Search for: All records

In this essay, we review how counter-stereotypical scientists have been featured in life science courses and discuss the benefits and costs of developing and interacting with these materials from the perspectives of three groups: students, instructors, and the featured scientists. 

An important aspect of how viruses spread and infect is the viral burst size, or the number of new viruses produced by each infected cell. Surprisingly, this value remains poorly characterized for influenza A virus (IAV), commonly known as the flu. In this study, we screened tens of thousands of cells using a microfluidic method called droplet quantitative PCR (dqPCR). The high-throughput capability of dqPCR enabled the measurement of a large population of infected cells producing progeny virus. By measuring the fully assembled and successfully released viruses from these infected cells, we discover that the viral burst sizes for both the seasonal H3N2 and the 2009 pandemic H1N1 strains vary significantly, with H3N2 ranging from 10¹to 10⁴viruses per cell, and H1N1 ranging from 10¹to 10³viruses per cell. Some infected cells produce average numbers of new viruses, while others generate extensive number of viruses. In fact, we find that only 10% of the single-cell infections are responsible for creating a significant portion of all the viruses. This small fraction produced approximately 60% of new viruses for H3N2 and 40% for H1N1. On average, each infected cell of the H3N2 flu strain produced 709 new viruses, whereas for H1N1, each infected cell produced 358 viruses. This novel method reveals insights into the flu virus and can lead to improved strategies for managing and preventing the spread of viruses. 

ABSTRACT Drop-based microfluidics has revolutionized single-cell studies and can be applied toward analyzing tens of thousands to millions of single cells and their products contained within picoliter-sized drops. Drop-based microfluidics can shed insight into single-cell virology, enabling higher-resolution analysis of cellular and viral heterogeneity during viral infection. In this work, individual A549, MDCK, and siat7e cells were infected with influenza A virus (IAV) and encapsulated into 100-μm-size drops. Initial studies of uninfected cells encapsulated in drops demonstrated high cell viability and drop stability. Cell viability of uninfected cells in the drops remained above 75%, and the average drop radii changed by less than 3% following cell encapsulation and incubation over 24 h. Infection parameters were analyzed over 24 h from individually infected cells in drops. The number of IAV viral genomes and infectious viruses released from A549 and MDCK cells in drops was not significantly different from bulk infection as measured by reverse transcriptase quantitative PCR (RT-qPCR) and plaque assay. The application of drop-based microfluidics in this work expands the capacity to propagate IAV viruses and perform high-throughput analyses of individually infected cells. IMPORTANCE Drop-based microfluidics is a cutting-edge tool in single-cell research. Here, we used drop-based microfluidics to encapsulate thousands of individual cells infected with influenza A virus within picoliter-sized drops. Drop stability, cell loading, and cell viability were quantified from three different cell lines that support influenza A virus propagation. Similar levels of viral progeny as determined by RT-qPCR and plaque assay were observed from encapsulated cells in drops compared to bulk culture. This approach enables the ability to propagate influenza A virus from encapsulated cells, allowing for future high-throughput analysis of single host cell interactions in isolated microenvironments over the course of the viral life cycle. 

Abstract Neural circuit function is shaped both by the cell types that comprise the circuit and the connections between those cell types¹. Neural cell types have previously been defined by morphology^{2, 3}, electrophysiology^{4, 5}, transcriptomic expression^6–8, connectivity^9–13, or even a combination of such modalities^14–16. More recently, the Patch-seq technique has enabled the characterization of morphology (M), electrophysiology (E), and transcriptomic (T) properties from individual cells^17–20. Using this technique, these properties were integrated to define 28, inhibitory multimodal, MET-types in mouse primary visual cortex²¹. It is unknown how these MET-types connect within the broader cortical circuitry however. Here we show that we can predict the MET-type identity of inhibitory cells within a large-scale electron microscopy (EM) dataset and these MET-types have distinct ultrastructural features and synapse connectivity patterns. We found that EM Martinotti cells, a well defined morphological cell type^{22, 23}known to be Somatostatin positive (Sst+)^{24, 25}, were successfully predicted to belong to Sst+ MET-types. Each identified MET-type had distinct axon myelination patterns and synapsed onto specific excitatory targets. Our results demonstrate that morphological features can be used to link cell type identities across imaging modalities, which enables further comparison of connectivity in relation to transcriptomic or electrophysiological properties. Furthermore, our results show that MET-types have distinct connectivity patterns, supporting the use of MET-types and connectivity to meaningfully define cell types. 

Corals are in decline worldwide due to local anthropogenic stressors, such as nutrient loading, and global stressors, such as ocean warming. Anthropogenic nutrient loading, which is often rich in nitrate, inhibits coral growth and worsens corals’ response to warming while natural sources of nitrogen, such as ammonium from fish excretion, promotes coral growth. Although the effects of nutrient loading and ocean warming have been well-studied, it remains unclear how these factors may interact with biotic processes, such as corallivory, to alter coral health and the coral microbiome. This study examined how nitrate vs. ammonium enrichment altered the effects of increased seawater temperature and simulated parrotfish corallivory on the health of Pocillopora meandrina and its microbial community. We tested the effects of nitrogen source on the response to corallivory under contrasting temperatures (control: 26 °C, warming: 29 °C) in a factorial mesocosm experiment in Moorea, French Polynesia. Corals were able to maintain growth rates despite simultaneous stressors. Seawater warming suppressed wound healing rates by nearly 66%. However, both ammonium and nitrate enrichment counteracted the effect of higher temperatures on would healing rates. Elevated seawater temperature and ammonium enrichment independently increased Symbiodiniaceae densities relative to controls, yet there was no effect of nitrate enrichment on algal symbiont densities. Microbiome variability increased with the addition of nitrate or ammonium. Moreover, microbial indicator analysis showed that Desulfovibrionaceae Operational taxonomic units (OTUs) are indicators of exclusively temperature stress while Rhodobacteraceae and Saprospiraceae OTUs were indicators of high temperature, wounding, and nitrogen enrichment. Overall, our results suggest that nitrogen source may not alter the response of the coral host to simultaneous stressors, but that the associated microbial community may be distinct depending on the source of enrichment. 

Climate change is increasing the frequency and magnitude of temperature anomalies that cause coral bleaching, leading to widespread mortality of stony corals that can fundamentally alter reef structure and function. However, bleaching often is spatially variable for a given heat stress event, and drivers of this heterogeneity are not well resolved. While small-scale experiments have shown that excess nitrogen can increase the susceptibility of a coral colony to bleaching, we lack evidence that heterogeneity in nitrogen pollution can shape spatial patterns of coral bleaching across a seascape. Using island-wide surveys of coral bleaching and nitrogen availability within a Bayesian hierarchical modeling framework, we tested the hypothesis that excess nitrogen interacts with temperature anomalies to alter coral bleaching for the two dominant genera of branching corals in Moorea, French Polynesia. For both coral genera,PocilloporaandAcropora, heat stress primarily drove bleaching prevalence (i.e., the proportion of colonies on a reef that bleached). In contrast, the severity of bleaching (i.e., the proportion of an individual colony that bleached) was positively associated with both heat stress and nitrogen availability for both genera. Importantly, nitrogen interacted with heat stress to increase bleaching severity up to twofold when nitrogen was high and heat stress was relatively low. Our finding that excess nitrogen can trigger severe bleaching even under relatively low heat stress implies that mitigating nutrient pollution may enhance the resilience of coral communities in the face of mounting stresses from global climate change.