Evaluation of Sort Recovery via Rmax.

Cell sorting performance can be evaluated in regard to the purity and recovery of the sorted fractions. The purity provides checks on sample quality, acquisition settings, gating strategy, and the sort decisions made by the instrument, but alone it is not sufficient to evaluate sorting performance. Recovery, defined here as the number of target particles sorted relative to the number of original target particles to be sorted, is a key metric of sort fitness and performance but is often neglected due to difficulties in its measurement.

Assessing the contribution of bacteria to the heat tolerance of experimentally evolved coral photosymbionts.

Coral reefs are extremely vulnerable to ocean warming, which triggers coral bleaching-the loss of endosymbiotic microalgae (Symbiodiniaceae) from coral tissues, often leading to death. To enhance coral climate resilience, the symbiont, Cladocopium proliferum was experimentally evolved for >10 years under elevated temperatures resulting in increased heat tolerance. Bacterial 16S rRNA gene metabarcoding showed the composition of intra- and extracellular bacterial communities of heat-evolved strains was significantly different from that of wild-type strains, suggesting bacteria responded to elevated temperatures, and may even play a role in C.

Comparing the Role of ROS and RNS in the Thermal Stress Response of Two Cnidarian Models, and .

Coral reefs are threatened by climate change, because it causes increasingly frequent and severe summer heatwaves, resulting in mass coral bleaching and mortality. Coral bleaching is believed to be driven by an excess production of reactive oxygen (ROS) and nitrogen species (RNS), yet their relative roles during thermal stress remain understudied. Here, we measured ROS and RNS net production, as well as activities of key enzymes involved in ROS scavenging (superoxide dismutase and catalase) and RNS synthesis (nitric oxide synthase) and linked these metrics to physiological measurements of cnidarian holobiont health during thermal stress.

Label-free macrophage phenotype classification using machine learning methods.

Macrophages are heterogeneous innate immune cells that are functionally shaped by their surrounding microenvironment. Diverse macrophage populations have multifaceted differences related to their morphology, metabolism, expressed markers, and functions, where the identification of the different phenotypes is of an utmost importance in modelling immune response. While expressed markers are the most used signature to classify phenotypes, multiple reports indicate that macrophage morphology and autofluorescence are also valuable clues that can be used in the identification process.

Characterization of Mucosal-Associated Invariant T Cells in Oral Lichen Planus.

Oral lichen planus (OLP) is an inflammatory condition of unknown cause that has been associated with concurrent candidal infection. Mucosal-associated invariant T (MAIT) cells express the T cell receptor TCRVα7.2 and are activated by riboflavin intermediates produced by microbes.

Colonization and metabolite profiles of homologous, heterologous and experimentally evolved algal symbionts in the sea anemone Exaiptasia diaphana.

The sea anemone, Exaiptasia diaphana, is a model of coral-dinoflagellate (Symbiodiniaceae) symbiosis. However, little is known of its potential to form symbiosis with Cladocopium-a key Indo-Pacific algal symbiont of scleractinian corals, nor the host nutritional consequences of such an association. Aposymbiotic anemones were inoculated with homologous algal symbionts, Breviolum minutum, and seven heterologous strains of Cladocopium C1 (wild-type and heat-evolved) under ambient conditions.

Unlocking autofluorescence in the era of full spectrum analysis: Implications for immunophenotype discovery projects.

Understanding the complex elements affecting signal resolution in cytometry is key for quality experimental design and data. In this study, we incorporate autofluorescence as a contributing factor to our understanding of resolution in cytometry and corroborate its impact in fluorescence signal detection through mathematical predictions supported by empirical evidence. Our findings illustrate the critical importance of autofluorescence extraction via full spectrum unmixing in unmasking dim signals and delineating the expression and subset distribution of low abundance markers in discovery projects.

Hierarchical modelling of immunoglobulin coated bacteria in dogs with chronic enteropathy shows reduction in coating with disease remission but marked inter-individual and treatment-response variability.

Chronic enteropathies are a common problem in dogs, but many aspects of the pathogenesis remain unknown, making the therapeutic approach challenging in some cases. Environmental factors are intimately related to the development and perpetuation of gastrointestinal disease and the gut microbiome has been identified as a contributing factor. Previous studies have identified dysbiosis and reduced bacterial diversity in the gastrointestinal microbiota of dogs with chronic enteropathies.

Intracellular bacteria are common and taxonomically diverse in cultured and in hospite algal endosymbionts of coral reefs.

Corals house a variety of microorganisms which they depend on for their survival, including endosymbiotic dinoflagellates (Symbiodiniaceae) and bacteria. While cnidarian-microorganism interactions are widely studied, Symbiodiniaceae-bacteria interactions are only just beginning to receive attention. Here, we describe the localization and composition of the bacterial communities associated with cultures of 11 Symbiodiniaceae strains from nine species and six genera.

Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion and .

Outer membrane vesicles (OMVs) are proteoliposomes blebbed from the surface of Gram-negative bacteria. Chronic periodontitis is associated with an increase in subgingival plaque of Gram-negative bacteria, , and . In this study, we investigated the immune-modulatory effects of , and OMVs on monocytes and differentiated macrophages.

Rmax: A systematic approach to evaluate instrument sort performance using center stream catch.

Sorting performance can be evaluated with regard to Purity, Yield and/or Recovery of the sorted fraction. Purity is a check on the quality of the sample and the sort decisions made by the instrument. Recovery and Yield definitions vary with some authors regarding both as how efficient the instrument is at sorting the target particles from the original sample, others distinguishing Recovery from Yield, where the former is used to describe the accuracy of the instrument's sort count.

Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.

Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B.

Sequencing of a patient with balanced chromosome abnormalities and neurodevelopmental disease identifies disruption of multiple high risk loci by structural variation.

Balanced chromosome abnormalities (BCAs) occur at a high frequency in healthy and diseased individuals, but cost-efficient strategies to identify BCAs and evaluate whether they contribute to a phenotype have not yet become widespread. Here we apply genome-wide mate-pair library sequencing to characterize structural variation in a patient with unclear neurodevelopmental disease (NDD) and complex de novo BCAs at the karyotype level. Nucleotide-level characterization of the clinically described BCA breakpoints revealed disruption of at least three NDD candidate genes (LINC00299, NUP205, PSMD14) that gave rise to abnormal mRNAs and could be assumed as disease-causing.

Cell type-specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP.

This protocol describes the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) for analysis of histone modifications, transcription factor binding, or polymerase occupancy within the context of a multicellular organism or tissue. Embryos expressing a cell type-specific nuclear marker are formaldehyde cross-linked and then subjected to dissociation. Fixed nuclei are isolated and sorted using FACS on the basis of the cell type-specific nuclear marker.

Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development.

Chromatin modifications are associated with many aspects of gene expression, yet their role in cellular transitions during development remains elusive. Here, we use a new approach to obtain cell type-specific information on chromatin state and RNA polymerase II (Pol II) occupancy within the multicellular Drosophila melanogaster embryo. We directly assessed the relationship between chromatin modifications and the spatio-temporal activity of enhancers.

Sphingosine kinase-1 (SphK-1) regulates Mycobacterium smegmatis infection in macrophages.

Sphingosine kinase-1 is known to mediate Mycobacterium smegmatis induced inflammatory responses in macrophages, but its role in controlling infection has not been reported to date. We aimed to unravel the significance of SphK-1 in controlling M. smegmatis infection in RAW 264.

Role of lipids in killing mycobacteria by macrophages: evidence for NF-kappaB-dependent and -independent killing induced by different lipids.

We have shown that several lipids can modulate the macrophage innate immune response against mycobacteria and enhance their killing. Since NF-kappaB is required for mycobacterial killing, we tested the ability of lipids to activate NF-kappaB in uninfected macrophages and those infected with mycobacteria. In uninfected cells, sphingomyelin (SM), phosphatidylinositol-4-phosphate (PIP) and arachidonic acid (AA) enhanced NF-kappaB activation and the cell surface expression of CD69, a macrophage activation marker regulated by NF-kappaB.