress or drug exposure, for example. The endocannabinoid (eCB) system modulates stress response, reward circuits, and other physiological functions, including hypothalamus-pituitary-adrenal axis activation and immune response. eCBs, for example, act retrogradely at presynaptic neurons, limiting the release of neurotransmitters, a mechanism implicated in the antidepressant and anxiolytic effects after stress. Epigenetic mechanisms can impact the expression of eCB system molecules, which in turn can regulate epigenetic mechanisms. This review will present evidence of how the eCB system and epigenetic mechanisms interact and the consequences of this interaction in modulating behavioral changes after stress exposure in preclinical studies or psychiatric conditions. Moreover, evidence that correlates the involvement of the eCB system and epigenetic mechanisms in drug abuse contexts will be discussed.

Neurite outgrowth in human induced pluripotent stem cell-derived neurons as a high-throughput screen for developmental neurotoxicity or neurotoxicity