The gut microbiota, or the microorganisms that live in the gastrointestinal tract (GI), play a significant role in both gut and overall health. In LMICs (Low-Middle Income Countries), diarrheal diseases are among the leading causes of death in children under five. The ongoing EcoMiD (Enteropatógenos, Crecimiento, Microbioma, y Diarrea) project aims to study the interaction between infants' gut microbiomes and viral, bacterial, and parasitic enteric pathogens along a rural-urban gradient in Ecuador through a cohort study of 600 mother-child dyads. The objective of this portion of the study was to validate a method for assaying multiple enteric pathogens in stool samples simultaneously using TaqMan Array Cards (TAC). Before samples can be analyzed using TAC, each of the 56 pathogenic targets of interest must be validated to determine the limit of detection, limit of quantification, inhibition effect of the stool matrix on detection, repeatability and reproducibility. The TAC Positive Control validation process initially involves growing up each of the organisms of interest, extracting its' total nucleic acids (TNA) and quantifying this through (RT)-qPCR and Qubit. Analysis of this data enables seeding the positive controls at known concentrations to quantify target TNA for TAC. Of the 56 targets, we have completed this work with 35 targets (26 reference organisms, 9 gBlocks). For targets that are time/cost intensive to work with or not culturable, gBlocks are utilized in place of reference organisms. gBlocks are synthetic gene fragments with a known sequence. Next steps of this process includes validation of the 21 remaining pathogens, some of which involves working with anaerobic organisms. Analysis of this data will inform seeding concentrations to validate the developed TAC and enable quantification of these targets in stool samples from Ecuador. 

Life depends on a series of well-orchestrated biochemical reactions facilitated by proteins, which are differentially transcribed and activated in response to changing conditions. Hydrogels, water-swollen polymeric biomaterials, have proven useful as synthetic platforms to probe and direct biological activities by enabling researchers to recapitulate many aspects of the native cell environment. Though current hydrogel protein patterning techniques are capable of driving specific cell fates in individual cells in time and space (i.e. 4D), the timescales for patterning place dramatic limits on the types of biological functions that can be controlled. Furthermore, current techniques rely on slowly diffusing bioactive proteins into materials prior to immobilization within gels, so complete temporal control of protein activation within hydrogels remains out of reach. To address these limitations, my project focuses on directly photoactivating proteins within hydrogels using cytocompatible light. We predict that the extent of protein activation can be controlled dose-dependently by varying light exposure duration and intensity. We intend to use this platform to direct stem cell migration, differentiation, and proliferation in 4D on physiologically relevant timescales, which has tremendous utility in stem cell biology and regenerative medicine.

Cells in the body grow inside the extracellular matrix (ECM), which is composed of a combination of carbohydrates and proteins, presenting chemical and mechanical cues to the cells inside. Nearly all cell types are sensitive to the mechanics of the ECM and respond to cues such as stress, strain, and curvature, which influence organism development and disease progression. Hydrogel biomaterials are water-swollen polymer networks that mimic the properties of the ECM in vitro, allowing researchers to study cellular behavior in a controlled environment. In this project, we aim to develop a hydrogel platform where strain on the material, generated by contractile cells embedded within it, can be activated externally by a researcher in order to induce curvature in an engineered tissue, which we will use to investigate the effects of mechanical cues on cells encapsulated inside the hydrogel. We have synthesized a peptide crosslinker that acts as a two-input Boolean AND gate, with one half degradable by cell-secreted enzymes and the other half degradable by sortase, a researcher-added enzyme. We predict that when a cell-adhesive hydrogel is made with this crosslinker, contractile cells will be unable to expand until the addition of sortase; after sortase degrades one arm of the cyclic AND-type crosslinker, they will be able to locally degrade the hydrogel, spread within the gel, and then contract to generate stress and strain. We intend to encapsulate immature cardiac stem cells partway through differentiation, predicting that curvature alone will trigger further specification of these cells into their mature subtypes. Understanding the mechanism by which mechanical cues affect development will help identify new therapeutic targets for diseases where tissue curvature is important, and it will also inform new stimuli to improve the similarity of tissue grown in vitro to native tissue.

Individuals experiencing homelessness and housing instability in King County have inadequate access to water, sanitation, and hygiene (WASH) services. This urgent matter of community health has only been exacerbated by the coronavirus disease (COVID-19) pandemic in recent years and can necessitate open defecation or unsafe disposal of wastewater. Particularly among those residing in recreational vehicles (RVs) and use their bathroom facilities, unsafe sanitation practices increase the risks of intestinal pathogen transmission and infection in densely populated communities. In a continuation of efforts to address this, I have developed a study that aims to identify an optimal method of RV wastewater concentration for downstream detection of pathogens. With informed consent from RV residents and sampling assistance from Seattle Public Utilities, I have began collecting a representative collection of samples of RV wastewater from multiple neighborhoods across Seattle. I have utilized a split-and-seed approach in which half the samples were seeded with known amounts of target bacterial, viral, and spore-forming organisms, and the other half left unseeded as a control. These samples will be used to compare two methods of sample concentration, skimmed milk flocculation and membrane filtration, using a weighted rubric that evaluates biosafety, seeded organism recovery efficiency, personnel time, and cost. Upon observation in the lab, I have found that skimmed milk flocculation led to inhibition during detection via molecular assays. Additionally, high turbidity of the samples yielded significant logistical challenges with processing skimmed milk samples, leading my team and I to instead favor the membrane filtration technique. As we move forward, I anticipate detecting elevated levels of pathogens in wastewater from RVs when compared to samples from control locations chosen to represent populations living in sewered and unsewered environments representing populations. Upon development of the optimized protocol, my work will be prepared for publication in a peer-reviewed journal and shared with the City of Seattle.