Extraction of Aerosol-Deposited Yersinia pestis from Indoor Surfaces To Determine Bacterial Environmental Decay.

UNLABELLED: Public health and decontamination decisions following an event that causes indoor contamination with a biological agent require knowledge of the environmental persistence of the agent. The goals of this study were to develop methods for experimentally depositing bacteria onto indoor surfaces via aerosol, evaluate methods for sampling and enumerating the agent on surfaces, and use these methods to determine bacterial surface decay. A specialized aerosol deposition chamber was constructed, and methods were established for reproducible and uniform aerosol deposition of bacteria onto four coupon types. The deposition chamber facilitated the control of relative humidity (RH; 10 to 70%) following particle deposition to mimic the conditions of indoor environments, as RH is not controlled by standard heating, ventilation, and air conditioning (HVAC) systems. Extraction and culture-based enumeration methods to quantify the viable bacteria on coupons were shown to be highly sensitive and reproducible. To demonstrate the usefulness of the system for decay studies,Yersinia pestis persistence as a function of surface type at 21 °C and 40% RH was determined to be >40%/min for all surfaces. Based upon these results, at typical indoor temperature and RH, a 6-log reduction in titer would expected to be achieved within 1 h as the result of environmental decay on surfaces without active decontamination. The developed approach will facilitate future persistence and decontamination studies with a broad range of biological agents and surfaces, providing agent decay data to inform both assessments of risk to personnel entering a contaminated site and decontamination decisions following biological contamination of an indoor environment. IMPORTANCE: Public health and decontamination decisions following contamination of an indoor environment with a biological agent require knowledge of the environmental persistence of the agent. Previous studies on Y. pestis persistence have utilized large liquid droplet deposition to provide persistence data. As a result, methods were developed to deposit aerosols containing bacteria onto indoor surfaces, reproducibly enumerate bacteria harvested from coupons, and determine surface decay utilizing Y. pestis The results of this study provide foundational methods required to evaluate surface decay of bacteria and potentially other biological agents, such as viruses, in aerosol particles as a function of surface type and environment. Integrating the data from both aerosol and liquid deposition surface decay studies will provide medical and public health personnel with a more complete understanding of agent persistence on surfaces in contaminated areas for assessment of health risks and to inform decontamination decisions.

A novel method to prioritize RNAseq data for post-hoc analysis based on absolute changes in transcript abundance.

The use of fold-change (FC) to prioritize differentially expressed genes (DEGs) for post-hoc characterization is a common technique in the analysis of RNA sequencing datasets. However, the use of FC can overlook certain population of DEGs, such as high copy number transcripts which undergo metabolically expensive changes in expression yet fail to exceed the ratiometric FC cut-off, thereby missing potential important biological information. Here we evaluate an alternative approach to prioritizing RNAseq data based on absolute changes in normalized transcript counts (ΔT) between control and treatment conditions. In five pairwise comparisons with a wide range of effect sizes, rank-ordering of DEGs based on the magnitude of ΔT produced a power curve-like distribution, in which 4.7-5.0% of transcripts were responsible for 36-50% of the cumulative change. Thus, differential gene expression is characterized by the high production-cost expression of a small number of genes (large ΔT genes), while the differential expression of the majority of genes involves a much smaller metabolic investment by the cell. To determine whether the large ΔT datasets are representative of coordinated changes in the transcriptional program, we evaluated large ΔT genes for enrichment of gene ontologies (GOs) and predicted protein interactions. In comparison to randomly selected DEGs, the large ΔT transcripts were significantly enriched for both GOs and predicted protein interactions. Furthermore, enrichments were were consistent with the biological context of each comparison yet distinct from those produced using equal-sized populations of large FC genes, indicating that the large ΔT genes represent an orthagonal transcriptional response. Finally, the composition of the large ΔT gene sets were unique to each pairwise comparison, indicating that they represent coherent and context-specific responses to biological conditions rather than the non-specific upregulation of a family of genes. These findings suggest that the large ΔT genes are not a product of random or stochastic phenomenon, but rather represent biologically meaningful changes in the transcriptional program. They furthermore imply that high abundance transcripts are associated with particularly cellular states, and as cells change in response to internal or external conditions, the relative distribution of the abundant transcripts changes accordingly. Thus, prioritization of DEGs based on the concept of metabolic cost is a simple yet powerful method to identify biologically important transcriptional changes and provide novel insights into cellular behaviors.

Longitudinal RNA sequencing of the deep transcriptome during neurogenesis of cortical glutamatergic neurons from murine ESCs.

Using paired-end RNA sequencing, we have quantified the deep transcriptional changes that occur during differentiation of murine embryonic stem cells into a highly enriched population of glutamatergic cortical neurons. These data provide a detailed and nuanced account of longitudinal changes in the transcriptome during neurogenesis and neuronal maturation, starting from mouse embryonic stem cells and progressing through neuroepithelial stem cell induction, radial glial cell formation, neurogenesis, neuronal maturation and cortical patterning. Understanding the transcriptional mechanisms underlying the differentiation of stem cells into mature, glutamatergic neurons of cortical identity has myriad applications, including the elucidation of mechanisms of cortical patterning; identification of neurogenic processes; modeling of disease states; detailing of the host cell response to neurotoxic stimuli; and determination of potential therapeutic targets. In future work we anticipate correlating changes in longitudinal gene expression to other cell parameters, including neuronal function as well as characterizations of the proteome and metabolome. In this data article, we describe the methods used to produce the data and present the raw sequence read data in FASTQ files, sequencing run statistics and a summary flatfile of raw counts for 22,164 genes across 31 samples, representing 3-5 biological replicates at each timepoint. We propose that this data will be a valuable contribution to diverse research efforts in bioinformatics, stem cell research and developmental neuroscience studies.

Novel application of stem cell-derived neurons to evaluate the time- and dose-dependent progression of excitotoxic injury.

Glutamate receptor (GluR)-mediated neurotoxicity is implicated in a variety of disorders ranging from ischemia to neural degeneration. Under conditions of elevated glutamate, the excessive activation of GluRs causes internalization of pathologic levels of Ca(2+), culminating in bioenergetic failure, organelle degradation, and cell death. Efforts to characterize cellular and molecular aspects of excitotoxicity and conduct therapeutic screening for pharmacologic inhibitors of excitogenic progression have been hindered by limitations associated with primary neuron culture. To address this, we evaluated glutamate-induced neurotoxicity in highly enriched glutamatergic neurons (ESNs) derived from murine embryonic stem cells. As of 18 days in vitro (DIV 18), ESNs were synaptically coupled, exhibited spontaneous network activity with neurotypic mEPSCs and expressed NMDARs and AMPARs with physiological current:voltage behaviors. Addition of 0.78-200 µM glutamate evoked reproducible time- and dose-dependent metabolic failure in 6 h, with a calculated EC50 value of 0.44 µM at 24 h. Using a combination of cell viability assays and electrophysiology, we determined that glutamate-induced toxicity was specifically mediated by NMDARs and could be inhibited by addition of NMDAR antagonists, increased extracellular Mg(2+) or substitution of Ba(2+) for Ca(2+). Glutamate treatment evoked neurite fragmentation and focal swelling by both immunocytochemistry and scanning electron microscopy. Presentation of morphological markers of cell death was dose-dependent, with 0.78-200 µM glutamate resulting in apoptosis and 3000 µM glutamate generating a mixture of necrosis and apoptosis. Addition of neuroprotective small molecules reduced glutamate-induced neurotoxicity in a dose-dependent fashion. These data indicate that ESNs replicate many of the excitogenic mechanisms observed in primary neuron culture, offering a moderate-throughput model of excitotoxicity that combines the verisimilitude of primary neurons with the flexibility and scalability of cultured cells. ESNs therefore offer a physiologically relevant platform that exhibits characteristic NMDAR responses, and appears suitable to evaluate molecular mechanisms of glutamate-induced excitotoxicity and screen for candidate therapeutics.

High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research.

BACKGROUND: Recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. Derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. However, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available. METHODS: With the objective of developing a more facile, robust and efficient method to generate enriched glutamatergic neuronal cultures, we evaluated the neurogenic capacity of three mouse embryonic stem cell (ESC) lines (R1, C57BL/6 and D3) adapted to feeder-independent suspension culture. Neurogenesis and neuronal maturation were characterized as a function of time in culture using immunological, genomic, morphological and functional metrics. The functional responses of ESNs to neurotropic toxins with distinctly different targets and mechanisms of toxicity, such as glutamate, α-latrotoxin (LTX), and botulinum neurotoxin (BoNT), were also evaluated. RESULTS: Suspension-adapted ESCs expressed markers of pluripotency through at least 30 passages, and differentiation produced 97×106 neural progenitor cells (NPCs) per 10-cm dish. Greater than 99% of embryonic stem cell-derived neurons (ESNs) expressed neuron-specific markers by 96 h after plating and rapidly developed complex axodendritic arbors and appropriate compartmentalization of neurotypic proteins. Expression profiling demonstrated the presence of transcripts necessary for neuronal function and confirmed that ESN populations were predominantly glutamatergic. Furthermore, ESNs were functionally receptive to all toxins with sensitivities and responses consistent with primary neurons. CONCLUSIONS: These findings demonstrate a cost-effective, scalable and flexible method to produce a highly enriched glutamatergic neuron population. The functional characterization of pathophysiological responses to neurotropic toxins and the compatibility with multi-well plating formats were used to demonstrate the suitability of ESNs as a discovery platform for molecular mechanisms of action, moderate-throughput analytical approaches and diagnostic screening. Furthermore, for the first time we demonstrate a cell-based model that is sensitive to all seven BoNT serotypes with EC50 values comparable to those reported in primary neuron populations. These data providing compelling evidence that ESNs offer a neuromimetic platform suitable for the evaluation of molecular mechanisms of neurotoxicity.

Compatibility of SYTO 13 and Hoechst 33342 for longitudinal imaging of neuron viability and cell death.

BACKGROUND: Simultaneous use of cell-permeant and impermeant fluorescent nuclear dyes is a common method to study cell viability and cell death progression. Although these assays are usually conducted as end-point studies, time-lapse imaging offers a powerful technique to distinguish temporal changes in cell viability at single-cell resolution. SYTO 13 and Hoechst 33342 are two commonly used cell-permeant nuclear dyes; however their suitability for live imaging has not been well characterized. We compare end-point assays with time-lapse imaging studies over a 6 h period to evaluate the compatibility of these two dyes with longitudinal imaging, using both control neurons and an apoptotic neuron model. FINDINGS: In longitudinal assays of untreated neurons, SYTO 13 addition caused acute necrosis within 3 h, whereas neurons imaged with Hoechst remained viable for at least 6 h. In a staurosporine-induced apoptotic model of neurotoxicity, determinations of the mode of cell death and measurements of nuclear size were identical between longitudinal studies using Hoechst and end-point assays. Alternatively, longitudinal studies using 500 nM or 5 nM SYTO 13 were not consistent with end-point assays. CONCLUSIONS: SYTO 13 is acutely neurotoxic and when used in longitudinal studies, masked end-stage morphologic evidence of apoptotic cell death. In contrast, a single application of Hoechst evoked no evidence of toxicity over a 6 h period, and was consistent with end-point characterizations of cell viability and nuclear morphology. For longitudinal characterization of acute cell death, Hoechst is a superior option.

Mechanism of inhibition of Bacillus anthracis spore outgrowth by the lantibiotic nisin.

The lantibiotic nisin inhibits growth of vegetative Gram-positive bacteria by binding to lipid II, which disrupts cell wall biosynthesis and facilitates pore formation. Nisin also inhibits the outgrowth of bacterial spores, including spores of Bacillus anthracis, whose structural and biochemical properties are fundamentally different from those of vegetative bacteria. The molecular basis of nisin inhibition of spore outgrowth had not been identified, as previous studies suggested that inhibition of spore outgrowth involved either covalent binding to a spore target or loss of membrane integrity; disruption of cell wall biosynthesis via binding to lipid II had not been investigated. To provide insights into the latter possibility, the effects of nisin were compared with those of vancomycin, another lipid II binding antibiotic that inhibits cell wall biosynthesis but does not form pores. Nisin and vancomycin both inhibited the replication of vegetative cells, but only nisin inhibited the transition from a germinated spore to a vegetative cell. Moreover, vancomycin prevented nisin's activity in competition studies, suggesting that the nisin-lipid II interaction is important for inhibition of spore outgrowth. In experiments with fluorescently labeled nisin, no evidence was found for a covalent mechanism for inhibition of spore outgrowth. Interestingly, mutants in the hinge region (N20P/M21P and M21P/K22P) that still bind lipid II but cannot form pores had potent antimicrobial activity against vegetative B. anthracis cells but did not inhibit spore outgrowth. Therefore, pore formation is essential for the latter activity but not the former. Collectively, these studies suggest that nisin utilizes lipid II as the germinated spore target during outgrowth inhibition and that nisin-mediated membrane disruption is essential to inhibit spore development into vegetative cells.

Bacillus anthracis spore interactions with mammalian cells: relationship between germination state and the outcome of in vitro.

BACKGROUND: During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium. RESULTS: Using an in vitro model of infection, we evaluated the influence of the germination state of B. anthracis spores, as controlled by defined culture conditions, on the outcome of infection. Spores prepared from B. anthracis Sterne 7702 germinated in a variety of common cell culture media supplemented with fetal bovine serum (FBS) while, in the absence of FBS, germination was strictly dependent on medium composition. RAW264.7 macrophage-like cells internalized spores to the same extent in either germinating or non-germinating media. However, significantly more viable, intracellular B. anthracis were recovered from cells infected under non-germinating conditions compared to germinating conditions. At the same time, RAW264.7 cells demonstrated a significant loss in viability when infected under non-germinating conditions. CONCLUSIONS: These results suggest that the outcome of host cell infection is sensitive to the germination state of spores at the time of uptake. Moreover, this study demonstrates the efficacy of studying B. anthracis spore infection of host cells within a defined, non-germinating, in vitro environment.

Inhibition of Bacillus anthracis spore outgrowth by nisin.

The lantibiotic nisin has previously been reported to inhibit the outgrowth of spores from several Bacillus species. However, the mode of action of nisin responsible for outgrowth inhibition is poorly understood. By using B. anthracis Sterne 7702 as a model, nisin acted against spores with a 50% inhibitory concentration (IC(50)) and an IC(90) of 0.57 microM and 0.90 microM, respectively. Viable B. anthracis organisms were not recoverable from cultures containing concentrations of nisin greater than the IC(90). These studies demonstrated that spores lose heat resistance and become hydrated in the presence of nisin, thereby ruling out a possible mechanism of inhibition in which nisin acts to block germination initiation. Rather, germination initiation is requisite for the action of nisin. This study also revealed that nisin rapidly and irreversibly inhibits growth by preventing the establishment of oxidative metabolism and the membrane potential in germinating spores. On the other hand, nisin had no detectable effects on the typical changes associated with the dissolution of the outer spore structures (e.g., the spore coats, cortex, and exosporium). Thus, the action of nisin results in the uncoupling of two critical sequences of events necessary for the outgrowth of spores: the establishment of metabolism and the shedding of the external spore structures.