Summary High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S 3 (“Second-Strand Synthesis”), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S 3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S 3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω-monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses. Substitution of alanine for arginine at position 146 is associated with slow cell growth, suggesting that the amino acid identity at this site may influence ribosomal function and/or biogenesis. Analysis of the three-dimensional structure of Rps3 in S. cerevisiae shows that arginine-146 makes contacts with the small subunit rRNA. Screening of deletion mutants encoding potential yeast methyltransferases revealed that the loss of the YOR021C gene results in the absence of methylation on Rps3. We demonstrated that recombinant Yor021c catalyzes ω-monomethylarginine formation when incubated with S-adenosylmethionine and hypomethylated ribosomes prepared from a YOR021C deletion strain. Interestingly, Yor021c belongs to the family of SPOUT methyltransferases that, to date, have only been shown to modify RNA substrates. Our findings suggest a wider role for SPOUT methyltransferases in nature. Finally, we have demonstrated the presence of a stoichiometrically methylated cysteine residue at position 39 of Rps27a in a zinc-cysteine cluster. The discovery of these three novel sites of protein modification within the small ribosomal subunit will now allow for an analysis of their functional roles in translation and possibly other cellular processes.

Human CD8 cytotoxic T lymphocytes (CTLs) contribute to antimicrobial defense against intracellular pathogens through secretion of cytotoxic granule proteins granzyme B, perforin, and granulysin. However, CTLs are heterogeneous in the expression of these proteins, and the subset(s) responsible for antimicrobial activity is unclear. Studying human leprosy, we found that the subset of CTLs coexpressing all three cytotoxic molecules is increased in the resistant form of the disease, can be expanded by interleukin-15 (IL-15), and is differentiated from naïve CD8 T cells by Langerhans cells. RNA sequencing analysis identified that these CTLs express a gene signature that includes an array of surface receptors typically expressed by natural killer (NK) cells. We determined that CD8 CTLs expressing granzyme B, perforin, and granulysin, as well as the activating NK receptor NKG2C, represent a population of "antimicrobial CTLs" (amCTLs) capable of T cell receptor (TCR)-dependent and TCR-independent release of cytotoxic granule proteins that mediate antimicrobial activity.

1The development of high-throughput single-cell RNA-sequencing (scRNA-Seq) methodologies 2 has empowered the characterization of complex biological samples by dramatically increasing the 3 number of constituent cells that can be examined concurrently. Nevertheless, these approaches 4 typically recover substantially less information per-cell as compared to lower-throughput microtiter 5 plate-based strategies. To uncover critical phenotypic differences among cells and effectively link 6 scRNA-Seq observations to legacy datasets, reliable detection of phenotype-defining transcripts 7 -such as transcription factors, affinity receptors, and signaling molecules -by these methods is 8 essential. Here, we describe a substantially improved massively-parallel scRNA-Seq protocol we 9 term Seq-Well S^3 ("Second-Strand Synthesis") that increases the efficiency of transcript capture 10 and gene detection by up to 10-and 5-fold, respectively, relative to previous iterations, surpassing 11 best-in-class commercial analogs. We first characterized the performance of Seq-Well S^3 in cell 12 lines and PBMCs, and then examined five different inflammatory skin diseases, illustrative of 13 distinct types of inflammation, to explore the breadth of potential immune and parenchymal cell 14 states. Our work presents an essential methodological advance as well as a valuable resource 15 for studying the cellular and molecular features that inform human skin inflammation. 109Mann-Whitney U Test & Linear Regression; Figure 1B-C). To confirm that these overall 110 improvements were not driven by changes in the relative frequencies of different cell types 111 captured by each technology, we also examined each subset independently (Figure S2A-B). For 112 each cell type detected, we observed significant increases in the numbers of transcripts captured 113 and genes detected using S^3 for each pairwise comparison between techniques (P < 0.05, 114 Mann-Whitney U Test; CD4 + T cells, Seq-Well V1: 1,044 ± 62.3 UMIs/cell; 10x v2: 7,671 ± 103.9 115 UMIs/cell; Seq-Well S^3: 13,390 ± 253.4 UMIs/cell; Mean ± Standard Error of the Median (SEM); 116 Figure S2). Both Seq-Well S^3 and 10x v2 displayed increased sensitivity for transcripts and 117 genes relative to Seq-Well v1, but Seq-Well S^3 showed the greatest efficiency (defined as genes 118 recovered at matched read depth) to detect genes for each cell type (Figure 1D-E; Figure S2). 119We sought to further understand whether these improvements resulted in enhanced 120 detection of biologically relevant genes typically under-represented in high-throughput single-cell 121 sequencing libraries (Tabula Muris Consortium et al., 2018). Importantly, genes that were 122 differentially detected (i.e., higher in S^3) within each cell type include numerous transcription 123 factors, cytokines and cell-surface receptors (Figure 1D-E). For example, among CD4 + T cells, 124we observe significantly increased detection of cytokines (e.g., TGFB1 and TNF), surface 125 receptors (e.g., TGFBR and CCR4) and transcription fact...

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Candida albicans is an opportunistic pathogen capable of causing life-threatening infections in immunocompromised individuals. Despite its significant health impact, our understanding of C. albicans pathogenicity is limited, particularly at the molecular level. One of the largely understudied enzyme families in C. albicans is small molecule AdoMet-dependent methyltransferases (smMTases), which are important for maintenance of cellular homeostasis by clearing toxic chemicals, generating novel cellular intermediates and regulating intra- and interspecies interactions. Putative smMTase orf19.633 has little homology to any known protein and was previously identified based on its ability to functionally complement a baker’s yeast crg1 mutant in response to protein phosphatase inhibitor cantharidin. In this study, we demonstrated that C. albicans Crg1 (CaCrg1) is a bona fide smMTase that interacts with the toxin in vitro and in vivo. We report that CaCrg1 is important for virulence-related processes such as adhesion, hyphal elongation and membrane trafficking in response to this toxin. Using biochemical and genetic analysis we also found that CaCrg1 plays a role in complex sphingolipid pathway: it binds to exogenous short-chain ceramides in vitro, it interacts genetically with genes of glucosylceramide pathway and the deletion of CaCRG1 leads to significant changes in the abundance of phytoceramides. Finally we found that this novel lipid-related smMTase is required for virulence in the waxmoth Galleria mellonella, a model of infection.

Th17 cells play a critical role in the adaptive immune response against extracellular bacteria, and the possible mechanisms by which they can protect against infection are of particular interest. In this study, we describe, to our knowledge, a novel IL-1b dependent pathway for secretion of the antimicrobial peptide IL-26 from human Th17 cells that is independent of and more rapid than classical TCR activation. We find that IL-26 is secreted 3 hours after treating PBMCs with Mycobacterium leprae as compared with 48 hours for IFN-g and IL-17A. IL-1b was required for microbial ligand induction of IL-26 and was sufficient to stimulate IL-26 release from Th17 cells. Only IL-1RI + Th17 cells responded to IL-1b, inducing an NF-kB-regulated transcriptome. Finally, supernatants from IL-1b-treated memory T cells killed Escherichia coli in an IL-26-dependent manner. These results identify a mechanism by which human IL-1RI + "antimicrobial Th17 cells" can be rapidly activated by IL-1b as part of the innate immune response to produce IL-26 to kill extracellular bacteria.

Mucosal-associated Invariant T (MAIT) cells are an innate-like T cell subset that recognize a broad array of microbial pathogens, including respiratory pathogens. Here we investigate the transcriptional profile of MAIT cells localized to the human lung, and postulate that MAIT cells may play a role in maintaining homeostasis at this mucosal barrier. Using the MR1/5-OP-RU tetramer, we identified MAIT cells and non-MAIT CD8+ T cells in lung tissue not suitable for transplant from human donors. We used RNA-sequencing of MAIT cells compared to non-MAIT CD8+ T cells to define the transcriptome of MAIT cells in the human lung. We show that, as a population, lung MAIT cells are polycytotoxic, secrete the directly antimicrobial molecule IL-26, express genes associated with persistence, and selectively express cytokine and chemokine- related molecules distinct from other lung-resident CD8+ T cells, such as interferon-γ- and IL-12- receptors. These data highlight MAIT cells’ predisposition to rapid pro-inflammatory cytokine responsiveness and antimicrobial mechanisms in human lung tissue, concordant with findings of blood-derived counterparts, and support a function for MAIT cells as early sensors in the defense of respiratory barrier function.