Jeff Yoder

Per- and polyfluoroalkyl substances (PFAS) are emerging as a major public health problem in North Carolina and across the United States. PFAS comprise a class of over 5,000 compounds. Their unique chemical properties have been harnessed to make consumer and industrial products more water, stain, and grease resistant; they are found in products as diverse as cosmetics and flame-retardants. PFAS are resistant to degradation, move easily through the environment, and accumulate in living organisms. Exposure to PFAS has been associated with health effects including cancer and toxicity to the liver, reproductive development, and thyroid and immune systems. Despite widespread detection in the environment and evidence of increasing human exposure, understanding about PFAS toxicity, its bioaccumulative potential in dietary sources such as aquatic organisms, and effective remediation remain notably understudied. The recent discovery by this proposed Center������������������s Deputy Director, Dr. Detlef Knappe, of widespread PFAS contamination in the Cape Fear River watershed in NC underscores that these compounds are in need of immediate investigation.. The goal of our Center is to advance understanding about the environmental and health impacts of PFAS. To meet this goal we are employing a highly trans-disciplinary approach that will integrate leaders in diverse fields (epidemiology, environmental science and engineering, biology, toxicology, immunology, data science, and advanced analytics); all levels of biological organization (biomolecule, pathway, cell, tissue, organ, model organism, human, and human population); state-of-the-art analytical technologies; cutting-edge data science approaches; a recognized track record in interdisciplinary, environmental health science (EHS) training; and well-established partnerships with government and community stakeholders.

Per- and polyfluoroalkyl substances (PFASs) can be detected in the serum of 98% of Americans and have been linked to several severe health effects, including cancer and inflammatory diseases. Many of these health effects are linked to dysregulation of innate immune function, but how PFASs impact innate immunology is widely unknown. Preliminary data show that PFAS exposure suppresses innate immune function in vitro and in vivo. Further, PFAS exposure is associated with thyroid disease, and normal thyroid hormone levels are essential for innate immune function. Therefore, the central hypothesis is that PFAS-induced immunotoxicity is mediated by decreased thyroid hormone signaling. This hypothesis will be tested for multiple structurally diverse PFASs in both human innate immune cell lines and a hypothyroid zebrafish model. Aim 1 will determine if PFAS-induced suppression of natural killer (NK) cell function requires thyroid hormone signaling in vitro. Aim 2 will determine if PFAS-induced suppression of phagocyte cell function requires thyroid hormone signaling in vivo and in vitro. This work will begin to fill the knowledge gap surrounding PFAS-induced toxicity, specifically as it relates to their immunotoxicity and their potential mechanisms of action. Successful completion of this project will inform hazard identification and risk assessment processes and may lead to improved regulation of PFASs in the environment.

The College of Veterinary Medicine (CVM) at North Carolina State University (NCSU) is requesting funds to acquire a Multi Camera Array Microscope (MCAM) Kestrel from Ramona Optics Inc. (Durham, NC) for image capture and analysis of high throughput chemical and genetic screens. The MCAM Kestrel can acquire synchronous high-speed video (>160 frames-per-second) over an entire multi-well plate, and also screen an entire multi-well plate within seconds in fluorescence or brightfield - a 5X increase in temporal resolution and 24X increase in throughput compared with competing systems. The MCAM will be used to investigate the impact of chemical exposure or genetic variability on an observable phenotype with multiple variables being studied in parallel (e.g. high throughput). Such studies are fundamental to the research of a considerable number of investigators at NCSU, but there is currently no equipment on campus that offers affordable access to a microscopy system that can perform unbiased high-throughput screening and analysis over the area of an entire well-plate. The acquisition of the MCAM Kestrel will hence meet a significant demand from eight Major Users from multiple departments and colleges whose research spans a broad variety of fields including developmental biology, toxicology, immunology, neurobiology, textiles and high throughput screening. In the same vein, implementing easy and affordable access to the MCAM���s novel capabilities on our campus will authorize and stimulate the development of a broad variety of innovative research projects that have been hampered until now. While a significant percentage of the eight Major Users and four Additional Users are already federally funded investigators (NIH, NSF, DoD), the acquisition of the MCAM will undoubtedly increase competitiveness for additional federal funding. The high level of qualification of the persons involved, the space availability, the cutting edge technology of the MCAM, and the strong support from our institution further guarantee the success of the MCAM implementation, which will undoubtedly have positive and measurable repercussions for the development of innovative research at NCSU.

Current microscopes cannot form images with cellular-scale resolution over an area larger than a few square centimeters, which fundamentally limits our ability to monitor the detailed movements of living systems. Ramona Optics Inc. has recently developed a new micro-camera array microscope (MCAM) that overcomes these limitations to offer cellular-level detail over an 8x12 cm area, matching the size of a full 96 well-plate. This allows scientists, for the first time, to image nearly 100 individually moving organisms in parallelized assay experiments. The large datasets that the MCAM produces, however, remain time consuming and challenging for researchers to efficiently annotate and analyze. In this Phase IIB effort, we will develop automated software to eliminate the need for tedious hand-annotation of the MCAM������������������s image data. This new software will rapidly detect, identify and quantify key biological metrics such as body length, curvature, and eye size across many individual zebrafish larvae simultaneously, enabling scientists to draw insights about their work in less time and with higher confidence.

Per- and polyfluoroalkyl substances (PFASs) are used to produce nonstick coatings, food wrappers, and fire-fighting foams. PFASs are environmentally persistent, ubiquitous and can be detected in the serum of 99% of Americans. Despite well-established immunotoxicity, few studies have investigated the effects of PFASs on the innate immune system. We propose combining primary human neutrophils and zebrafish larvae with innovative functional assays to investigate the impact of PFASs on neutrophil function and on the ability to recover from a bacterial infection. Our preliminary data reveal that two PFASs, PFHxA and GenX, suppress the ability of a neutrophil-like cell line to generate reactive oxygen species (ROS). Generation of ROS, along with phagocytosis, are hallmark features of activated neutrophils and play important roles during infection. We have also observed that these PFAS suppress the ability of zebrafish larvae to generate ROS, highlighting the potential of these compounds to negatively impact an organism��������s ability to recover from infections. In this proposal, primary human neutrophils will be exposed to PFHxA or GenX and their ability to generate ROS and undergo phagocytosis will be quantified. In addition, zebrafish larvae will be exposed to PFHxA or GenX, infected with a bacterial pathogen, and the bacterial burden quantified. We anticipate that these PFASs will suppress ROS generation and phagocytosis in primary neutrophils, and increase the bacterial burden in infected zebrafish larvae. Successful completion of this project will lay the groundwork for future research on deciphering the cellular and molecular mechanisms by which these PFASs suppress immune function.

The use of molecular markers of self-identity as a basis for immunity marks a major evolutionary innovation in the early history of vertebrates. It is well established that self versus non-self recognition has spurred a co-evolutionary competition between vertebrate hosts and pathogens driving both high levels of inter- and intra-specific immune gene sequence diversity. Although immune gene diversification is likely essential for a species to survive new pathogens, the origin and evolutionary dynamics of vertebrate self versus non-self recognition remain poorly understood. As a group, ray-finned fish (Actinopterygii) constitute over half of the extant vertebrates on earth and display greater species diversity than any other group of vertebrates making them a powerful system for understanding the genetic and functional evolution of immune genes. Fish not only share certain immune gene families with mammals, but also encode a number of "fish-specific" immune gene families. This project will integrate new transcriptome and genomic sequence data from multiple early diverging lineages of ray-finned fishes with established sequence data from other fishes and a phylogenetic comparative framework to 1) establish the evolutionary origins of fish-specific immune receptors, 2) determine if genomic organization influences rates of immune gene family evolution and 3) define co-evolutionary relationships between markers of "self" and their candidate receptors. This study will not only provide a perspective on the early history of the vertebrate immune system, but will also reveal novel molecular innovations to pathogen resistance in vertebrates.

The Foreign Animal Disease Diagnostic Laboratory (FADDL) is a national reference laboratory for USDA Veterinary Services and the National Animal Health Laboratory Network (NAHLN), and an international reference laboratory for the Food and Agriculture Organization (FAO) of the United Nations and the World Organization for Animal Health (OIE). FADDL is currently located at the Plum Island Animal Disease Center (PIADC), the only U.S. location approved for handling high-consequence foreign animal diseases* (FAD), including foot and mouth disease (FMD) and Rinderpest viruses. The majority of the U.S. FAD subject matter diagnostic expertise (SME) for livestock diseases resides at PIADC-FADDL, within approximately 20 scientists that include microbiologists, veterinarians, and veterinary scientists (DVM/PhD). Based on anecdotal data from discussions with these FADDL employees, it is likely that most (>80%) of FADDL������������������s subject matter experts (SMEs) will not relocate to the new National Bio and Agro-Defense Facility (NBAF) in Kansas, creating an FAD SME gap throughout the transition process and during stand-up of FADDL at NBAF. This gap is compounded by the fact that few new veterinary school graduates are pursuing careers in FADs or other high-consequence animal diseases. According to 2017 market research statistics on the employment of U.S. veterinarians, less than 0.016% of all positions are with the federal government (AVMA). Within that group, even fewer veterinarians work with livestock and FADs. The trending emphasis of new veterinarians exclusively pursuing small animal private practice is expected to continue. In addition to the need to recruit FAD SMEs, the FADDL mission will expand at NBAF to include zoonotic and emerging diseases, with a new emphasis on biosafety level (BSL)-4 pathogens. SMEs knowledgeable in these agents and with expertise in working in BSL-4 laboratories are critical to develop BSL-4 programs at NBAF. APHIS has developed a graduate training program, APHIS NBAF Scientist Training Program (NSTP), to minimize the SME gap and identify highly qualified candidates to fill key roles in the new NBAF facility. NSTP fellows will receive full tuition and supplementary support to complete a MS, PhD or DVM/PhD program in target laboratory-based fields of study, including but not limited to microbiology, virology, molecular biology, diagnostics, and bioinformatics. APHIS will work with partner universities and laboratories to ensure the fellows������������������ research projects address specific FADs and capability needs. Upon successful completion of the programs, each fellow will be required to fulfill a service commitment at NBAF and/or PIADC-FADDL, dependent on agency needs and timing of degree completion. NCSU, located in Raleigh, NC, has a close association and long-term collaborations within USDA APHIS. NCSU faculty have significant subject matter expertise in infectious diseases, diagnostics and animal health.

The current view of cell extravasation, diapedesis, suffers significant limitation, and cannot explain how infused large multicellular spheres are effectively translocated without vascular damage. We have discovered a novel mechanism of cell extravasation we named as angiopellosis, whereby infused cells are expelled into the surrounding parenchyma via extensive microvascular remodeling. The goal of this grant study is to understand the cellular and molecular basis of angiopellosis and translate the findings into development of new strategies to boost therapeutic cell transplantation or, conversely, mitigate tumor cell metastasis.

Exposure to polycyclic aromatic hydrocarbons (PAHs), which are components of fossil fuels and by-products of combustion, can lead to serious health concerns including immune-suppression, birth defects, cancer and death. PAHs are present as mixtures in the environment and the EPA lists 16 PAHs as priority pollutants. However, most research has focused on a single PAH, benzo(a)pyrene, and little work has investigated the toxic effects of other, more abundant PAHs, such as pyrene. Although multiple PAHs are known to be immune suppressive, little research has been reported on the cellular and molecular basis of PAH-mediated immunotoxicity. Surprisingly, preliminary data indicate that exposure to pyrene concentrations that do not induce morphological defects and are environmentally relevant, is highly immune suppressive in zebrafish embryos, more so than three other PAHs including the well-studied benzo(a)pyrene. Remarkably, we find that pyrene exposure increases transcript levels of glucose metabolism-related genes in both zebrafish and human neutrophils. Based on these observations, we hypothesize that pyrene exposure alters the transcriptome causing a metabolic change that leads to immune suppression. In order to collect additional data to support this hypothesis, this project will i) quantify the impact of pyrene exposure on immune function, ii) define the molecular signaling pathways induced by pyrene exposure and iii) determine if pyrene exposure alters organismal and neutrophil metabolism. Successful completion of the proposed research will provide preliminary data essential for a NIH/NIEHS R01 proposal focusing on the links between pyrene exposure, immune function, glucose metabolism and disease.

In this analysis, Dr. Yoder's laboratory will treat zebrafish embryos with control and test compound at the 3-day and earlier embryonic stage of development. These vertebrates will then be assessed for normal fin and skeletal development. As a preliminary experiment, the effect of lower doses of control and active compounds will be determined in human xenografts of the fli1:GFP zebrafish line to detect changes in vasculature as proof-of-concept for future collaborative funding. Establish assays necessary to quantify toxicological effects of compounds in zebrafish including dose and kinetic studies along with quantification of abnormalities. Drugs will be provided by Moffittt Cancer Center.