Arthropods represent 29 percent of the world’s animal biomass and comprise over 500 million years of evolutionary diversity. Arthropods such as spiders and moths constitute a perfect milieu for the discovery of multitudes of highly diverse RNA viruses of unknown pathogenic potential. Sequencing and characterizing these viruses is of immense value for understanding many basic science questions about viruses. The sequences obtained and described can also provide great insight into future zoonotic outbreaks. We sequenced RNA extracted from 10 moths and 6 spiders collected from western Washington. We developed a custom bioinformatics pipeline for automatic discovery and characterization of completely new viruses. The custom software also automates the process of depositing viral sequences into the National Center for Biotechnology Information’s Genbank archives, the main database in which DNA and RNA sequences are shared among scientists and researchers. A total of 12 novel viral genomes were discovered in these 16 samples. These new viruses belong to 4 highly different viral families, all of which contain species known to infect humans. We also present what is likely a completely new viral clade within Picornavirales, the viral family that contains polio and many other diseases afflicting humans. This new viral clade has a never seen before genome organization and contains a highly divergent RNA dependent RNA polymerase. New and highly divergent strains such as the ones outlined here provide extremely valuable insight into the inner workings of viral biology and genetics.

Staphylococcus aureus is responsible for large numbers of patient morbidity worldwide. However, studies of this important pathogen have suffered from the lack of a robust toolset for scalable genetic and genomic manipulation in the organism. Our goal for this study was to develop an efficient, simple, and inexpensive system for precision genome engineering in S. aureus. Our strategy employs ssDNA oligonucleotide recombineering coupled with CRISPR/Cas9-mediated counterselection to address these needs. We first identified a single-stranded recombinase protein, EF2132, derived from Enterococcus faecalis, which can successfully integrate single-stranded DNA oligonucleotides into the S. aureus genome. Our results showed that EF2132 readily mediates recombineering across many different laboratory strains and primary clinical strains, normally yielding thousands of recombinants per transformation. We proceeded to expand the generality of our genome editing system by incorporating lethal counterselection, enabling efficient recovery of recombineered isolates lacking a selectable phenotype. We constructed a temperature sensitive, two-vector system that enables conditional recombineering and CRISPR/Cas9- mediated counterselection in S. aureus without permanently introducing exogenous genetic material or unintended genetic lesions. Our findings indicate that our system effectively targets and edits genes with both specific mutations and large-scale knockouts by utilizing inexpensive, commercially synthesized oligonucleotides as substrates for inducing and counterselecting precise genomic changes. Since our system utilizes cheap synthetic DNA oligonucleotides as substrates for recombineering and counterselection, it provides a scalable, precise and inexpensive tool for producing isogenic strain in S.aureus. Because of this, our system can be used to effectively facilitate future studies to investigate the genetic mechanisms of many different biological phenomena. As our study progresses, we hope to introduce refinements to further improve the scalability and efficiency of this methodology.

Concomitant conditions in pediatric patients have caused discordances between the activated partial thromboplastin time (APTT) and the anti-Xa assay, complicating the management of heparin therapy. This raises concerns in administering heparin for the desired therapeutic effect. We tested the protamine assay to determine baseline protamine partial thromboplastin time (PrPTT) and the new plasma hemoglobin and total bilirubin assays to correct the false measurements. In doing this, we found a notable decrease in discordance in APTT and anti-Xa values. These findings are potentially significant in that it could alter therapeutic decisions in the patient.

Extracorporeal Membrane Oxygenation (ECMO) is a complex technology used to support patients with severe respiratory and/or cardiac failures. Because blood contacts with foreign non-biologic surfaces that result in thrombus formation within the extracorporeal circuit, the management of patients placed on ECMO requires monitoring anticoagulation – heparin (UFH), which is used to balance between clot formation and bleeding difficulties. Though Activated Clotting Time (ACT), which is a test used to monitor high doses of unfractionated heparin therapy, is the preferred method of monitoring, it is imprecise and insensitive to lower dose (0.2-0.4 aXa U/mL) of heparin. The goal of this study was to evaluate the effects of other factors than heparin – hematocrit, coagulation factor levels, platelet count, contact activators, and antithrombin – on ACT and raise awareness of how the use of ACT in monitoring low dose UFH under ECMO support was controversial. Results of experiments showed when only antithrombin levels or heparin concentration increased, ACT values were prolonged but only for heparin concentration > 0.5 U/mL. Decrease in single factor change of coagulation factors, Factor XII, and platelet count led to rise in ACT results. However, no correlation was observed between ACT values and hematocrit changes (r² = 0.03). When different levels of all factors of interest and heparin were mixed together randomly, heparin concentration and ACT values didn’t show any correlation (r² = 0.00), suggesting the interference of other factors on its results. However, aXa heparin activity assay demonstrated a strong correlation (r² = 0.99) with heparin concentration even in multiple random factors change. Thus, further research should be performed to evaluate impacts of other factors in clinical relevance and aid in a change to the preferred method of monitoring low dose UFH under ECMO support.

Zika is a virus in the Flaviviridae family, along with other established viruses such as Dengue virus and West Nile virus. These viruses have been shown to infect humans, often with dire consequences. Zika, specifically, has been linked to fetal microcephaly as a consequence of infection during pregnancy. While the mechanism Flaviviruses employ upon infecting cells has been researched, many details of this process remain unknown, especially for Zika. Flaviviruses cause endoplasmic reticulum stress in cells, which in turn elicits the cellular unfolded protein response. This is a common cellular response to Flavivirus infection and provided the basis for the experiments conducted in this research. One of the components of the unfolded protein response is the activation of IRE1, which is an enzyme in the membrane of the endoplasmic reticulum that is activated by endoplasmic reticulum stress. Activated IRE1 functions as a nuclease, which is responsible for processing the mRNA encoding a transcription factor called XBP1. IRE1 elicits a cascade which results in splicing of the mRNA encoding this transcription factor and leads to the expression of a variety of downstream gene products. The mRNA of these genes can be measured through laboratory techniques such as quantitative PCR. Our research goal was to identify whether Zika virus activates this cascade, and whether its activation aids in viral replication. Our experiments have shown that spliced XBP1 mRNA is present in increased levels in Zika virus infected cells. Our experiments have also detected the induction of XBP1 target genes, suggesting that IRE1 activation is present in cells infected with Zika virus. Additionally, we have found decreased levels of Zika virus RNA in cells which are genetic knockouts for the enzymatic activity of IRE1. These data suggest that IRE1 is a potential drug target for suppressing viral replication.

Natural products are often studied as treatments for chronic inflammation. In this study, the activity of the nuclear factor NF-κB pathway, a pro-inflammatory pathway, was analyzed after treatment with three different combination therapies between natural products Artesunate, Cantharidin, Parthenolide, and Withaferin A, which have all been separately shown to inhibit the NF- κB pathway. The combination therapies chosen to test were determined by Systems Biology, a bioinformatics system that uses a collaborative and integrative design from many scientific disciplines to predict how the cells’ biological system change over time and under varying conditions. The combination therapies of interest were: Artesunate and Cantharidin, Parthenolide and Withaferin A, and Cantharidin and Withaferin A. Exposure of Huh7, a human liver cell line, to the combination therapies of interest and TNF-α induced NF-κB activation. Western blot and luciferase reaction were then used to measure how different combination therapies changed NF-κB activation. The three combinations demonstrated different effects of NF-κB. Our findings will have important implications in understanding the human immune responses to diseases and how known treatments could be develop to be more effective.