This study describes the pharmacokinetic (PK) and pharmacodynamic (PD) profile of GSK2292767A, a novel low solubility inhaled PI3Kδ inhibitor developed as an alternative to nemiralisib, which is a highly soluble inhaled inhibitor of PI3Kδ with a lung profile consistent with once-daily dosing. GSK2292767A has a similar in vitro cellular profile to nemiralisib and reduces eosinophilia in a murine PD model by 63% (n=5, p<0.05). To explore whether a low soluble compound results in effective PI3Kδ inhibition in humans, a first time in human study was conducted with GSK2292767A in healthy volunteers who smoke. GSK2292767A was generally well tolerated with headache being the most common reported adverse event. PD changes in induced sputum were measured in combination with drug concentrations in plasma from single (0.05-2 mg, n=37), and 14-day repeat (2 mg, n=12) doses of GSK2292767A. Trough bronchoalveolar lavage (BAL) for PK was taken after 14 days repeat dosing. GSK2292767A displayed a linear increase in plasma exposure with dose, with marginal accumulation after 14 days. Induced sputum showed a 27% (90% CI 15, 37) reduction in phosphatidylinositol-trisphosphate (PIP3, the product of PI3K activation) 3 h after a single dose. Reduction was not maintained 24 h after single or repeat dosing. BAL analysis confirmed presence of GSK2292767A in lung at 24 h, consistent with the preclinical lung retention profile. Despite good lung retention, target engagement was only present at 3 h. This exposure-response disconnect is an important observation for future inhaled drug design strategies considering low solubility to drive lung retention.

Stem cell differentiation involves major chromatin reorganisation, heterochromatin formation and genomic relocalisation of structural proteins, including heterochromatin protein 1 gamma (HP1γ). As the principal reader of the repressive histone marks H3K9me2/3, HP1 plays a key role in numerous processes including heterochromatin formation and maintenance.

Macrophages possess intrinsic tumoricidal activity, yet tumor-associated macrophages (TAMs) rapidly adopt an alternative phenotype within the tumor microenvironment that is marked by tumor-promoting immunosuppressive and trophic functions. The mechanisms that promote such TAM polarization remain poorly understood, but once identified, they may represent important therapeutic targets to block the tumor-promoting functions of TAMs and restore their anti-tumor potential. Here, we have characterized TAMs in a mouse model of metastatic ovarian cancer. We show that ovarian cancer cells promote membrane-cholesterol efflux and depletion of lipid rafts from macrophages. Increased cholesterol efflux promoted IL-4-mediated reprogramming, including inhibition of IFNγ-induced gene expression. Genetic deletion of ABC transporters, which mediate cholesterol efflux, reverts the tumor-promoting functions of TAMs and reduces tumor progression. These studies reveal an unexpected role for membrane-cholesterol efflux in driving TAM-mediated tumor progression while pointing to a potentially novel anti-tumor therapeutic strategy.

Cell-cell communication in multicellular organisms depends on the dynamic and reversible phosphorylation of protein tyrosine residues. The receptor-linked protein tyrosine phosphatases (RPTPs) receive cues from the extracellular environment and are well placed to influence cell signaling. However, the direct events downstream of these receptors have been challenging to resolve. We report here that the homophilic receptor PTPRK is stabilized at cell-cell contacts in epithelial cells. By combining interaction studies, quantitative tyrosine phosphoproteomics, proximity labeling and dephosphorylation assays we identify high confidence PTPRK substrates. PTPRK directly and selectively dephosphorylates at least five substrates, including Afadin, PARD3 and δ-catenin family members, which are all important cell-cell adhesion regulators. In line with this, loss of PTPRK phosphatase activity leads to disrupted cell junctions and increased invasive characteristics. Thus, identifying PTPRK substrates provides insight into its downstream signaling and a potential molecular explanation for its proposed tumor suppressor function.

A paradox of tumor immunology is that tumor-infiltrating lymphocytes are dysfunctional in situ, yet are capable of stem cell-like behavior including self-renewal, expansion, and multipotency, resulting in the eradication of large metastatic tumors. We find that the overabundance of potassium in the tumor microenvironment underlies this dichotomy, triggering suppression of T cell effector function while preserving stemness. High levels of extracellular potassium constrain T cell effector programs by limiting nutrient uptake, thereby inducing autophagy and reduction of histone acetylation at effector and exhaustion loci, which in turn produces CD8 T cells with improved in vivo persistence, multipotency, and tumor clearance. This mechanistic knowledge advances our understanding of T cell dysfunction and may lead to novel approaches that enable the development of enhanced T cell strategies for cancer immunotherapy.

T cell tolerance depends upon Aire-expressing cells to purge the T cell repertoire of autoreactive clones. Once thought to be the exclusive domain of thymic epithelial cells, a new study by Yamano et al. (https://doi.org/10.1084/jem.20181430) in this issue of identifies ILC3-like cells in the lymph nodes with similar properties.

Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis.

Protein misfolding in the cell is linked to an array of diseases, including cancers, cardiovascular disease, type II diabetes, and numerous neurodegenerative disorders. Therefore, investigating cellular pathways by which misfolded proteins are trafficked and cleared ("protein quality control") is of both mechanistic and therapeutic importance. The clearance of most misfolded proteins involves the covalent attachment of one or more ubiquitin molecules; however, the precise fate of the ubiquitinated protein varies greatly, depending on the linkages present in the ubiquitin chain. Here, we discuss approaches for quantifying linkage-specific ubiquitination and clearance of misfolded proteins in the budding yeast Saccharomyces cerevisiae-a model organism used extensively for interrogation of protein quality control pathways, but which presents its own unique challenges for cell and molecular biology experiments. We present a fluorescence microscopy-based assay for monitoring the clearance of misfolded protein puncta, a cycloheximide-chase assay for calculating misfolded protein half-life, and two antibody-based methods for quantifying specific ubiquitin linkages on tagged misfolded proteins, including a 96-well plate-based ELISA. We hope these methods will be of use to the protein quality control, protein degradation, and ubiquitin biology communities.

Metazoan cell death mechanisms are diverse and include numerous non-apoptotic programs. One program called entosis involves the invasion of live cells into their neighbors and is known to occur in cancers. Here, we identify a developmental function for entosis: to clear the male-specific linker cell in C. elegans. The linker cell leads migration to shape the gonad and is removed to facilitate fusion of the gonad to the cloaca. We find that the linker cell is cleared in a manner involving cell-cell adhesions and cell-autonomous control of uptake through linker cell actin. Linker cell entosis generates a lobe structure that is deposited at the site of gonad-to-cloaca fusion and is removed during mating. Inhibition of lobe scission inhibits linker cell death, demonstrating that the linker cell invades its host while alive. Our findings demonstrate a developmental function for entosis: to eliminate a migrating cell and facilitate gonad-to-cloaca fusion, which is required for fertility.

The origin of the autophagosomal membrane started to be debated by scientists working in the field within one year of the modern definition of autophagy in 1963. There is now converging evidence from older and newer studies that the endoplasmic reticulum is involved in formation of autophagosomes. Thus, it is possible to trace from early morphological work - done without the benefit of molecular descriptions - to recent studies - dissecting how specific proteins nucleate autophagosome biogenesis - a long series of experimental findings that are beginning to answer the 55-year old question with some confidence. The view that has emerged is that specialised regions of the endoplasmic reticulum, in dynamic cross talk with most intracellular organelles via membrane contact sites, provide a platform for autophagosome biogenesis.

Nemiralisib (GSK2269557) is a potent inhaled inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) which is being developed for the treatment of respiratory disorders including COPD (Chronic Obstructive Pulmonary Disease). Determining the pharmacokinetic (PK) and pharmacodynamic (PD) responses of inhaled drugs early during drug development is key to informing the appropriate dose and preferred dose regimen in patients. We set out to measure PD changes in induced sputum in combination with drug concentrations in plasma and bronchoalveolar lavage (BAL) taken from healthy smokers (n=56) treated for up to 14 days with increasing doses of inhaled nemiralisib (0.1 mg to 6.4 mg). Induced sputum analysis demonstrated a dose-dependent reduction in phosphatidylinositol-trisphosphate (PIP3, the product of PI3K activation), with a maximum placebo-corrected reduction of 23% (90% CI 11-34%) and 36% (90% CI 11-64%) following single dose or 14 days of treatment with nemiralisib respectively (2 mg, once daily). Plasma analysis suggested a linear PK relationship with an observed accumulation of ~3-4.5-fold (peak vs. trough) in plasma exposure following 14 days of nemiralisib treatment. BAL analysis at trough confirmed higher levels of drug in lung vs. plasma (32-fold in the BAL fluid component, and 214-fold in the BAL cellular fraction). Comparison of drug levels in plasma and reductions in sputum PIP3 show a direct relationship between exposure and PIP3 reduction. In conclusion, these results demonstrate target engagement upon treatment with inhaled nemiralisib and provide confidence for a once-daily dosing regimen.

Juvenile idiopathic arthritis (JIA) is the most common class of childhood rheumatic diseases, with distinct disease subsets that may have diverging pathophysiological origins. Both adaptive and innate immune processes have been proposed as primary drivers, which may account for the observed clinical heterogeneity, but few high-depth studies have been performed.

In mammals, the B-cell lineage arises from pluripotent progenitors in the bone marrow. During their development, B-cells undergo lineage specification and commitment, followed by expansion and selection. These processes are mediated by regulated changes in gene expression programmes, rearrangements of immunoglobulin (Ig) genes, and well-timed rounds of proliferation and apoptosis. Many of these processes are initiated by environmental factors including cytokines, chemokines, and cell-cell contacts. Developing B-cells process these environmental cues into stage-specific functions via signalling pathways including the PI3K, MAPK, or JAK-STAT pathway. The cytokines FLT3-Ligand and c-Kit-Ligand are important for the early expansion of the B-cell precursors at different developmental stages and conditions. Interleukin 7 is essential for commitment to the B-cell lineage and for orchestrating the Ig recombination machinery. After rearrangement of the immunoglobulin heavy chain, proliferation and apoptosis, and thus selection, are mediated by the clonal pre-B-cell receptor, and, following light chain rearrangement, by the B-cell receptor.

Syntaxin 17 (Stx17) has been implicated in autophagosome-lysosome fusion. Here, we report that Stx17 functions in assembly of protein complexes during autophagy initiation. Stx17 is phosphorylated by TBK1 whereby phospho-Stx17 controls the formation of the ATG13FIP200 mammalian pre-autophagosomal structure (mPAS) in response to induction of autophagy. TBK1 phosphorylates Stx17 at S202. During autophagy induction, Stx17 transfers from the Golgi, where its steady-state pools localize, to the ATG13FIP200 mPAS. Stx17 was in complexes with ATG13 and FIP200, whereas its non-phosphorylatable mutant Stx17 was not. Stx17 or TBK1 knockouts blocked ATG13 and FIP200 puncta formation. Stx17 or TBK1 knockouts reduced the formation of ATG13 protein complexes with FIP200 and ULK1. Endogenous Stx17 colocalized with LC3B following induction of autophagy. Stx17 knockout diminished LC3 response and reduced sequestration of the prototypical bulk autophagy cargo lactate dehydrogenase. We conclude that Stx17 is a TBK1 substrate and that together they orchestrate assembly of mPAS.

Following publication of the original article [1], it was reported that the incorrect "Additional file 3" was published. The correct additional file is given below.

Complex multicellular organisms, such as mammals, express two complete sets of chromosomes per nucleus, combining the genetic material of both parents. However, epigenetic studies have demonstrated violations to this rule that are necessary for mammalian physiology; the most notable parental allele expression phenomenon is genomic imprinting. With the identification of endogenous imprinted genes, genomic imprinting became well-established as an epigenetic mechanism in which the expression pattern of a parental allele influences phenotypic expression. The expanding study of genomic imprinting is revealing a significant impact on brain functions and associated diseases. Here, we review key milestones in the field of imprinting and discuss mechanisms and systems in which imprinted genes exert a significant role.

In this contribution, we describe a multi-omics systems biology study of the metabolic changes that occur during aging in . Sampling several time points from young adulthood until early old age, our study covers the full duration of aging and include transcriptomics, and targeted MS-based metabolomics. In order to focus on the metabolic changes due to age we used two strains that are metabolically close to wild-type, yet are conditionally non-reproductive. Using these data in combination with a whole-genome model of the metabolism of and mathematical modeling, we predicted metabolic fluxes during early aging. We find that standard Flux Balance Analysis does not accurately predict measured fluxes nor age-related changes associated with the Citric Acid cycle. We present a novel Flux Balance Analysis method where we combined biomass production and targeted metabolomics information to generate an objective function that is more suitable for aging studies. We validated this approach with a detailed case study of the age-associated changes in the Citric Acid cycle. Our approach provides a comprehensive time-resolved multi-omics and modeling resource for studying the metabolic changes during normal aging in .

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1 chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8 T cell memory compartment. Following viral infection, CD8 T cells lacking Myb underwent terminal differentiation and generated fewer stem cell-like central memory cells than did Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8 T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8 T cell stemness and highlight its therapeutic potential.

Metastatic progression is a major cause of mortality in cervical cancers, but factors regulating migratory and pre-metastatic cell populations remain poorly understood. Here, we sought to assess whether a SUV39H1-low chromatin state promotes migratory cell populations in cervical cancers, using meta-analysis of data from The Cancer Genome Atlas (TCGA), immunohistochemistry, genomics and functional assays. Cervical cancer cells sorted based on migratory ability in vitro have low levels of SUV39H1 protein, and SUV39H1 knockdown in vitro enhanced cervical cancer cell migration. Further, TCGA SUV39H1-low tumours correlated with poor clinical outcomes and showed gene expression signatures of cell migration. SUV39H1 expression was examined within biopsies, and SUV39H1 cells within tumours also demonstrated migratory features. Next, to understand genome scale transcriptional and chromatin changes in migratory populations, cell populations sorted based on migration in vitro were examined using RNA-Seq, along with ChIP-Seq for H3K9me3, the histone mark associated with SUV39H1. Migrated populations showed SUV39H1-linked migratory gene expression signatures, along with broad depletion of H3K9me3 across gene promoters. We show for the first time that a SUV39H1-low chromatin state associates with, and promotes, migratory populations in cervical cancers. Our results posit SUV39H1-low cells as key populations for prognosis estimation and as targets for novel therapies.

Low temperatures are required to regulate the transition from vegetative to reproductive growth via a pathway called vernalization. In wheat, vernalization predominantly involves the cold upregulation of the floral activator (). Here, we have used an extreme vernalization response, identified through studying ambient temperature responses, to reveal the complexity of temperature inputs into , with allelic inter-copy variation at a gene expansion of modulating these effects. We find that the repressors of the reproductive transition, () and , are re-activated when plants experience high temperatures during and after vernalization. In addition, this re-activation is regulated by photoperiod for but was independent of photoperiod for We also find this warm temperature interruption affects flowering time and floret number and is stage specific. This research highlights the important balance between floral activators and repressors in coordinating the response of a plant to temperature, and that the absence of warmth is essential for the completion of vernalization. This knowledge can be used to develop agricultural germplasm with more predictable vernalization responses that will be more resilient to variable growth temperatures.

Chronic graft-versus-host disease is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T-cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center -promoting Tfollicular helper cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ, a lipid kinase, is critical for activated T-cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T-cells that become Tfhs is required for cGVHD in a non-sclerodermatous multi-organ system disease model that includes bronchiolitis obliterans, dependent upon GC B-cells, Tfhs, and counterbalanced by Tfollicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B-cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow derived GC B-cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443 that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of FDA-approved PI3Kδ inhibitors for cGVHD therapy in patients. This article is protected by copyright. All rights reserved.

Alternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products. Using bulk RNA sequencing, splicing variation has been studied across human tissues and in genetically diverse populations. This has identified disease-relevant splicing events, as well as associations between splicing and genomic features, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue or cell type and its determinants remains poorly understood.