Prior to the outbreak of SARS CoV-2, enveloped RNA viruses had been successfully detected in municipal wastewater. Since SARS CoV-2 emergence, researchers have further validated this technique. By March 2020, research suggested viral concentration via wastewater sampling was predictive, by 3-5 days of a subsequent rise in COVID-19 infections. Thus, measuring municipal wastewater quickly became a useful epidemiological tool for COVID-19 management. Despite relative validation, these data provided population level trends, leaving officials little time, or location-specificity, to make use of this warning signal. To address this problem, we partnered with the City of Tacoma Environmental Services Division and Tacoma Pierce County Department of Health (TPCDH) on behalf of RAIN, a Tacoma based biotechnology non-profit, and designed a neighborhood-level rapid response plan. By analyzing health disparities data, wastewater infrastructure and active case rates we sought to illuminate sites of active transmission. To conduct this study, we sampled weekly from two wastewater treatment plants in Tacoma, WA, and five geographically disparate neighborhood sites to monitor SARS CoV-2 levels. We collected population-specific biological samples in each neighborhood for a population-specific SARS-CoV-2 analysis. Using quantitative real-time polymerase chain reaction (qRT-PCR), we successfully isolated SARS-CoV-2 at variable levels in all sample populations between April 2020 and March 2021. Taken together, our data reveal infection trends within a municipal sub-population in absence of individual testing. Additionally, by this method we collect information on the outstanding question of asymptomatic infections. Overall, these data provide health officials geographically specific information to best mobilize limited healthcare resources.

Synapses between neurons are crucial for all of human behavior as they underlie the ability to form thoughts, make precise movements, learn skills, and form new memories. Spinules are finger like projections from one neuron that are embedded within the information-sending end (i.e., presynaptic bouton) of another neuron. While recent data suggests that spinules are abundant within synapses across the brain, we have a limited understanding of their synaptic specificity or function. It is speculated that synaptic spinules may act as anchoring mechanisms or as a novel form of neuronal communication. Here, we sought to determine how presynaptic boutons that contain spinules differ from those that do not, as a first step towards uncovering whether spinules target specific subsets of synapses in the brain. We performed extensive quantitative 3D reconstructions and analyses of 138 excitatory (i.e., releasing glutamate) presynaptic boutons within the CA1 Hippocampus (center for memory formation) of an adult rat, including 87 spinule-bearing boutons (SBBs), and 51 presynaptic boutons without spinules (Non-SBBs). We found that on average, SBB volumes were 2.6-times larger than Non-SBB volumes (p<.00001), and that the size of the synapses made by SBBs were 1.6-times larger than those made by Non-SBBs (p<.00001). Since the anatomical size of a bouton and its synapse are highly correlated with its physiological strength, these data suggest that spinules may increase the physiological strength of excitatory synaptic connections in the CA1 hippocampus, and may therefore play an important role in memory formation.

COVID-19, the viral disease caused by the novel coronavirus SARS-CoV-2, is associated with cardiovascular complications such as arrhythmias, myocarditis, and even cardiac arrest. There are two possible mechanisms of SARS-CoV-2 entry into human cells; the endosomal-mediated pathway which requires intracellular processing by intracellular proteases, and the membrane fusion pathway mediated by secreted proteases. Importantly, SARS-CoV-2 entry relies on the expression of the transmembrane receptor ACE2, which interacts with the viral spike protein. It’s still unclear if ACE2 is required for both viral entry pathways. Cardiomyocytes express ACE2, thus SARS-CoV-2 can enter heart tissue; however, the mechanism by which this occurs and how it may lead to cardiac dysfunction is unknown. We previously demonstrated that SARS-CoV-2 significantly impairs mechanical and electrical function of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our goal is to understand if ACE2 is required for viral entry into the heart, using hiPSC-CMs as a model, in order to better understand COVID-19 pathology affecting the heart. Using a CRISPR/Cas9 system, we targeted the ACE2 gene at three loci to effectively knockout (KO) gene expression from WTC11 iPSCs. Two KO clones were selected and isolated after sequencing. Wild type (WT) and KO iPSCs were directly differentiated into CMs over a 17-day period. Preliminary results confirmed the absence of ACE2 protein expression in both KO clones by western blot. Fluorescent imaging of CMs infected with GFP-tagged SARS-CoV-2 showed severe infection and cell death at varied time points and multiplicities of infection (MOI) in WT WTC-CMs, while ACE2 KO-CMs showed absence of prominent infection and cell death. These data indicate that the lack of ACE2 markedly prevents SARS-CoV-2 entry into CMs, and understanding if blocking viral entry is sufficient to prevent functional impairment will provide key insights into the development of cardiomyopathies in COVID-19 patients.