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Microbiome dysbiosis across the respiratory tract has been implicated in the development and exacerbations of asthma. Little is known regarding the short-term temporal variability of the respiratory microbiome in health and asthma. In this study, we evaluate six scenarios of dysbiosis by investigating the inter- and intra-individual variability of the nasopharyngeal microbiome, measured with metagenomic sequencing. We characterized 239 microbiome samples from thirteen healthy individuals and from seventeen patients with well-controlled mild to moderate persistent asthma collected during a one-month period in which patients experienced no asthma exacerbations or infections. We controlled for sex, age (range 5 to 38 years), population (Greece and Poland), and microbiome type, for multiple levels of species incidence (expanded to core). Microbiomes collected from the same individual at different time points were more similar than those collected from the same group (health or asthma), but some individuals’ microbiomes were more variable than others. Microbiomes drawn from asthma patients were less likely to resemble each other than the microbiomes of healthy individuals, suggesting increased inter-sample variability and susceptibility to stochastic changes in asthma. In addition, the microbiomes of asthma patients became increasingly dissimilar over time, suggesting increased intra-individual temporal dynamics. Finally, microbiomes of individuals with asthma were more diverse than those of healthy individuals, while species diversity was negatively associated with temporal change in both the asthma and health groups. We propose that the increased diversity, inter-sample variability and intra-individual temporal dynamics during stable disease state(s) are intrinsic properties of the microbial system in asthma and characterise dysbiosis. Our work has significant implications for the relevance of the respiratory microbiome in asthma.

Ice cream is essentially a foam consisting of air bubbles dispersed in a mixture of fat, water and ice crystals. The air fraction is typically around 50% by volume, and this is crucial for the product to have the consistency and texture desired by customers. A common manufacturing method involves mixing the air and other ingredients under high pressure before discharging the product to atmospheric pressure for further processing. As the pressure is released, air may escape from the foam into the atmosphere. The Study Group was asked to quantify this air loss and to predict how it might depend on, for example, the rheological properties of the foam mixture, the bubble size distribution and the rate at which the pressure is released.

The Romer Labs RapidChek®Listeria monocytogenes test system (Performance Tested Method 011805) was validated against the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (USDA-FSIS/MLG), U.S. Food and Drug Association Bacteriological Analytical Manual (FDA/BAM), and AOAC Official Methods of Analysis (AOAC/OMA) cultural reference methods for the detection of L. monocytogenes on selected foods including hot dogs, frozen cooked breaded chicken, frozen cooked shrimp, cured ham, and ice cream, and environmental surfaces including stainless steel and plastic in an unpaired study design. The RapidChek method uses a proprietary enrichment media system, a 44–48 h enrichment at 30 ± 1°C, and detects L. monocytogenes on an immunochromatographic lateral flow device within 10 min. Different L. monocytogenes strains were used to spike each of the matrixes. Samples were confirmed based on the reference method confirmations and an alternate confirmation method. A total of 140 low-level spiked samples were tested by the RapidChek method after enrichment for 44–48 h in parallel with the cultural reference method. There were 88 RapidChek presumptive positives. One of the presumptive positives was not confirmed culturally. Additionally, one of the culturally confirmed samples did not exhibit a presumptive positive. No difference between the alternate confirmation method and reference confirmation method was observed. The respective cultural reference methods (USDA-FSIS/MLG, FDA/BAM, and AOAC/OMA) produced a total of 63 confirmed positive results. Nonspiked samples from all foods were reported as negative for L. monocytogenes by all methods. Probability of detection analysis demonstrated no significant differences in the number of positive samples detected by the RapidChek method and the respective cultural reference method.

Summary Single-cell gene expression is inherently variable, but how this variability is controlled in response to stimulation remains unclear. Here, we use single-cell RNA-seq and single-molecule mRNA counting (smFISH) to study inducible gene expression in the immune toll-like receptor system. We show that mRNA counts of tumor necrosis factor α conform to a standard stochastic switch model, while transcription of interleukin-1β involves an additional regulatory step resulting in increased heterogeneity. Despite different modes of regulation, systematic analysis of single-cell data for a range of genes demonstrates that the variability in transcript count is linearly constrained by the mean response over a range of conditions. Mathematical modeling of smFISH counts and experimental perturbation of chromatin state demonstrates that linear constraints emerge through modulation of transcriptional bursting along with gene-specific relationships. Overall, our analyses demonstrate that the variability of the inducible single-cell mRNA response is constrained by transcriptional bursting.

Toll-like receptor (TLR) signaling regulates macrophage activation and effector cytokine propagation in the constrained environment of a tissue. In macrophage populations, TLR4 stimulates the dose-dependent transcription of nuclear factor B (NF-B) target genes. However, using single-RNA counting, we found that individual cells exhibited a wide range (three orders of magnitude) of expression of the gene encoding the proinflammatory cytokine tumor necrosis factor  (TNF-). The TLR4-induced TNFA transcriptional response correlated with the extent of NF-B signaling in the cells and their size. We compared the rates of TNF- production and uptake in macrophages and mouse embryonic fibroblasts and generated a mathematical model to explore the heterogeneity in the response of macrophages to TLR4 stimulation and the propagation of the TNF- signal in the tissue. The model predicts that the local propagation of the TLR4-dependent TNF- response and cellular NF-B signaling are limited to small distances of a few cell diameters between neighboring tissue-resident macrophages. In our predictive model, TNF- propagation was constrained by competitive uptake of TNF- from the environment, rather than by heterogeneous production of the cytokine. We propose that the highly constrained architecture of tissues enables effective localized propagation of inflammatory cues, while avoiding out-of-context responses at longer distances.