A targeted approach to enrich host-associated bacteria for metagenomic sequencing.

Multicellular eukaryotic organisms are hosts to communities of bacteria that reside on or inside their tissues. Often the eukaryotic members of the system contribute to high proportions of metagenomic sequencing reads, making it challenging to achieve sufficient sequencing depth to evaluate bacterial ecology. Stony corals are one such complex community; however, separation of bacterial from eukaryotic (primarily coral and algal symbiont) cells has so far not been successful. Using a combination of hybridization chain reaction fluorescence in situ hybridization and fluorescence activated cell sorting (HCR-FISH + FACS), we sorted two populations of bacteria from five genotypes of the coral Acropora loripes, targeting (i) Endozoicomonas spp, and (ii) all other bacteria. NovaSeq sequencing resulted in 67-91 M reads per sample, 55%-90% of which were identified as bacterial. Most reads were taxonomically assigned to the key coral-associated family, Endozoicomonadaceae, with Vibrionaceae also abundant. Endozoicomonadaceae were 5x more abundant in the 'Endozoicomonas' population, highlighting the success of the dual-labelling approach. This method effectively enriched coral samples for bacteria with <1% contamination from host and algal symbionts. The application of this method will allow researchers to decipher the functional potential of coral-associated bacteria. This method can also be adapted to accommodate other host-associated communities.

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. proliferum thermal tolerance. To assess whether microbiome transplantation could enhance heat tolerance of the sensitive wild-type C. proliferum, we transplanted bacterial communities from heat-evolved to the wild-type strain and subjected it to acute heat stress. Microbiome transplantation resulted in the incorporation of only 30 low-abundance strains into the microbiome of wild-type cultures, while the relative abundance of 14 pre-existing strains doubled in inoculated versus uninoculated samples. Inoculation with either wild-type or heat-evolved bacterial communities boosted C. proliferum growth, although no difference in heat tolerance was observed between the two inoculation treatments. This study provides evidence that Symbiodiniaceae-associated bacterial communities respond to heat selection and may contribute to coral adaptation to climate change.

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. We apply our findings to the precise definition of the tissue and cellular distribution of a weakly expressed fluorescent protein that reports on a low-abundance immunological gene. Exploiting the full spectrum coverage enabled by Aurora 5L, we describe a novel approach to the isolation of pure cell subset-specific autofluorescence profiles based on high dimensionality reduction algorithms. This method can also be used to unveil differences in the autofluorescent fingerprints of tissues in homeostasis and after immunological challenges.

An integrated analysis of human myeloid cells identifies gaps in in vitro models of in vivo biology.

The Stemformatics myeloid atlas is an integrated transcriptome atlas of human macrophages and dendritic cells that systematically compares freshly isolated tissue-resident, cultured, and pluripotent stem cell-derived myeloid cells. Three classes of tissue-resident macrophage were identified: Kupffer cells and microglia; monocyte-associated; and tumor-associated macrophages. Culture had a major impact on all primary cell phenotypes. Pluripotent stem cell-derived macrophages were characterized by atypical expression of collagen and a highly efferocytotic phenotype. Myeloid subsets, and phenotypes associated with derivation, were reproducible across experimental series including data projected from single-cell studies, demonstrating that the atlas provides a robust reference for myeloid phenotypes. Implementation in Stemformatics.org allows users to visualize patterns of sample grouping or gene expression for user-selected conditions and supports temporary upload of your own microarray or RNA sequencing samples, including single-cell data, to benchmark against the atlas.

Small molecule-directed specification of sclerotome-like chondroprogenitors and induction of a somitic chondrogenesis program from embryonic stem cells.

Pluripotent embryonic stem cells (ESCs) generate rostral paraxial mesoderm-like progeny in 5-6 days of differentiation induced by Wnt3a and Noggin (Nog). We report that canonical Wnt signaling introduced either by forced expression of activated ß-catenin, or the small-molecule inhibitor of Gsk3, CHIR99021, satisfied the need for Wnt3a signaling, and that the small-molecule inhibitor of BMP type I receptors, LDN193189, was able to replace Nog. Mesodermal progeny generated using such small molecules were chondrogenic in vitro, and expressed trunk paraxial mesoderm markers such as Tcf15 and Meox1, and somite markers such as Uncx, but failed to express sclerotome markers such as Pax1. Induction of the osteochondrogenically committed sclerotome from somite requires sonic hedgehog and Nog. Consistently, Pax1 and Bapx1 expression was induced when the isolated paraxial mesodermal progeny were treated with SAG1 (a hedgehog receptor agonist) and LDN193189, then Sox9 expression was induced, leading to cartilaginous nodules and particles in the presence of BMP, indicative of chondrogenesis via sclerotome specification. By contrast, treatment with TGFß also supported chondrogenesis and stimulated Sox9 expression, but failed to induce the expression of Pax1 and Bapx1. On ectopic transplantation to immunocompromised mice, the cartilage particles developed under either condition became similarly mineralized and formed pieces of bone with marrow. Thus, the use of small molecules led to the effective generation from ESCs of paraxial mesodermal progeny, and to their further differentiation in vitro through sclerotome specification into growth plate-like chondrocytes, a mechanism resembling in vivo somitic chondrogenesis that is not recapitulated with TGFß.