Locomotion and brain gene expression exhibit sex-specific non-monotonic dose-response to HFPO-DA during Drosophila melanogaster lifespan.

BACKGROUND: Legacy per- and polyfluoroalkyl substances (PFAS), known for their environmental persistence and bio-accumulative properties, have been phased out in the U.S. due to public health concerns. A newer polymerization aid used in the manufacture of some fluoropolymers, hexafluoropropylene oxide-dimer acid (HFPO-DA), has lower reported bioaccumulation and toxicity, but is a potential neurotoxicant implicated in dopaminergic neurodegeneration. OBJECTIVE: We investigated HFPO-DA's bio-accumulative potential and sex-specific effects on lifespan, locomotion, and brain gene expression in fruit flies. METHODS: We quantified bioaccumulation of HFPO-DA in fruit flies exposed to 8.7 × 104 µg/L of HFPO-DA in the fly media for 14 days via UHPLC-MS. Long-term effect on lifespan was determined by exposing both sexes to 8.7 × 102 - 8.7 × 105 µg/L of HFPO-DA in media. Locomotion was measured following 3, 7, and 14 days of exposures at 8.7 × 101 - 8.7 × 105 µg/L of HFPO-DA in media, and high-throughput 3'-end RNA-sequencing was used to quantify gene expression in fly brains across the same time points. RESULTS: Bioaccumulation of HFPO-DA in fruit flies was not detected. HFPO-DA-induced effects on lifespan, locomotion, and brain gene expression, and lowest adverse effect level (LOAEL) showed sexually dimorphic patterns. Locomotion scores significantly decreased in at least one dose at all time points for females and only at 3-day exposure for males, while brain gene expression exhibited non-monotonic dose-response. Differentially expressed genes correlated to locomotion scores revealed sex-specific numbers of positively and negatively correlated genes per functional category. CONCLUSION: Although HFPO-DA effects on locomotion and survival were significant at doses higher than the US EPA reference dose, the brain transcriptomic profiling reveals sex-specific changes and neurological molecular targets; gene enrichments highlight disproportionately affected categories, including immune response: female-specific co-upregulation suggests potential neuroinflammation. Consistent sex-specific exposure effects necessitate blocking for sex in experimental design during HFPO-DA risk assessment.

Life Science Research Immersion Program Improves STEM-Specific Skills and Science Attitudes among Precollege Students.

A predicted rapid growth in science, technology, engineering, and math (STEM) careers demands a vast and talented workforce, but students most commonly abandon STEM majors within the first 2 years of college. Performance in introductory courses, scientific literacy, and the ability to critically reason are main predictors of retention in STEM, highlighting the importance of precollege and early college experience. The Life Science Research Immersion Program (LSRIP) is a novel science education model that focuses on the development of scientific research skills, thus preparing students for introductory college courses and beyond. To evaluate the efficacy of the LSRIP, pre- and postprogram assessments and surveys were administered to three precollege student cohorts. Scientific reasoning assessment scores improved by 4.70% in Summer 2019 (P < 0.01), 9.44% in Fall 2019 (P < 0.05), and 0.97% in Winter 2020 cohorts, with two of five questions showing statistically significant improvement. Surveyed attitudes toward science improved in 62.9% of questions across all cohorts. These results suggest that research immersion experiences are an effective educational instrument for improving and promoting scientific reasoning and attitudes among precollege students. To better prepare students for success in STEM higher education and careers, we recommend implementing LSRIPs to complement traditional precollege science curricula.

Imagining, designing, and interpreting experiments: Using quantitative assessment to improve instruction in scientific reasoning.

Effectively teaching scientific reasoning requires an understanding of the challenges students face when learning these skills. We designed an assessment that measures undergraduate student abilities to form hypotheses, design experiments, and interpret data from experiments in cellular and molecular biology. The assessment uses intermediate-constraint free-response questions with a defined rubric to facilitate use with large classes, while identifying common reasoning errors that may prevent students from becoming proficient at designing and interpreting experiments. The assessment measured a statistically significant improvement in a senior-level biochemistry laboratory course, and a larger improvement between the biochemistry lab students and a separate cohort in a first-year introductory biology lab course. Two common errors were identified for forming hypotheses and using experimental controls. Students frequently constructed a hypothesis that was a restatement of the observation it was supposed to explain. They also often made comparisons to control conditions not included in an experiment. Both errors were most frequent among first-year students, and decreased in frequency as students completed the senior-level biochemistry lab. Further investigation of the absent controls error indicated that difficulties with reasoning about experimental controls may be widespread in undergraduate students. The assessment was a useful instrument for measuring improvement in scientific reasoning at different levels of instruction, and identified errors that can be targeted to improve instruction in the process of science.

Toxicity assessment of hexafluoropropylene oxide-dimer acid on morphology, heart physiology, and gene expression during zebrafish (Danio rerio) development.

Hexafluoropropylene oxide-dimer acid (HFPO-DA) is one of the emerging replacements for the "forever" carcinogenic and toxic long-chain PFAS. HFPO-DA is a polymerization aid used for manufacturing fluoropolymers, whose global distribution and undetermined toxic properties are a concern regarding human and ecological health. To assess embryotoxic potential, zebrafish embryos were exposed to HFPO-DA at concentrations of 0.5-20,000 mg/L at 24-, 48-, and 72-h post-fertilization (hpf). Heart rate increased significantly in embryos exposed to 2 mg/L and 10 mg/L HFPO-DA across all time points. Spinal deformities and edema phenotypes were evident among embryos exposed to 1000-16,000 mg/L HFPO-DA at 72 hpf. A median lethal concentration (LC50) was derived as 7651 mg/L at 72 hpf. Shallow RNA sequencing analysis of 9465 transcripts identified 38 consistently differentially expressed genes at 0.5 mg/L, 1 mg/L, 2 mg/L, and 10 mg/L HFPO-DA exposures. Notably, seven downregulated genes were associated with visual response, and seven upregulated genes were expressed in or regulated the cardiovascular system. This study identifies biological targets and molecular pathways affected during animal development by an emerging, potentially problematic, and ubiquitous industrial chemical.

Physiological and transcriptomic effects of hexafluoropropylene oxide dimer acid in Caenorhabditis elegans during development.

Per- and polyfluoroalkyl substances (PFAS) are chemicals resistant to degradation. While such a feature is desirable in consumer and industrial products, some PFAS, including perfluorooctanoic acid (PFOA), are toxic and bioaccumulate. Hexafluoropropylene oxide dimer acid (HFPO-DA), an emerging PFAS developed to replace PFOA, has not been extensively studied. To evaluate the potential toxicity of HFPO-DA with a cost- and time-efficient approach, we exposed C. elegans larvae for 48 h to 4 × 10-9-4 g/L HFPO-DA in liquid media and measured developmental, behavioral, locomotor, and transcriptional effects at various exposure levels. Worms exposed to 1.5-4 g/L HFPO-DA were developmentally delayed, and progeny production was significantly delayed (p < 0.05) in worms exposed to 2-4 g/L HFPO-DA. Statistically significant differential gene expression was identified in all fourteen HFPO-DA exposure groups ranging from 1.25 × 10-5 to 4 g/L, except for 6.25 × 10-5 g/L. Among 10298 analyzed genes, 2624 differentially expressed genes (DEGs) were identified in the developmentally delayed 4 g/L group only, and 78 genes were differentially expressed in at least one of the thirteen groups testing 1.25 × 10-5-2 g/L HFPO-DA exposures. Genes encoding for detoxification enzymes including cytochrome P450 and UDP glucuronosyltransferases were upregulated in 0.25-4 g/L acute exposure groups. DEGs were also identified in lower exposure level groups, though they did not share biological functions except for six ribosomal protein-coding genes. While our transcriptional data is inconclusive to infer mechanisms of toxicity, the significant gene expression differences at 1.25 × 10-5 g/L, the lowest concentration tested for transcriptional changes, calls for further targeted analyses of low-dose HFPO-DA exposure effects.

U.S. national, regional, and state-specific socioeconomic factors correlate with child and adolescent ADHD diagnoses pre-COVID-19 pandemic.

Attention-deficit/hyperactivity disorder (ADHD), the most diagnosed emerging neurodevelopmental disorder in children, is a growing health crisis in the United States. Due to the potential increase in ADHD severity during and post the COVID-19 pandemic, we analyzed recent national and two state-specific ADHD data distribution among U.S. children and adolescents by investigating a broad range of socioeconomic status (SES) factors. Child and adolescent ADHD diagnosis and treatment data were parent-reported via National Survey of Children's Health (NSCH). The nationwide childhood prevalence of ADHD is 8.7%, and 62.1% of diagnosed children are taking medication. Louisiana (15.7%) has the highest percentage of children diagnosed with ADHD and California (5.6%) has the lowest, followed by Nevada (5.9%). Multiple correspondence analysis (MCA, n = 51,939) examining 30 factors highlights four areas of interest at the national and state level: race/ethnicity, financial status, family structure, and neighborhood characteristics. Positive correlations between ADHD diagnosis and unsafe school, unsafe neighborhood, and economic hardship are evident nationally and statewide, while the association between a lack of ADHD diagnosis and higher urban neighborhood amenities are evident nationally, but not in two opposing outlier states-Louisiana or Nevada. National and state-specific hierarchical analyses demonstrate significant correlations between the various SES factors and ADHD outcomes. Since the national analysis does not account for the demographic heterogeneity within regions or individual states, the U.S. should rely on comprehensive, county-specific, near real-time data reporting to effectively model and mitigate the ADHD epidemic and similar national health crises.