Polychlorinated biphenyls (PCBs) are lipophilic environmental pollutants previously used in industrial and consumer products that are being found in fatty compartments of many aquatic species consumed by humans today. The gut microbiome is increasingly recognized to influence the metabolism and biotransformation of many substances including those linked to neurodevelopmental disorders (NDDs) such as PCBs. To test the hypothesis that the gut-brain axis mediates PCB neurotoxicity, we studied how maternal PCB exposure modulates gut microbiome in offspring. C57BL/6 mouse dams were randomly assigned to receive either vehicle (peanut butter and peanut oil mixture) or the Fox River PCB mixture at a daily oral dose of 0.1, 1.0, or 6.0 mg/kg body weight. To study changes in the gut microbiome, shotgun metagenomic sequencing was used to sequence the DNA of the large intestinal contents of male and female pups collected at postnatal day (PND) 28 and PND 35 (n>5 per group). The sequences were then aligned against a curated database (includes RefSeq, cell-cultured, and mouse-specific high-quality metagenomically assembled genomes) used for downstream analysis, and also the Kyoto Encyclopedia of Genes and Genomes Orthology groups (KEGG KOs) to predict functional changes of the microbiome. The analysis confirmed that maternal PCB exposure produce gut dysbiosis in the offsprings. Female pups were found to be more susceptible than males at both ages and for both sexes, PND 28 groups were more susceptible than PND 35 groups. Functional predictions of the microbiome also showed PCB-mediated increase in pathways involving ammonia production and cytochrome oxidases, but a decrease in sugar transport systems (which all may negatively influences the brain) in an age-, sex-, and PCB dose-dependent manner. In conclusion, maternal exposure to PCBs produced gut disruption in gut microbiome and may be implicated in developmental neurotoxicity through the gut-brain axis.

It is well-established that mutations have impacts on an organism’s fitness; however, the fitness effects of mutations are not static, and can vary depending on environmental contexts, such as the species in which a mutation is found. Evolution of the same gene in different species could thus lead to the evolution of different phenotypes, as different species would favour different sets of mutations. If that gene could be exchanged between species, it could lead to increased evolutionary possibilities, as high-fitness genotypes that require a prerequisite deleterious mutation in one species could become accessible if the mutation is not deleterious in another. Our research examines how the presence of two bacterial hosts, Escherichia coli and Klebsiella pneumoniae, could affect the evolution of an antibiotic resistance-conferring TEM-1 β-lactamase gene located on a conjugative plasmid. If different hosts confer different mutational effects to TEM-1, the process of horizontal gene transfer (HGT) that allows mutations to be shared between species could open up more mutational possibilities than those accessible in either single-species population alone. We tested this hypothesis through three rounds of experimental evolution in the presence of the antibiotic cefotaxime, where we evolved two single-species E. coli and K. pneumoniae populations and one multi-species population where HGT was simulated with a shared plasmid pool. We are now reconstructing the genotypes found in all three populations after each round to assess how much antibiotic resistance they confer in both species, and hope to see if the genotypes acquired under HGT treatment provide higher resistance compared to the single-species populations. Our results have practical implications for the predictability and nature of antibiotic resistance development in the real world, a current global health crisis, and potentially motivate further study in predicting resistance emergence in clinically encountered multi-species populations.

 Werner's Syndrome (WS) is a rare genetic disease that is characterized by accelerated aging and cancer susceptibility. WS is known to be caused by a mutation in the WRN gene, which encodes a helicase/exonuclease involved in DNA metabolism, including DNA repair, replication, transcription, recombination, and telomere maintenance. The WRN gene has recently been implicated in maintaining constitutive heterochromatin, and loss of heterochromatin has been observed in aging cells and is associated with a pro-inflammatory state. To better characterize WRN’s role in maintaining constitutive heterochromatin, I am conducting experiments to assess the status of pericentromeric heterochromatin in cultured human cells with and without the WRN protein. I have applied fluorescence in situ hybridization (FISH), which is used to visualize specific DNA sequences in intact cells, to detect a subset of pericentromeric heterochromatin known as SATII, and I will now perform SATII FISH to see the effect of WRN deficiency on the microscopic appearance of SATII loci. I have also conducted proximity ligation assays (PLA), which allow me to assess interactions between two specific proteins. Using PLA, I observed interactions between HP1a and KAP1, two proteins that are constituents of pericentromeric heterochromatin that help to maintain its compact state. In future experiments, I will observe the interaction of HP1a and KAP1 in cells that are not expressing WRN and determine if disruption of this interaction leads to a change in state of the SATII heterochromatin. Based on previous findings showing that WRN helps maintain constitutive heterochromatin, I am anticipating a difference in pericentromeric heterochromatin between the cells with and without WRN, with the cells not expressing WRN having distended heterochromatin as opposed to compact heterochromatin. Through these experiments, I hope to gain a better understanding of the mechanisms of WS to help in the development of potential therapies.

Joubert syndrome (JS) is a neurodevelopmental condition diagnosed by the appearance of the “molar tooth sign” on axial brain magnetic imaging (MRI). Patients display hypotonia, abnormal eye movements, and ataxia. Substantial progress has been made on identifying the genetic causes of JS, which typically displays recessive inheritance. Nonetheless, the cause cannot be identified in ~25% of our cohort of JS-affected families. The contribution of variants that impact RNA splicing remains unknown. Our goal is to evaluate the role of noncanonical splice variants in the pathogenesis of JS. Canonical splice variants impact RNA splicing by disrupting the splice site directly, whereas noncanonical splice variants may affect it through alternative mechanisms, which need to be validated by RNA analysis. We previously identified genetic causes in 520 of 679 families with JS. To identify additional causes, we used SpliceAI (SpliceAI score >0.5) to identify candidate variants that impact splicing. We extracted RNA from patient cell lines and converted it into complementary DNA (cDNA). Then we used polymerase chain reaction (PCR) to amplify the affected exons with two sets of primers flanking the relevant splice junction. We evaluated PCR product size and sequence using gel electrophoresis and Sanger sequencing. We found 74 families with ≥1 canonical splice variant. An additional 34 families have ≥1 candidate noncanonical splice variant. We confirmed the pathogenicity of two of the candidate noncanonical splice variants by demonstrating an abnormal splicing event in AHI1 and MKS1 in two patient samples. By extrapolation from our data in JS, noncanonical splice variants may contribute as much as 10% to the genetic causes of recessive conditions. A precise genetic diagnosis informs prognosis, avoids unnecessary work-up, guides monitoring for associated complications, and opens the door to gene-specific treatments.

Osteoporosis is a polygenic disease defined by low bone mineral density and is associated with increased rates for fractures and mortality. This condition commonly occurs in concert with sarcopenia, which is characterized by loss of muscle mass and function. When these occur in conjunction, a condition termed osteosarcopenia, there is an increased risk of falls which heightens the risk of fracture of already fragile osteoporotic bone. Genome wide association studies have identified genetic variants which influence osteosarcopenia-related traits. One such study identified pleiotropic effects on bone mineral density and lean mass at the CPED1/WNT16 lLocus. CPED1 has been hypothesized to be a causal gene, however there are very few studies characterizing CPED1, and it has no confirmed functions in humans or zebrafish. The goal of this study was twofold: to investigate the necessity of CPED1 for bone and lean mass in zebrafish. We analyzed a single-cell atlas of embryonic development and found that CPED1 is most strongly expressed in muscle. We generated two mutant alleles, CPED1w1003 and CPED1sa20221 via CRISPR gene editing. For analysis, 3 or 14 month old zebrafish were scanned using microCT, and ImageJ and FishCuT software were utilized to measure vertebral morphology and mineralization, lean mass, and standard length. Results showed no significant differences between mutant and control groups for both mutant alleles. The results of this study do not support CPED1 as being a causative gene underlying bone and muscle pleiotropy at the CPED1/WNT16 locus. This study also raises questions regarding the function of CPED1 in muscle and whether its loss may be compensated for by other genes.

Osteoporosis, a polygenic disease characterized by low bone mineral density (BMD) and increased fracture risk, is the most prevalent bone disease impacting over 200 million people worldwide. Because of the associated financial burdens and reductions in quality of life, there is an urgent need to determine the genetic causes of osteoporosis. The WNT family of proteins has been implicated in numerous developmental and disease pathways, with WNT16 specifically being linked to osteoporosis risk. WNT proteins contain 24 conserved cysteines, and mutations involving half of these cysteines are associated with human diseases or disrupted development in animal models. While modifications in cysteines 1-6, 8-9, 11-12, and 24 in different WNT proteins have been defined in vivo, the impact of cysteine 10 (c10) alteration remains unknown. Wnt crystal structure suggests that c10 creates a disulfide linkage with cysteine 11 and resides in a region that directly interacts with Frizzled receptors to initiate WNT signaling pathways. My hypothesis is that loss of c10 in WNT16 will result in altered BMD indicative of elevated osteoporosis risk. To test this, I outcrossed CRISPR-generated somatic zebrafish mutants harboring mutations at the wnt16 locus that target the c10 position, and isolated wnt16^w1012 mutants. Sanger sequencing and sequence alignment revealed a three amino acid deletion at Cys214, corresponding to c10 (p.Cys214_Gly216del). Analysis of micro-computed tomography scans showed significant decreases in wnt16^w1012 mutant centrum length, which matches wnt16 knockouts. My data indicates that wnt16^w1012 mutants phenocopy wnt16 knockouts, suggesting that c10 plays an essential role in WNT16 secretion and/or activity. My data further suggests that mutations that alter c10 have potential to contribute to osteoporosis pathogenesis. My ongoing studies are focused on further characterizing musculoskeletal phenotypes in wnt16^w1012 mutants and understanding the consequences of the mutation on protein structure through computational modeling.