CUREing biochemistry lab monotony.

Undergraduate research experience is critical to success in post-graduate research settings. The recent movement away from "cookbook" style labs to course-based undergraduate research experiences (CUREs) in undergraduate laboratories has allowed universities to provide inclusive research experience while bypassing the limitations of extracurricular apprenticeships. This paper describes an upper-level biochemistry CURE designed to provide students with an introductory experience to graduate-level research by studying a suspected DNA helicase. This CURE is designed to span multiple semesters, where each student cohort builds upon the work of previous semesters. Pre- and post-course surveys were employed to assess student confidence in bench skills, perceptions of the course, and project ownership. The results show that the incorporation of lab meeting-style recitations and poster presentations led to higher project ownership, while overcoming troubleshooting was a significant challenge. Furthermore, confidence in every experimental technique increased significantly in all but one instance. Based on these results, this CURE is providing students with a realistic experience in graduate-level research.

Xenobiotic metabolism and transport in Caenorhabditis elegans.

Caenorhabditis elegans has emerged as a major model in biomedical and environmental toxicology. Numerous papers on toxicology and pharmacology in C. elegans have been published, and this species has now been adopted by investigators in academic toxicology, pharmacology, and drug discovery labs. C. elegans has also attracted the interest of governmental regulatory agencies charged with evaluating the safety of chemicals. However, a major, fundamental aspect of toxicological science remains underdeveloped in C. elegans: xenobiotic metabolism and transport processes that are critical to understanding toxicokinetics and toxicodynamics, and extrapolation to other species. The aim of this review was to initially briefly describe the history and trajectory of the use of C. elegans in toxicological and pharmacological studies. Subsequently, physical barriers to chemical uptake and the role of the worm microbiome in xenobiotic transformation were described. Then a review of what is and is not known regarding the classic Phase I, Phase II, and Phase III processes was performed. In addition, the following were discussed (1) regulation of xenobiotic metabolism; (2) review of published toxicokinetics for specific chemicals; and (3) genetic diversity of these processes in C. elegans. Finally, worm xenobiotic transport and metabolism was placed in an evolutionary context; key areas for future research highlighted; and implications for extrapolating C. elegans toxicity results to other species discussed.

Proteasome Inhibition in Brassica napus Roots Increases Amino Acid Synthesis to Offset Reduced Proteolysis.

Cellular homeostasis is maintained by the proteasomal degradation of regulatory and misfolded proteins, which sustains the amino acid pool. Although proteasomes alleviate stress by removing damaged proteins, mounting evidence indicates that severe stress caused by salt, metal(oids), and some pathogens can impair the proteasome. However, the consequences of proteasome inhibition in plants are not well understood and even less is known about how its malfunctioning alters metabolic activities. Lethality causes by proteasome inhibition in non-photosynthetic organisms stem from amino acid depletion, and we hypothesized that plants respond to proteasome inhibition by increasing amino acid biosynthesis. To address these questions, the short-term effects of proteasome inhibition were monitored for 3, 8 and 48 h in the roots of Brassica napus treated with the proteasome inhibitor MG132. Proteasome inhibition did not affect the pool of free amino acids after 48 h, which was attributed to elevated de novo amino acid synthesis; these observations coincided with increased levels of sulfite reductase and nitrate reductase activities at earlier time points. However, elevated amino acid synthesis failed to fully restore protein synthesis. In addition, transcriptome analysis points to perturbed abscisic acid signaling and decreased sugar metabolism after 8 h of proteasome inhibition. Proteasome inhibition increased the levels of alternative oxidase but decreased aconitase activity, most sugars and tricarboxylic acid metabolites in root tissue after 48 h. These metabolic responses occurred before we observed an accumulation of reactive oxygen species. We discuss how the metabolic response to proteasome inhibition and abiotic stress partially overlap in plants.

Lack of detectable sex differences in the mitochondrial function of Caenorhabditis elegans.

BACKGROUND: Sex differences in mitochondrial function have been reported in multiple tissue and cell types. Additionally, sex-variable responses to stressors including environmental pollutants and drugs that cause mitochondrial toxicity have been observed. The mechanisms that establish these differences are thought to include hormonal modulation, epigenetic regulation, double dosing of X-linked genes, and the maternal inheritance of mtDNA. Understanding the drivers of sex differences in mitochondrial function and being able to model them in vitro is important for identifying toxic compounds with sex-variable effects. Additionally, understanding how sex differences in mitochondrial function compare across species may permit insight into the drivers of these differences, which is important for basic biology research. This study explored whether Caenorhabditis elegans, a model organism commonly used to study stress biology and toxicology, exhibits sex differences in mitochondrial function and toxicant susceptibility. To assess sex differences in mitochondrial function, we utilized four male enriched populations (N2 wild-type male enriched, fog-2(q71), him-5(e1490), and him-8(e1498)). We performed whole worm respirometry and determined whole worm ATP levels and mtDNA copy number. To probe whether sex differences manifest only after stress and inform the growing use of C. elegans as a mitochondrial health and toxicologic model, we also assessed susceptibility to a classic mitochondrial toxicant, rotenone. RESULTS: We detected few to no large differences in mitochondrial function between C. elegans sexes. Though we saw no sex differences in vulnerability to rotenone, we did observe sex differences in the uptake of this lipophilic compound, which may be of interest to those utilizing C. elegans as a model organism for toxicologic studies. Additionally, we observed altered non-mitochondrial respiration in two him strains, which may be of interest to other researchers utilizing these strains. CONCLUSIONS: Basal mitochondrial parameters in male and hermaphrodite C. elegans are similar, at least at the whole-organism level, as is toxicity associated with a mitochondrial Complex I inhibitor, rotenone. Our data highlights the limitation of using C. elegans as a model to study sex-variable mitochondrial function and toxicological responses.

The use of race terms in epigenetics research: considerations moving forward.

The field of environmental epigenetics is uniquely suited to investigate biologic mechanisms that have the potential to link stressors to health disparities. However, it is common practice in basic epigenetic research to treat race as a covariable in large data analyses in a way that can perpetuate harmful biases without providing any biologic insight. In this article, we i) propose that epigenetic researchers open a dialogue about how and why race is employed in study designs and think critically about how this might perpetuate harmful biases; ii) call for interdisciplinary conversation and collaboration between epigeneticists and social scientists to promote the collection of more detailed social metrics, particularly institutional and structural metrics such as levels of discrimination that could improve our understanding of individual health outcomes; iii) encourage the development of standards and practices that promote full transparency about data collection methods, particularly with regard to race; and iv) encourage the field of epigenetics to continue to investigate how social structures contribute to biological health disparities, with a particular focus on the influence that structural racism may have in driving these health disparities.

rol-6 and dpy-10C. elegans mutants have normal mitochondrial function after normalizing to delayed development.

Collagen mutations are commonly used in the creation of Caenorhabditis elegans transgenic strains, but their secondary effects are not fully characterized . We compared the mitochondrial function of N2, dpy-10, rol-6, and PE255 C. elegans . N2 worms exhibited ~2-fold greater volume, mitochondrial DNA copy number, and nuclear DNA copy number than collagen mutants (p<0.05). Whole-worm respirometry and ATP levels were higher in N2 worms, but differences in respirometry largely disappeared after normalization to mitochondrial DNA copy number. This data suggests that rol-6 and dpy-10 mutants are developmentally delayed but have comparable mitochondrial function to N2 worms once the data is normalized to developmental stage.

Increased cytotoxicity of Pb2+ with co-exposures to a mitochondrial uncoupler and mitochondrial calcium uniporter inhibitor.

Lead (Pb2+) is an important developmental toxicant. The mitochondrial calcium uniporter (MCU) imports calcium ions using the mitochondrial membrane potential (MMP), and also appears to mediate the influx of Pb2+ into the mitochondria. Since our environment contains mixtures of toxic agents, it is important to consider multi-chemical exposures. To begin to develop generalizable, predictive models of interactive toxicity, we developed mechanism-based hypotheses about interactive effects of Pb2+ with other chemicals. To test these hypotheses, we exposed HepG2 (human liver) cells to Pb2+ alone and in mixtures with other mitochondria-damaging chemicals: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler that reduces MMP, and Ruthenium Red (RuRed), a dye that inhibits the MCU. After 24 hours, Pb2+ alone, the mixture of Pb2+ and RuRed, and the mixture of Pb2+ and FCCP caused no decrease in cell viability. However, the combination of all three exposures led to a significant decrease in cell viability at higher Pb2+ concentrations. After 48 hours, the co-exposure to elevated Pb2+ concentrations and FCCP caused a significant decrease in cell viability, and the mixture of all three showed a clear dose-response curve with significant decreases in cell viability across a range of Pb2+ concentrations. We performed ICP-MS analyses on isolated mitochondrial and cytosolic fractions and found no differences in Pb2+ uptake across exposure groups, ruling out altered cellular uptake as the mechanism for interactive toxicity. We assessed MMP following exposure and observed a decrease in membrane potential that corresponds to loss of cell viability but is likely not sufficient to be the causative mechanistic driver of cell death. This research provides a mechanistically-based framework for understanding Pb2+ toxicity in mixtures with mitochondrial toxicants.

Cannabis alters DNA methylation at maternally imprinted and autism candidate genes in spermatogenic cells.

Cannabis use in the United States is increasing, with highest consumption among men at their peak reproductive years. We previously demonstrated widespread changes in sperm DNA methylation with cannabis exposure in humans and rats, including genes important in neurodevelopment. Here, we use an in vitro human spermatogenesis model to recapitulate chronic cannabis use and assess DNA methylation at imprinted and autism spectrum disorder (ASD) candidate genes in spermatogonial stem cell (SSC)- and spermatid-like cells. Methylation at maternally imprinted genes SGCE and GRB10 was significantly altered in SSC- and spermatid-like cells, respectively, while PEG3 was significantly differentially methylated in spermatid-like cells. Two of ten randomly selected ASD candidate genes, HCN1 and NR4A2, had significantly altered methylation with cannabis exposure in SSC-like cells. These results support our findings in human cohorts and provide a new tool with which to gain mechanistic insights into the association between paternal cannabis use and risk of ASD in offspring.

Sex-specific DNA methylation and associations with in utero tobacco smoke exposure at nuclear-encoded mitochondrial genes.

Sex-linked differences in mitochondrial ATP production, enzyme activities, and reactive oxygen species generation have been reported in multiple tissue and cell types. While the effects of reproductive hormones underlie many of these differences, regulation of sexually dimorphic mitochondrial function has not been fully characterized. We hypothesized that sex-specific DNA methylation contributes to sex-specific expression of nuclear genes that influence mitochondrial function. Herein, we analysed DNA methylation data specifically focused on nuclear-encoded mitochondrial genes in 191 males and 190 females. We found 596 differentially methylated sites (DMSs) (FDR p < 0.05), corresponding to 324 genes, with at least a 1% difference in methylation between sexes. To investigate the potential functional significance, we utilized gene expression microarray data. Of the 324 genes containing DMSs, 17 showed differences in gene expression by sex. Particularly striking was that ATP5G2, encoding subunit C of ATP synthase, contains seven DMSs and exhibits a sex difference in expression (p = 0.04). Finally, we also found that alterations in DNA methylation associated with in utero tobacco smoke exposure were sex-specific in these nuclear-encoded mitochondrial genes. Interestingly, the level of sex differences in DNA methylation at nuclear-encoded mitochondrial genes and the level of methylation changes associated with smoke exposure were less prominent than that of other genes. This suggests more conservative regulation of DNA methylation at these nuclear-encoded mitochondrial genes as compared to others. Overall, our findings suggest that sex-specific DNA methylation may help establish sex differences in expression and function and that sex-specific alterations in DNA methylation in response to exposures could contribute to sex-variable toxicological responses.

Direct comparisons of bisulfite pyrosequencing versus targeted bisulfite sequencing.

DNA methylation is an important epigenetic mechanism involved in proper genome function. Bisulfite pyrosequencing (PSQ) is a commonly used technique to quantify DNA methylation. Although very accurate, bisulfite pyrosequencing can be expensive and time consuming for large-scale quantitative DNA methylation analysis at the single nucleotide level. High throughput DNA methylation sequencing has the potential to address these limitations, but its comparability to other methylation detection methods has not been well studied. We compared QIAseq Targeted Methyl Panel technologies (QMS) and PSQ by analyzing four CpG sites within four genes involved in neurodevelopment. QMS and PSQ had an average 5.6% difference in the detected level of DNA methylation for the same four CpG sites. However, we observed a strong correlation in the levels of methylation across all four CpG sites between the two technologies. These findings demonstrate the comparability of QMS relative to PSQ in the ability to accurately quantify DNA methylation at specific CpG sites.

Refraining from use diminishes cannabis-associated epigenetic changes in human sperm.

Cannabis use alters sperm DNA methylation, but the potential reversibility of these changes is unknown. Semen samples from cannabis users and non-user controls were collected at baseline and again following a 77-day period of cannabis abstinence (one spermatogenic cycle). Users and controls did not significantly differ by demographics or semen analyses. Whole-genome bisulfite sequencing identified 163 CpG sites with significantly different DNA methylation in sperm between groups (P < 2.94 × 10-9). Genes associated with altered CpG sites were enriched with those involved in development, including cardiogenesis and neurodevelopment. Many of the differences in sperm DNA methylation between groups were diminished after cannabis abstinence. These results indicate that sustained cannabis abstinence significantly reduces the number of sperm showing cannabis-associated alterations at genes important for early development.