The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production.

RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine-rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

A semi-invariant Vα10+ T cell antigen receptor defines a population of natural killer T cells with distinct glycolipid antigen-recognition properties.

Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(α)14-J(α)18) T cell antigen receptor (TCR) α-chain and recognition of the glycolipid α-galactosylceramide (α-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of α-GalCer-reactive NKT cells that expressed a canonical V(α)10-J(α)50 TCR α-chain, which showed a preference for α-glucosylceramide (α-GlcCer) and bacterial α-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCRα sequence identity, the V(α)10 TCR-CD1d-α-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-α-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.

i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4.

CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an "i-body," the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor.

Niche Extracellular Matrix Components and Their Influence on HSC.

Maintenance of hematopoietic stem cells (HSC) takes place in a highly specialized microenvironment within the bone marrow. Technological improvements, especially in the field of in vivo imaging, have helped unravel the complexity of the niche microenvironment and have completely changed the classical concept from what was previously believed to be a static supportive platform, to a dynamic microenvironment tightly regulating HSC homeostasis through the complex interplay between diverse cell types, secreted factors, extracellular matrix molecules, and the expression of different transmembrane receptors. To add to the complexity, non-protein based metabolites have also been recognized as a component of the bone marrow niche. The objective of this review is to discuss the current understanding on how the different extracellular matrix components of the niche regulate HSC fate, both during embryonic development and in adulthood. Special attention will be provided to the description of non-protein metabolites, such as lipids and metal ions, which contribute to the regulation of HSC behavior. J. Cell. Biochem. 118: 1984-1993, 2017. © 2017 Wiley Periodicals, Inc.

Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere.

Pharmaceutical and agrochemical discovery programs are under considerable pressure to meet increasing global demand and thus require constant innovation. Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market place, but an obvious omission is that of the Platonic solid cubane. Eaton, however, suggested that this molecule has the potential to act as a benzene bioisostere. Herein, we report the validation of Eaton's hypothesis with cubane derivatives of five molecules that are used clinically or as agrochemicals. Two cubane analogues showed increased bioactivity compared to their benzene counterparts whereas two further analogues displayed equal bioactivity, and the fifth one demonstrated only partial efficacy. Ramifications from this study are best realized by reflecting on the number of bioactive molecules that contain a benzene ring. Substitution with the cubane scaffold where possible could revitalize these systems, and thus expedite much needed lead candidate identification.

Design, synthesis and binding properties of a fluorescent α9ß1/α4ß1 integrin antagonist and its application as an in vivo probe for bone marrow haemopoietic stem cells.

The α9ß1 and α4ß1 integrin subtypes are expressed on bone marrow haemopoietic stem cells and have important roles in stem cell regulation and trafficking. Although the roles of α4ß1 integrin have been thoroughly investigated with respect to HSC function, the role of α9ß1 integrin remains poorly characterised. Small molecule fluorescent probes are useful tools for monitoring biological processes in vivo, to determine cell-associated protein localisation and activation, and to elucidate the mechanism of small molecule mediated protein interactions. Herein, we report the design, synthesis and integrin-dependent cell binding properties of a new fluorescent α9ß1 integrin antagonist (R-BC154), which was based on a series of N-phenylsulfonyl proline dipeptides and assembled using the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. Using transfected human glioblastoma LN18 cells, we show that R-BC154 exhibits high nanomolar binding affinities to α9ß1 integrin with potent cross-reactivity against α4ß1 integrin under physiological mimicking conditions. On-rate and off-rate measurements revealed distinct differences in the binding kinetics between α9ß1 and α4ß1 integrins, which showed faster binding to α4ß1 integrin relative to α9ß1, but more prolonged binding to the latter. Finally, we show that R-BC154 was capable of binding rare populations of bone marrow haemopoietic stem and progenitor cells when administered to mice. Thus, R-BC154 represents a useful multi-purpose fluorescent integrin probe that can be used for (1) screening small molecule inhibitors of α9ß1 and α4ß1 integrins; (2) investigating the biochemical properties of α9ß1 and α4ß1 integrin binding and (3) investigating integrin expression and activation on defined cell phenotypes in vivo.

The activity of early-life gene regulatory elements is hijacked in aging through pervasive AP-1-linked chromatin opening.

A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening.

Synthesis, structural elucidation, and biochemical analysis of immunoactive glucuronosyl diacylglycerides of mycobacteria and corynebacteria.

Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop "rules" allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg(2+)/Mn(2+)-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.

Late fetal hematopoietic failure results from ZBTB11 deficiency despite abundant HSC specification.

Hematopoiesis produces diverse blood cell lineages to meet the basal needs and sudden demands of injury or infection. A rapid response to such challenges requires the expansion of specific lineages and a prompt return to balanced steady-state levels, necessitating tightly coordinated regulation. Previously we identified a requirement for the zinc finger and broad complex, tramtrak, bric-a-brac domain-containing 11 (ZBTB11) transcription factor in definitive hematopoiesis using a forward genetic screen for zebrafish myeloid mutants. To understand its relevance to mammalian systems, we extended these studies to mice. When Zbtb11 was deleted in the hematopoietic compartment, embryos died at embryonic day (E) 18.5 with hematopoietic failure. Zbtb11 hematopoietic knockout (Zbtb11hKO) hematopoietic stem cells (HSCs) were overabundantly specified from E14.5 to E17.5 compared with those in controls. Overspecification was accompanied by loss of stemness, inability to differentiate into committed progenitors and mature lineages in the fetal liver, failure to seed fetal bone marrow, and total hematopoietic failure. The Zbtb11hKO HSCs did not proliferate in vitro and were constrained in cell cycle progression, demonstrating the cell-intrinsic role of Zbtb11 in proliferation and cell cycle regulation in mammalian HSCs. Single-cell RNA sequencing analysis identified that Zbtb11-deficient HSCs were underrepresented in an erythroid-primed subpopulation and showed downregulation of oxidative phosphorylation pathways and dysregulation of genes associated with the hematopoietic niche. We identified a cell-intrinsic requirement for Zbtb11-mediated gene regulatory networks in sustaining a pool of maturation-capable HSCs and progenitor cells.

Stabilization and improved functionality of three-dimensional perfusable microvascular networks in microfluidic devices under macromolecular crowding.

BACKGROUND: There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro, which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability. METHODS: Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality. RESULTS: MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature. CONCLUSION: Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions.

High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production.

Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model (Pf4-Srsf3Δ/Δ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans.

Synthesis of glycoconjugate fragments of mycobacterial phosphatidylinositol mannosides and lipomannan.

Mycobacterium tuberculosis, the causitive agent of tuberculosis (TB), possesses a complex cell wall containing mannose-rich glycophospholids termed phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). These glycophospholipids play important roles in cell wall function and host-pathogen interactions. Synthetic PIM/LM/LAM substructures are useful biochemical tools to delineate and dissect the fine details of mannose glycophospholipid biosynthesis and their interactions with host cells. We report the efficient synthesis of a series of azidooctyl di- and trimannosides possessing the following glycan structures: α-Man-1,6-α-Man, α-Man-1,6-α-Man-1,6-α-Man, α-Man-1,2-α-Man-1,6-α-Man and 2,6-di-(α-Man)-α-Man. The synthesis includes the use of non-benzyl protecting groups compatible with the azido group and preparation of the branched trisaccharide structure 2,6-di-(α-Man)-α-Man through a double glycosylation of a 3,4-butanediacetal-protected mannoside. The azidooctyl groups of these synthetic mannans were elaborated to fluorescent glycoconjugates and squaric ester derivatives useful for further conjugation studies.

NogoA-expressing astrocytes limit peripheral macrophage infiltration after ischemic brain injury in primates.

Astrocytes play critical roles after brain injury, but their precise function is poorly defined. Utilizing single-nuclei transcriptomics to characterize astrocytes after ischemic stroke in the visual cortex of the marmoset monkey, we observed nearly complete segregation between stroke and control astrocyte clusters. Screening for the top 30 differentially expressed genes that might limit stroke recovery, we discovered that a majority of astrocytes expressed RTN4A/ NogoA, a neurite-outgrowth inhibitory protein previously only associated with oligodendrocytes. NogoA upregulation on reactive astrocytes post-stroke was significant in both the marmoset and human brain, whereas only a marginal change was observed in mice. We determined that NogoA mediated an anti-inflammatory response which likely contributes to limiting the infiltration of peripheral macrophages into the surviving parenchyma.

Bone Marrow Regulatory T Cells Are a Unique Population, Supported by Niche-Specific Cytokines and Plasmacytoid Dendritic Cells, and Required for Chronic Graft-Versus-Host Disease Control.

Regulatory T cell (Treg) reconstitution is essential for reestablishing tolerance and maintaining homeostasis following stem-cell transplantation. We previously reported that bone marrow (BM) is highly enriched in autophagy-dependent Treg and autophagy disruption leads to a significant Treg loss, particularly BM-Treg. To correct the known Treg deficiency observed in chronic graft-versus-host disease (cGVHD) patients, low dose IL-2 infusion has been administered, substantially increasing peripheral Treg (pTreg) numbers. However, as clinical responses were only seen in ∼50% of patients, we postulated that pTreg augmentation was more robust than for BM-Treg. We show that BM-Treg and pTreg have distinct characteristics, indicated by differential transcriptome expression for chemokine receptors, transcription factors, cell cycle control of replication and genes linked to Treg function. Further, BM-Treg were more quiescent, expressed lower FoxP3, were highly enriched for co-inhibitory markers and more profoundly depleted than splenic Treg in cGVHD mice. In vivo our data are consistent with the BM and not splenic microenvironment is, at least in part, driving this BM-Treg signature, as adoptively transferred splenic Treg that entered the BM niche acquired a BM-Treg phenotype. Analyses identified upregulated expression of IL-9R, IL-33R, and IL-7R in BM-Treg. Administration of the T cell produced cytokine IL-2 was required by splenic Treg expansion but had no impact on BM-Treg, whereas the converse was true for IL-9 administration. Plasmacytoid dendritic cells (pDCs) within the BM also may contribute to BM-Treg maintenance. Using pDC-specific BDCA2-DTR mice in which diptheria toxin administration results in global pDC depletion, we demonstrate that pDC depletion hampers BM, but not splenic, Treg homeostasis. Together, these data provide evidence that BM-Treg and splenic Treg are phenotypically and functionally distinct and influenced by niche-specific mediators that selectively support their respective Treg populations. The unique properties of BM-Treg should be considered for new therapies to reconstitute Treg and reestablish tolerance following SCT.

Nicotinamide riboside attenuates age-associated metabolic and functional changes in hematopoietic stem cells.

With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, damping of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.

Glycosylated Nanoparticles Derived from RAFT Polymerization for Effective Drug Delivery to Macrophages.

The functional group tolerance and simplicity of reversible addition fragmentation chain transfer (RAFT) polymerization enable its use in the preparation of a wide range of functional polymer architectures for a variety of applications, including drug delivery. Given the role of tumor-associated macrophages (TAMs) in cancer and their dependence on the tyrosine kinase receptor FMS (CSF-1R), the key aim of this work was to achieve effective delivery of an FMS inhibitor to cells using a polymer delivery system. Such a system has the potential to exploit biological features specific to macrophages and therefore provide enhanced selectivity. Building on our prior work, we have prepared RAFT polymers based on a poly(butyl methacrylate-co-methacrylic acid) diblock, which were extended with a hydrophilic block, a cross-linker, and a mannose-based monomer scaffold, exploiting the abundance of macrophage mannose receptors (MMRs, CD206) on the surface of macrophages. We demonstrate that the prepared polymers can be assembled into nanoparticles and are successfully internalized into macrophages, in part, via the MMR (CD206). Finally, we showcase the developed nanoparticles in the delivery of an FMS inhibitor to cells, resulting in inhibition of the FMS receptor. As such, this study lays the groundwork for further drug-delivery studies aimed at specifically targeting TAMs with molecularly targeted therapeutics.

Mouse Hematopoietic Stem Cell Modification and Labelling by Transduction and Tracking Posttransplantation.

The tracking of the hematopoietic potential of genetically manipulated fluorescent hematopoietic stem cells (HSC) in the bone marrow (BM) allows the assessment of regulatory processes involved in the re-establishment of hematopoiesis posttransplant. Herein, we describe the means to assess the consequence of expressing specific genes in HSC on their engraftment potential posttransplant.

Osteopontin is An Important Regulative Component of the Fetal Bone Marrow Hematopoietic Stem Cell Niche.

Osteopontin (OPN) is an important component in both bone and blood regulation, functioning as a bridge between the two. Previously, thrombin-cleaved osteopontin (trOPN), the dominant form of OPN in adult bone marrow (BM), was demonstrated to be a critical negative regulator of adult hematopoietic stem cells (HSC) via interactions with α4ß1 and α9ß1 integrins. We now demonstrate OPN is also required for fetal hematopoiesis in maintaining the HSC and progenitor pool in fetal BM. Specifically, we showed that trOPN is highly expressed in fetal BM and its receptors, α4ß1 and α9ß1 integrins, are both highly expressed and endogenously activated on fetal BM HSC and progenitors. Notably, the endogenous activation of integrins expressed by HSC was attributed to high concentrations of three divalent metal cations, Ca2+, Mg2+ and Mn2+, which were highly prevalent in developing fetal BM. In contrast, minimal levels of OPN were detected in fetal liver, and α4ß1 and α9ß1 integrins expressed by fetal liver HSC were not in the activated state, thereby permitting the massive expansion of HSC and progenitors required during early fetal hematopoiesis. Consistent with these results, no differences in the number or composition of hematopoietic cells in the liver of fetal OPN-/- mice were detected, but significant increases in the hematopoietic progenitor pool in fetal BM as well as an increase in the BM HSC pool following birth and into adulthood were observed. Together, the data demonstrates OPN is a necessary negative regulator of fetal and neonatal BM progenitors and HSC, and it exhibits preserved regulatory roles during early development, adulthood and ageing.