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.

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.

Cardiac physiology and metabolic gene expression during late organogenesis among F. heteroclitus embryo families from crosses between pollution-sensitive and -resistant parents.

BACKGROUND: The teleost fish Fundulus heteroclitus inhabit estuaries heavily polluted with persistent and bioaccumulative chemicals. While embryos of parents from polluted sites are remarkably resistant to toxic sediment and develop normally, embryos of parents from relatively clean estuaries, when treated with polluted sediment extracts, are developmentally delayed, displaying deformities characteristic of pollution-induced embryotoxicity. To gain insight into parental effects on sensitive and resistant phenotypes during late organogenesis, we established sensitive, resistant, and crossed embryo families using five female and five male parents from relatively clean and predominantly PAH-polluted estuaries each, measured heart rates, and quantified individual embryo expression of 179 metabolic genes. RESULTS: Pollution-induced embryotoxicity manifested as morphological deformities, significant developmental delays, and altered cardiac physiology was evident among sensitive embryos resulting from crosses between females and males from relatively clean estuaries. Significantly different heart rates among several geographically unrelated populations of sensitive, resistant, and crossed embryo families during late organogenesis and pre-hatching suggest site-specific adaptive cardiac physiology phenotypes relative to pollution exposure. Metabolic gene expression patterns (32 genes, 17.9%, at p < 0.05; 11 genes, 6.1%, at p < 0.01) among the embryo families indicate maternal pollutant deposition in the eggs and parental effects on gene expression and metabolic alterations. CONCLUSION: Heart rate differences among sensitive, resistant, and crossed embryos is a reliable phenotype for further explorations of adaptive mechanisms. While metabolic gene expression patterns among embryo families are suggestive of parental effects on several differentially expressed genes, a definitive adaptive signature and metabolic cost of resistant phenotypes is unclear and shows unexpected sensitive-resistant crossed embryo expression profiles. Our study highlights physiological and metabolic gene expression differences during a critical embryonic stage among pollution sensitive, resistant, and crossed embryo families, which may contribute to underlying resistance mechanisms observed in natural F. heteroclitus populations living in heavily contaminated estuaries.

Evolution of "On-Barcode" Luminescence Oxygen Channeling Immunoassay by Exploring the Barcode Structure and the Assay System.

The multiplexed luminescence oxygen channeling immunoassay (multi-LOCI) platform we developed recently that combines conventional LOCI and suspension array technology is capable of realizing facile "mix-and-measure" multiplexed assays without tedious washing steps. However, previous work lacks comprehensive studies of the structure-performance relationship of the host-guest-structured barcode, which may obstruct the evolution and further translation of this exciting new technology to practical applications. Accordingly, this work revealed that polyelectrolyte interlayers played a crucial role in tuning the packing density of guest acceptor beads (ABs). More interestingly, we noticed that "sparse" barcodes (barcodes with low ABs packing density) exhibited comparable assay performance with "compact" ones (barcodes with high ABs packing density). The high robustness of barcodes allows for multi-LOCI to be a more universal and flexible assay platform. Furthermore, through optimization of the assay system including the laser power, as well as the concentrations of donor beads and biotinylated detection antibodies, the multi-LOCI platform showed a significant improvement in sensitivity compared with our previous work, with the limit of detection decreasing to as low as ca. 1 pg/mL. Impressively, multi-LOCI that enabled simultaneous detection of multiple analytes exhibited comparable sensitivity with the classical single-plexed LOCI, due to the ingenious structural design of the multi-LOCI barcode and the unique "on-barcode" assay format.

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.

Reevaluate how to evaluate: Systemic assessment biases affect students' confidence in college upper-division biology laboratory courses.

Grades influence students' confidence and decisions to complete STEM degrees and pursue relevant careers. What affects students' confidence and performance in college upper-division biology laboratory courses and how relevant are evaluation methods to career success? STEM laboratory courses are an excellent model to address these issues because of the hybrid environment, combining traditional lecture course format and the practical application of knowledge. We surveyed 567 students in two upper-division laboratory molecular biology courses at a major research university to compare course-content self-assessment, students' predicted grades, and actual grades received. By analyzing students' confidence and correlating them to grades assigned by the instructor, we identified biases including student and Instructor Assistant (IA) gender, IA experience, and academic quarter. Considering the systemic effect of identified biases, a correlation (R2  = 0.37, p < 0.01) between predicted and actual grades, and weak but statistically significant correlation (R2  = 0.10, p < 0.01) between students' comprehensive course-content self-assessment and their predicted grade are not surprising. Our analysis suggests that students' quantifiable self-assessment, a relatively simple and data-rich resource, helps identify evaluation bias. If administered periodically throughout the course, these assessments can help mitigate biases, improve student learning, evaluation, and retention in STEM fields.

PAH-pollution effects on sensitive and resistant embryos: Integrating structure and function with gene expression.

Polycyclic aromatic hydrocarbons (PAHs) are among the most widespread natural and anthropogenic pollutants, and some PAHs are proven developmental toxicants. We chemically characterized clean and heavily polluted sites and exposed fish embryos to PAH polluted sediment extracts during four critical developmental stages. Embryos were collected from Fundulus heteroclitus populations inhabiting the clean and heavily polluted Superfund estuary. Embryos of parents from the clean sites are sensitive to PAH pollutants while those of parents from the heavily polluted site are resistant. Chemical analysis of embryos suggests PAH accumulation and pollution-induced toxicity among sensitive embryos during development that ultimately kills all sensitive embryos before hatching, while remarkably, the resistant embryos develop normally. The adverse effects on sensitive embryos are manifested as developmental delays, reduced heart rates, and severe heart, liver, and kidney morphological abnormalities. Gene expression analysis of early somitogenesis, heartbeat initiation, late organogenesis, and pre-hatching developmental stages reveals genes whose expression significantly differs between sensitive and resistant embryo populations and helps to explain mechanisms of sensitivity and resistance to polluted environments during vertebrate animal development.

Relative genetic diversity of the rare and endangered Agave shawii ssp. shawii and associated soil microbes within a southern California ecological preserve.

Shaw's Agave (Agave shawii ssp. shawii) is an endangered maritime succulent growing along the coast of California and northern Baja California. The population inhabiting Point Loma Peninsula has a complicated history of transplantation without documentation. The low effective population size in California prompted agave transplanting from the U.S. Naval Base site (NB) to Cabrillo National Monument (CNM). Since 2008, there are no agave sprouts identified on the CNM site, and concerns have been raised about the genetic diversity of this population. We sequenced two barcoding loci, rbcL and matK, of 27 individual plants from 5 geographically distinct populations, including 12 individuals from California (NB and CNM). Phylogenetic analysis revealed the three US and two Mexican agave populations are closely related and have similar genetic variation at the two barcoding regions, suggesting the Point Loma agave population is not clonal. Agave-associated soil microbes used significantly more carbon sources in CNM soil samples than in NB soil likely due to higher pH and moisture content; meanwhile, soil type and soil chemistry analysis including phosphorus, nitrate nitrogen, organic matter, and metals revealed significant correlations between microbial diversity and base saturation (p < 0.05, r 2 = 0.3676), lime buffer capacity (p < 0.01, r 2 = 0.7055), equilibrium lime buffer capacity (p < 0.01, r 2 = 0.7142), and zinc (p < 0.01, r 2 = 0.7136). Soil microbiome analysis within the CNM population revealed overall expected richness (H' = 5.647-6.982) for Agave species, while the diversity range (1 - D = 0.003392-0.014108) suggests relatively low diversity marked by high individual variation. The most prominent remaining US population of this rare species is not clonal and does not seem to be threatened by a lack of genetic and microbial diversity. These results prompt further efforts to investigate factors affecting Agave's reproduction and fitness.

Biological and clinical significance of flap endonuclease­1 in triple­negative breast cancer: Support of metastasis and a poor prognosis.

Flap endonuclease­1 (FEN1), a structure­specific nuclease participating in DNA replication and repair processes, has been confirmed to promote the proliferation and drug resistance of tumor cells. However, the biological functions of FEN1 in cancer cell migration and invasion have not been defined. In the present study, using online database analysis and immunohistochemistry of the specimens, it was found that FEN1 expression was associated with a highly invasive triple­negative breast cancer (TNBC) subtype in both breast cancer samples from the Oncomine database and from patients recruited into the study. Furthermore, FEN1 was an important biomarker of lymph node metastasis and poor prognosis in patients with TNBC. FEN1 promoted migration of TNBC cell lines and FEN1 knockdown reduced the number of spontaneous lung metastasis in vivo. Ingenuity Pathway Analysis of FEN1­related transcripts in 198 patients with TNBC demonstrated that the polo­like kinase family may be the downstream target of FEN1. PLK4 was further identified as a critical target of FEN1 mediating TNBC cell migration, by regulating actin cytoskeleton rearrangement. The results of the present study validate FEN1 as a therapeutic target in patients with TNBC and revealed a new role for FEN1 in regulating TNBC invasion and metastasis.