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To better understand the field ecology of stored grain beetles in southern Australia we monitored the flight activity of Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) over a 2 year period. We used target-specific pheromone lures and Lindgren funnel traps in open fields, at grain storages, and in remnant native vegetation. Over 35,000 R. dominica and 2,500 T. castaneum were caught. Significantly more R. dominica (46.4%) were caught in areas of remnant vegetation than at storage sites (29.2%) or in open fields (24.4%), suggesting this species may be utilising non-grain food sources in some areas. In contrast, the majority of T. castaneum (45.4%) were caught adjacent to storages. An intense summer heatwave (14 day mean maximum air temperature 41.9 °C) corresponded to captures of both species declining to near zero until the following spring. Sex ratios of both species varied significantly over time but were unaffected by habitat type. Two mark/release/recapture (MRR) trials using laboratory-reared R. dominica were conducted using a 1 km trap array and pheromone lures. The recovery rate for marked beetles in the first trial was 1.41%, however a subsequent experiment comparing fresh and weathered lures showed that lure attractancy declines to 50% between 3 and 4 days after deployment during hot weather (mean daily maximum air temperature 29.4 °C). When the MRR trial was repeated with lures replaced at shorter (2 day) intervals, the recovery rate rose to 3.27% and the number of wild R. dominica captured increased proportionately. The short half-life of rubber septum-based R. dominica pheromone lures needs to be considered when designing surveillance programs, particularly if attempts are being made to correlate R. dominica dispersal patterns to short-term weather events.

Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics. Keywords: Single-cell RNA-Seq, Mass, Growth, Serial suspended microchannel resonator, Multi-omics, Single cell, T cell activation, Glioblastoma, GBM, Drug response, Microfluidics, Biophysical properties

Methods to rapidly assess cell growth would be useful for many applications, including drug susceptibility testing, but current technologies have limited sensitivity or throughput. Here we present an approach to precisely and rapidly measure growth rates of many individual cells simultaneously. We flow cells in suspension through a microfluidic channel with 10-12 resonant mass sensors distributed along its length, weighing each cell repeatedly over the 4-20 min it spends in the channel. Because multiple cells traverse the channel at the same time, we obtain growth rates for >60 cells/h with a resolution of 0.2 pg/h for mammalian cells and 0.02 pg/h for bacteria. We measure the growth of single lymphocytic cells, mouse and human T cells, primary human leukemia cells, yeast, Escherichia coli and Enterococcus faecalis. Our system reveals subpopulations of cells with divergent growth kinetics and enables assessment of cellular responses to antibiotics and antimicrobial peptides within minutes., United States. Army Research Office (Grant W911NF-09-D-0001), National Science Foundation (U.S.) (Grant 1129359), National Cancer Institute (U.S.) (Grant U54CA143874), National Cancer Institute (U.S.) (Grant P30-CA14051), National Cancer Institute (U.S.) (Grant R33-CA191143), National Institutes of Health (U.S.) (Grant T32-GM008334), National Institute of General Medical Sciences (U.S.) (Grant T32-GM008334)

Multiple myeloma (MM) has benefited from significant advancements in treatment that have improved outcomes and reduced morbidity. However, the disease remains incurable and is characterized by high rates of drug resistance and relapse. Consequently, methods to select the most efficacious therapy are of great interest. Here we utilize a functional assay to assess the ex vivo drug sensitivity of single multiple myeloma cells based on measuring their mass accumulation rate (MAR). We show that MAR accurately and rapidly defines therapeutic susceptibility across human multiple myeloma cell lines to a gamut of standard-of-care therapies. Finally, we demonstrate that our MAR assay, without the need for extended culture ex vivo, correctly defines the response of nine patients to standard-of-care drugs according to their clinical diagnoses. This data highlights the MAR assay in both research and clinical applications as a promising tool for predicting therapeutic response using clinical samples.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016., Cataloged from PDF version of thesis., Includes bibliographical references (pages 117-123)., Cell growth is a fundamental process that is intrinsically linked to our understanding of biology. As a central process in the propagation of single cells, changes to cell growth are a reflection of changes to cell state, and to their robust internal programming. Furthermore, cell growth is a key characteristic in a variety of physiological situations, including development, immune response, and diseases such as cancer. Consequently, growth has been a critical metric used to understand a wide range of biological subjects, but our ability to measure it on short timescales matching those of newer molecular assays has been limited. Within this thesis we exhibit two applications of short-term growth measurements, demonstrating their value in both the study of fundamental biology of the cell and in translational applications. By using a suspended microchannel resonator to take precise mass measurements on single cells over time, we are able to describe for the first time the nature of single-cell growth response dynamics over minute-timescale intervals in response to perturbations. Metabolic depletion studies show that growth on short timescales is unique from canonical measurements of cell growth on longer timescales, and that the two timescales are not always directly coupled, suggesting that single-cell growth is far more plastic than generally acknowledged. Translational studies show that rapid growth measurements demonstrate promise in assessing single-cell growth heterogeneity and therapeutic susceptibility prior to loss of cell viability., by Mark M. Stevens., Ph. D.

Cytokine regulation of lymphocyte growth and proliferation is essential for matching nutrient consumption with cell state. Here, we examine how cellular biophysical changes that occur immediately after growth factor depletion promote adaptation to reduced nutrient uptake. After growth factor withdrawal, nutrient uptake decreases, leading to apoptosis. Bcl-xL expression prevents cell death, with autophagy facilitating long-term cell survival. However, autophagy induction is slow relative to the reduction of nutrient uptake, suggesting that cells must engage additional adaptive mechanisms to respond initially to growth factor depletion. We describe an acute biophysical response to growth factor withdrawal, characterized by a simultaneous decrease in cell volume and increase in cell density, which occurs before autophagy initiation and is observed in both FL5.12 Bcl-xL cells depleted of IL-3 and primary CD8+ T cells depleted of IL-2 that are differentiating toward memory cells. The response reduces cell surface area to minimize energy expenditure while conserving biomass, suggesting that the biophysical properties of cells can be regulated to promote survival under conditions of nutrient stress.

The coordination of metabolic processes to allow increased nutrient uptake and utilization for macromolecular synthesis is central for cell growth. Although studies of bulk cell populations have revealed important metabolic and signaling requirements that impact cell growth on long time scales, whether the same regulation influences short-term cell growth remains an open question. Here we investigate cell growth by monitoring mass accumulation of mammalian cells while rapidly depleting particular nutrients. Within minutes following the depletion of glucose or glutamine, we observe a growth reduction that is larger than the mass accumulation rate of the nutrient. This indicates that if one particular nutrient is depleted, the cell rapidly adjusts the amount that other nutrients are accumulated, which is consistent with cooperative nutrient accumulation. Population measurements of nutrient sensing pathways involving mTOR, AKT, ERK, PKA, MST1, or AMPK, or pro-survival pathways involving autophagy suggest that they do not mediate this growth reduction. Furthermore, the protein synthesis rate does not change proportionally to the mass accumulation rate over these time scales, suggesting that intracellular metabolic pools buffer the growth response. Our findings demonstrate that cell growth can be regulated over much shorter time scales than previously appreciated., National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051), National Cancer Institute (U.S.). Physical Sciences Oncology Center (U54CA143874), National Institutes of Health (U.S.) (Contract R01GM085457), National Cancer Institute (U.S.) (Fellowship F31CA167872), National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Program 5T32GM008334)