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The emergence of advanced and powerful neuroscientific technologies has greatly pushed forward the development of neurobiology in the last decade. Although neurotoxicology is an interdisciplinary subject sharing a mass of technologies with neurobiology, the implementation of these advanced technologies in neurotoxicology is merely seen. Here we describe the detailed methods and materials of some emerging neuroscientific technologies, including optogenetics, fiber photometry, in vivo two-photon Imaging, in vivo calcium imaging, and in vivo electrophysiological recording, hoping that the integration of technologies from neurotoxicology and neuroscience can lend weight to the development of neurotoxicology.Semi-quantitative reverse transcription and polymerase chain reaction (sqRT-PCR) is a simple and specific method for quantitative RNA in recent years. The relative quantity of a specific mRNA in the samples can be inferred by reverse transcription of mRNA into cDNA, and PCR amplification and determination of the quantity of PCR products. The semi-quantitative analysis is carried out under a fixed number of PCR cycles, and the total RNA concentration is kept in the exponential phase of the PCR. The method is to use a housekeeping gene (usually actin, GAPDH, and EF1α) as a reference standard in treated and control organisms to observe the expression of the interested genes (upregulated or downregulated) in toxicology. In this chapter, we describe a step-by-step method for determining the differential regulation of target genes in organisms exposed to environmental pollutants.Gene expression analysis has been becoming a popular method for studying gene function and response to different environmental stresses, including toxin/pollution exposure. Selection of a suitable reference gene is critically important for gene expression analysis due to that wrong reference genes will cause misleading and even wrong conclusion. A good reference gene should be a more stable reference gene, particularly during the toxicant exposure treatment and/or other investigation condition. In this chapter, a step-by-step protocol is present for primer design, reverse transcription PCR, primer efficiency and specificity test, qRT-PCR, and the strategy for identifying most stable reference genes for toxicogenomic and gene expression analysis. The detailed method for determining the primer gene specificity and primer efficiency are also presented in this chapter. Low primer efficiency will affect the fold changes during gene expression analysis; however, it does not affect the conclusion, up- or downregulation. Choosing a wrong reference gene may result in wrong conclusion.Exposure to multiple stressors often results in higher toxicity than one stressor alone. Examining joint effects of multiple stressors could provide more realistic exposure scenarios and a better understanding of the combined effects. In amphibian toxicology, simultaneous exposure to some pesticides and ultraviolet B (UVB) radiation has been suggested to be detrimental and more harmful in amphibian early-life stages than either stressor alone. Therefore, it is important to investigate the joint effects of these two stressors and provide data that could lead to more informed risk assessment. Here we describe how to set up a co-exposure to pesticides and ultraviolet B radiation to examine their joint toxicity in amphibian embryos and larvae, focusing on Xenopus laevis with notes on other amphibian species. With modifications, the methods may be applied to other types of chemicals or other aquatic organisms of interest.Drosophila melanogaster, the fruit fly, has been widely used in biological investigation as an invertebrate alternative to mammals for its various advantages compared to other model organisms, which include short life cycle, easy handling, high prolificacy, and great availability of substantial genetic information. The behavior of Drosophila melanogaster is closely related to its growth, which can reflect the physiological conditions of Drosophila. PF-05221304 We have optimized simple and robust behavioral assays for determining the larvae survival, adult climbing ability (mobility assay), reproductive behavior, and lifespan of Drosophila. In this chapter, we present the step-by-step detailed method for studying Drosophila behavior.Environmental pollutants inevitably exert adverse effects on humans and other species. Quick identification and in-depth characterization of the pollutants are requisite objectives for clinicians and environmental health scientists. The nematode Caenorhabditis elegans has been utilized as a model organism for toxicity evaluation of environmental pollutants, due to its transparency, short lifespan, entire genome sequencing, and economical characteristics. However, few researchers have systematically addressed mitochondrial toxicity in response to toxicants, despite the critical role mitochondria play in energy production and respiration, as well as the generation of reactive oxygen species. Mitochondria are vulnerable to environmental pollutants, and their dysfunction contributes to cellular damage and toxicity in plethora of diseases. Here, we describe methods in step-by-step for mitochondrial toxicity evaluation in response to pollutants, including exposure of C. elegans to toxicants, mitochondrial ROS detection, mitochondrial morphology analysis, mitochondrial function analysis, such as ATP production and oxygen consumption, and gene expression studies, with the application of corresponding genetically modified strains.The contamination of heavy metals, a class of naturally occurring and persistent toxicants, has become a major public health concern due to increasing industrial and anthropogenic activities. The use of COPAS Biosort, a flow cytometer capable of measuring thousands of nematodes in minutes via high-throughput assays, has been widely applied in C. elegans studies for assessing toxicity of individual metals; however, such application yet to be seen for metals or other chemical mixtures. In the present protocol, we investigated toxic effects of individual metals, Cd, Pb, and Mn, as well as their binary and ternary mixtures, using nematode C. elegans. The toxic outcomes, including effects on growth, reproduction, and feeding behavior, were measured using high-throughput platform analysis (COAPS Biosort).Crude oil disasters, such as the Deepwater Horizon accident, have caused severe environmental contamination and damage, affecting the health of marine and terrestrial organisms. Some previous studies have demonstrated cleanup efforts using chemical dispersant induced more potent toxicities than oil alone due to an increase in bioavailability of crude oil components, such as PAHs. However, there still lacks a systematic procedure that provides methods to determine genotypic and phenotypic changes following exposure to environmental toxicants or toxicant mixture, such as dispersed crude oil. Here, we describe methods for identifying a mechanism of dispersed crude oil-induced reproductive toxicity in the model organisms, Caenorhabditis elegans (C. elegans). Due to the genetic malleability of C. elegans, two mutant strains outlined in this chapter were used to identify a pathway responsible for inducing apoptosis MD701 bcIs39 [lim-7pced-1GFP + lin-15(+)], a mutant strain that allows visualization of apoptotic bodies via a green fluorescent protein fused to CED-1; and TJ1 (cep-1(gk138) I.), a p53/CEP-1 defective strain that is unable to activate apoptosis via the p53/CEP-1 pathway. In addition, qRT-PCR was utilized to demonstrate the aberrant expression of apoptosis (ced-13, ced-3, ced-4, ced-9, cep-1, dpl-1, efl-1, efl-2, egl-1, egl-38, lin-35, pax-2, and sir-2.1) and cytochrome P450 (cyp14a3, cyp35a1, cyp35a2, cyp35a5, and cyp35c1) protein-coding genes following exposure to dispersed crude oil. The procedure outlined here can be applicable to determine whether environmental contaminants, most of time contaminant mixture, cause reproductive toxicity by activation of the proapoptotic, p53/CEP-1 pathway.

Omecamtiv mecarbil (OM) is a novel cardiac myosin activator in development for the treatment of heart failure with reduced ejection fraction. The objective of this study was to evaluate the potential for OM to affect the pharmacokinetics of metformin.

This was anopen-label, fixed-sequencestudy in 14 healthy subjects.On Day 1, subjects received an 850 mg oral dose of metformin. From Days 4 to 9, subjects received twice-daily 25 mg oral doses of OM tablets. On Day 10, subjects received an 850 mg oral dose of metformin and a single 25 mg tablet of OM. Blood and urine samples were collected up to 36 h post-dose following administration of metformin on Days 1 and 10 to characterize concentrations of metformin in plasma and urine.

The ratios of the geometric least square means of metformin coadministered with OM compared to metformin alone were 98.7%, 99.3%, and 110.2% for AUC

, AUC

, and C

, respectively. The mean renal clearance of metformin was similar following metformin administered alone (34.2 L/h) compared to metformin coadministered with OM (32.9 L/h). All adverse events were mild in severity and resolved prior to the end of the study. No serious adverse events or treatment-emergent adverse events led to discontinuation from the study.

There was no clinically relevant effect of OM on the pharmacokinetics of metformin in healthy subjects.

There was no clinically relevant effect of OM on the pharmacokinetics of metformin in healthy subjects.Soyasapogenol B is an oleanane-type pentacyclic triterpene that has various applications in food and healthcare and has a higher biological activity than soyasaponin. Saccharomyces cerevisiae is a potential platform for terpenoid production with mature genetic tools for metabolic pathway manipulation. In this study, we developed a biosynthesis method to produce soyasapogenol B. First, we expressed β-amyrin synthase derived from Glycyrrhiza glabra in S. cerevisiae to generate β-amyrin, as the precursor of soyasapogenol B. Several different types of promoters were then used to regulate the expression of key genes in the mevalonate pathway (MVA), and this subsequently increased the yield of β-amyrin to 17.6 mg/L, 25-fold more than that produced in the original strain L01 (0.68 mg/L). Then, using the β-amyrin-producing strain, we expressed soyasapogenol B synthases from Medicago truncatula (CYP93E2 and CYP72A61V2) and from G. glabra (CYP93E3 and CYP72A566). Soyasapogenol B yields were then optimized by using soyasapogenol B synthases and cytochrome P450 reductase from G. glabra. The most effective soyasapogenol B production strain was used for fermentation, and the yield of soyasapogenol B reached 2.9 mg/L in flask and 8.36 mg/L in a 5-L bioreactor with fed glucose and ethanol. This study demonstrated the heterologous synthesis of soyasapogenol B in S. cerevisiae using the combined expression of CYP93E3 and CYP72A566 in the synthesis pathway, which significantly increased the production of soyasapogenol B and provides a reference method for the biosynthesis of other triterpenes.L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high-activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and kcat/Km of the mutant enzyme (V231A/K382G) is 4.36 mM-1∙s-1. The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.