ORCID Profile
0000-0002-5772-6051
Current Organisation
Olivia Newton-John Cancer Research Institute
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Protein Targeting And Signal Transduction | Biochemistry and Cell Biology | Optical Physics | Optics And Opto-Electronic Physics | Instruments And Techniques | Quantum Optics And Lasers
ANZSRC Socio-Economic Objective (SEO)
Biological sciences | Scientific instrumentation | Physical sciences |
Related Links
Publications
Fusing ToF-SIMS images for spatial-spectral resolution enhancement using a convolutional neural network
Publisher: Wiley
Date: 10-10-2022
Abstract: Hyperspectral data sets generated by time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) contain valuable spatial‐spectral information characterizing the distribution of atomic and molecular species across a s le surface. Modern ToF‐SIMS instruments have high spatial resolution (in the order of tens of nanometers) relative to most other mass spectrometry imaging (MSI) techniques. However, there is generally a trade‐off between spatial and mass resolution when using different instrument modes. In this study, a convolutional neural network (CNN) fusion method is used to fuse correlated high spatial and high mass resolution ToF‐SIMS hyperspectral data sets. This process generates resolution‐enhanced data, which exhibit both high spatial and mass resolution. The CNN fusion method is applied to ToF‐SIMS images of a simple, well‐characterized gold mesh s le and a significantly more complex biological (tumor) tissue section. The method is compared to another linear fusion method used in the broader MSI community and a substantial improvement is found. This comparison focuses on both visual quality observations as well as statistical similarity measures. This work demonstrates the utility of the CNN fusion method for ToF‐SIMS data, enabling investigation of the atomic and molecular characteristics of surfaces at high spatial and mass spectral resolution.
Dual requirement for the ETS transcription factors Fli-1 and Erg in hematopoietic stem cells and the megakaryocyte lineage
Publisher: Proceedings of the National Academy of Sciences
Date: 18-08-2009
Abstract: Fli-1 and Erg are closely related members of the Ets family of transcription factors. Both genes are translocated in human cancers, including Ewing's sarcoma, leukemia, and in the case of Erg , more than half of all prostate cancers. Although evidence from mice and humans suggests that Fli-1 is required for megakaryopoiesis, and that Erg is required for normal adult hematopoietic stem cell (HSC) regulation, their precise physiological roles remain to be defined. To elucidate the relationship between Fli-1 and Erg in hematopoiesis, we conducted an analysis of mice carrying mutations in both genes. Our results demonstrate that there is a profound genetic interaction between Fli-1 and Erg . Double heterozygotes displayed phenotypes more dramatic than single heterozygotes: severe thrombocytopenia, with a significant deficit in megakaryocyte numbers and evidence of megakaryocyte dysmorphogenesis, and loss of HSCs accompanied by a reduction in the number of committed hematopoietic progenitor cells. These results illustrate an indispensable requirement for both Fli-1 and Erg in normal HSC and megakaryocyte homeostasis, and suggest these transcription factors may coregulate common target genes.
The RNA polymerase I transcription inhibitor CX-5461 cooperates with topoisomerase 1 inhibition by enhancing the DNA damage response in homologous recombination-proficient high-grade serous ovarian ca
Publisher: Springer Science and Business Media LLC
Date: 11-11-2020
DOI: 10.1038/S41416-020-01158-Z
Abstract: Intrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC. Herein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC. We demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo. Our findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.
Targeting RNA Polymerase I transcription synergises with TOP1 inhibition in potentiating the DNA damage response in high-grade serous ovarian cancer
Publisher: Cold Spring Harbor Laboratory
Date: 20-11-2019
DOI: 10.1101/849307
Abstract: Limited effective therapeutic options are available for patients with recurrent high-grade serous carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. We have shown efficacy in poly-ADP ribose polymerase (PARP) inhibitor-resistant HGSC for the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 through its ability to activate a nucleolar-associated DNA damage response (DDR). Here, we screen the protein-coding genome to identify potential targets whose inhibition enhances the efficacy of CX-5461. We identify a network of cooperating inhibitory interactions, including components of homologous recombination (HR) DNA repair and DNA topoisomerase 1 (TOP1). We highlight that CX-5461 combined with topotecan, a TOP1 inhibitor used as salvage therapy in HGSC, induces robust cell cycle arrest and cell death in a panel of HR-proficient HGSC cell lines. The combination potentiates a nucleolar-associated DDR via recruitment of phosphorylated replication protein A (RPA) and ataxia telangiectasia and Rad3 related protein (ATR). CX-5461 plus low-dose topotecan cooperate to potently inhibit xenograft tumour growth, indicating the potential for this strategy to improve salvage therapeutic regimens to treat HGSC.
Influence of Scribble polarity complex on hematopoiesis and leukemia-a matter of where, when and how
Publisher: Impact Journals, LLC
Date: 05-10-2018
The identification and characterisation of transplanted hematopoietic stem cells in situ
Publisher: Oxford University Press (OUP)
Date: 08-2005
Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis.
Publisher: Springer Science and Business Media LLC
Date: 07-06-2013
DOI: 10.1038/CDD.2013.59
Megakaryocyte Response to Thrombin - Rebuttal
Publisher: Georg Thieme Verlag KG
Date: 1988
Heat-induced alterations in the localization of HSP72 and HSP73 as measured by indirect immunohistochemistry and immunogold electron microscopy
Publisher: SAGE Publications
Date: 03-2000
DOI: 10.1177/002215540004800302
Abstract: The heat shock proteins are a family of stress-inducible proteins that act as molecular chaperones for nascent proteins and assist in protection and repair of proteins whose conformation is altered by stress. HSP72 and HSP73 are two major cytosolic/nuclear stress proteins of mammalian cells, with extensive sequence homology. HSP73 is constitutively expressed, whereas HSP72 is highly stress-inducible. However, it is unclear why two isoforms are expressed and whether these two proteins have different functions in the cell. To assist in the delineation of function, we have completed a detailed study of the localization of HSP72 and HSP73 in the cell before and after heat stress, using two different methods of detection. By indirect immunohistochemistry, the localization of these two proteins is similar, cytoplasmic and nuclear in nonstressed cells with a translocation to nucleoli immediately after heat. By the more sensitive immunogold electron microscopy technique, differences in localization were noted. In nonstressed cells, HSP72 was primarily nuclear, localized in heterochromatic regions and in nucleoli. HSP73 was distributed throughout the cell, with most cytoplasmic label associated with mitochondria. Mitotic chromosomes were also heavily labeled. After stress, HSP72 concentrated in nuclei and nucleoli and HSP73 localized to nuclei, nucleoli, and cytoplasm, with increased label over mitochondria. These differences in localization suggest that the HSP72 and HSP73 may associate with different proteins or complexes and hence have different but overlapping functions in the cell.
Shared roles for Scl and Lyl1 in murine platelet production and function
Publisher: American Society of Hematology
Date: 05-09-2019
Abstract: The stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl. These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.
Granzyme M mediates a novel form of perforin-dependent cell death
Publisher: Elsevier BV
Date: 05-2004
Intrinsic apoptosis circumvents the functional decline of circulating platelets but does not cause the storage lesion
Publisher: American Society of Hematology
Date: 12-07-2018
DOI: 10.1182/BLOOD-2017-11-816355
Abstract: BAK/BAX depletion in murine platelets reveals that intrinsic apoptosis is not required for the development of the platelet storage lesion. Restriction of platelet life span by intrinsic apoptosis is pivotal to maintain a functional, hemostatically reactive platelet population.
Perturbed desmosomal cadherin expression in grainy head-like 1-null mice
Publisher: Wiley
Date: 21-02-2008
Mcl-1 and Bcl-xL coordinately regulate megakaryocyte survival
Publisher: American Society of Hematology
Date: 14-06-2012
DOI: 10.1182/BLOOD-2011-12-398834
Abstract: Mature megakaryocytes depend on the function of Bcl-xL, a member of the Bcl-2 family of prosurvival proteins, to proceed safely through the process of platelet shedding. Despite this, loss of Bcl-xL does not prevent the growth and maturation of megakaryocytes, suggesting redundancy with other prosurvival proteins. We therefore generated mice with a megakaryocyte-specific deletion of Mcl-1, which is known to be expressed in megakaryocytes. Megakaryopoiesis, platelet production, and platelet lifespan were unperturbed in Mcl-1Pf4Δ/Pf4Δ animals. However, treatment with ABT-737, a BH3 mimetic compound that inhibits the prosurvival proteins Bcl-2, Bcl-xL, and Bcl-w resulted in the complete ablation of megakaryocytes and platelets. Genetic deletion of both Mcl-1 and Bcl-xL in megakaryocytes resulted in preweaning lethality. Megakaryopoiesis in Bcl-xPf4Δ/Pf4ΔMcl-1Pf4Δ/Pf4Δ embryos was severely compromised, and these animals exhibited ectopic bleeding. Our studies indicate that the combination of Bcl-xL and Mcl-1 is essential for the viability of the megakaryocyte lineage.
A mutation in the translation initiation codon of Gata-1 disrupts megakaryocyte maturation and causes thrombocytopenia
Publisher: Proceedings of the National Academy of Sciences
Date: 19-09-2006
Abstract: We have generated mice from a N -ethyl- N -nitrosourea mutagenesis screen that carry a mutation in the translation initiation codon of Gata-1 , termed Plt13 , which is equivalent to mutations found in patients with acute megakaryoblastic leukemia and Down syndrome. The Gata-1 locus is present on the X chromosome in humans and in mice. Male mice hemizygous for the mutation ( Gata-1 Plt13 /Y) failed to produce red blood cells and died during embryogenesis at a similar stage to Gata-1 -null animals. Female mice that carry the Plt13 mutation are mosaic because of random inactivation of the X chromosome. Adult Gata-1 Plt13 /+ females were not anemic, but they were thrombocytopenic and accumulated abnormal megakaryocytes without a concomitant increase in megakaryocyte progenitor cells. Gata-1 Plt13 /+ mice contained large numbers of blast-like colony-forming cells, particularly in the fetal liver, but also in adult spleen and bone marrow, from which continuous mast cells lines were readily derived. Although the equivalent mutation to Gata-1 Plt13 in humans results in production of GATA-1s, a short protein isoform initiated from a start codon downstream of the mutated initiation codon, Gata-1s was not detected in Gata-1 Plt13 /+ mice.
Chronic rhinosinusitis patients display an aberrant immune cell localization with enhanced S aureus biofilm metabolic activity and biomass
Publisher: Elsevier BV
Date: 03-2007
Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity
Publisher: Wiley
Date: 22-01-2016
DOI: 10.1111/FEBS.13649
Abstract: The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.
Impaired lactation in mice expressing dominant‐negative FADD in mammary epithelium
Publisher: Wiley
Date: 19-03-2009
DOI: 10.1002/DVDY.21917
Abstract: The Fas-associated death domain (FADD/Mort1) adaptor protein was originally identified as a key mediator of apoptosis, although pleiotropic functions for FADD have also been reported. FADD-mediated tumoricidal effects have been described in breast cancer cells however, its physiological role in normal mammary gland epithelium is not well understood. To determine the role of FADD signaling during mammary gland development, we generated transgenic mice overexpressing dominant-negative FADD (DN-FADD) in mammary epithelium, using the steroid responsive mouse mammary tumor virus promoter. Transgenic mice exhibited a perturbation in lactation resulting in impaired milk production and pup growth retardation. Reduced expansion of alveoli was evident during early lactation with extensive shedding of luminal alveolar cells. Significantly more TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling)-positive cells were present at this time point and a subsequent increase in bromodeoxyuridine-positive cells was observed. These findings suggest a role for FADD in maintaining the survival of mammary secretory alveolar cells after the establishment of lactation.
Adult Neural Precursor Cells from the Subventricular Zone Contribute Significantly to Oligodendrocyte Regeneration and Remyelination
Publisher: Society for Neuroscience
Date: 15-10-2014
DOI: 10.1523/JNEUROSCI.3491-13.2014
Abstract: Parenchymal oligodendrocyte progenitor cells (pOPCs) are considered the principal cell type responsible for oligodendrogenesis and remyelinaton in demyelinating diseases. Recent studies have demonstrated that neural precursor cells (NPCs) from the adult subventricular zone (SVZ) can also generate new oligodendrocytes after demyelination. However, the relative contribution of NPCs versus pOPCs to remyelination is unknown. We used in vivo genetic fate mapping to assess the behavior of each progenitor type within the corpus callosi (CCs) of mice subjected to cuprizone-induced demyelination. Nestin-CreER T2 and Pdgfra-CreER T2 transgenic mice were crossed with fluorescent Cre reporter strains to map the fate of NPCs and pOPCs respectively. In cuprizone-challenged mice, substantial numbers of NPCs migrated into the demyelinated CC and contributed to oligodendrogenesis. This capacity was most prominent in rostral regions adjacent to the SVZ where NPC-derived oligodendrocytes significantly outnumbered those generated from pOPCs. Sixty-two percent of all nodes of Ranvier in this region were flanked by at least one paranode generated from an NPC-derived oligodendrocyte. Remarkably, g-ratios (ratio of the axon diameter to the diameter of the axon plus myelin sheath) of myelinated axons in regions subject to significant NPC-derived remyelination were equivalent to those of unchallenged controls, and immunoelectron microscopy revealed that NPC-derived myelin was significantly thicker than that generated by pOPCs, regardless of axonal caliber. We also demonstrate that a reduced efficiency of remyelination in the caudal CC was associated with long-term impairment in the maturation of oligodendrogenic NPCs but only transient delay in pOPC differentiation. Collectively, our data define a major distinct role for NPCs in remyelination, identifying them as a key target for enhancing myelin repair in demyelinating diseases.
Combined deficiency of PI3KC2α and PI3KC2β reveals a nonredundant role for PI3KC2α in regulating mouse platelet structure and thrombus stability
Publisher: Informa UK Limited
Date: 04-03-2016
DOI: 10.3109/09537104.2016.1145202
Abstract: The physiological functions and cellular signaling of Class II phosphoinositide 3-kinases (PI3Ks) remain largely unknown. Platelets express two Class II PI3Ks: PI3KC2α and PI3KC2β. PI3KC2α deficiency was recently reported to cause disruption of the internal membrane reserve structure of platelets (open canalicular system, OCS) that results in dysregulated platelet adhesion and impaired arterial thrombosis in vivo. Notably, these effects on platelets occurred despite normal agonist-induced 3-phosphorylated phosphoinositide (3-PPI) production and cellular activation in PI3KC2α-deficient platelets. However, the potential compensatory actions of PI3KC2β in platelets have not yet been investigated. Here, we report the first mice deficient in both PI3KC2α and PI3KC2β (no Class II PI3Ks in platelets) and reveal a nonredundant role for PI3KC2α in mouse platelet structure and function. Specifically, we show that the disrupted OCS and impaired thrombus stability observed in PI3KC2α-deficient platelets does not occur in PI3KC2β-deficient platelets and is not exaggerated in platelets taken from mice deficient in both enzymes. Furthermore, detailed examination of 3-PPI production in platelets from this series of mice revealed no changes in either unactivated or activated platelets, including those with a complete lack of Class II PI3Ks. These findings indicate a nonredundant role for PI3KC2α in regulating platelet structure and function, and suggest that Class II PI3Ks do not significantly contribute to the acute agonist-induced production of 3-PPIs in these cells.
Dual-specific Chimeric Antigen Receptor T Cells and an Indirect Vaccine Eradicate a Variety of Large Solid Tumors in an Immunocompetent, Self-antigen Setting
Publisher: American Association for Cancer Research (AACR)
Date: 14-05-2016
DOI: 10.1158/1078-0432.CCR-16-1860
Abstract: Purpose: While adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors for most cancers. We aimed to develop an immunotherapy approach effective against large tumors in an immunocompetent, self-antigen preclinical mouse model. Experimental Design: In this study, we generated dual-specific T cells expressing both a CAR specific for Her2 and a TCR specific for the melanocyte protein (gp100). We used a regimen of adoptive cell transfer incorporating vaccination (ACTIV), with recombinant vaccinia virus expressing gp100, to treat a range of tumors including orthotopic breast tumors and large liver tumors. Results: ACTIV therapy induced durable complete remission of a variety of Her2+ tumors, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Vaccinia virus induced extensive proliferation of T cells, leading to massive infiltration of T cells into tumors. Durable tumor responses required the chemokine receptor CXCR3 and exogenous IL2, but were independent of IFNγ. Mice were resistant to tumor rechallenge, indicating immune memory involving epitope spreading. Evidence of limited neurologic toxicity was observed, associated with infiltration of cerebellum by T cells, but was only transient. Conclusions: This study supports a view that it is possible to design a highly effective combination immunotherapy for solid cancers, with acceptable transient toxicity, even when the target antigen is also expressed in vital tissues. Clin Cancer Res 23(10) 2478–90. ©2016 AACR.
ULTRASTRUCTURE AND INNERVATION OF THE SWIMBLADDER OF TETRACTENOS-GLABER (TETRAODONTIDAE)
Publisher: Springer Science and Business Media LLC
Date: 08-1984
DOI: 10.1007/BF00217146
Abstract: The deactivated CRISPR/Cas9 (dCas9) is now the most widely used gene activator. However, current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, the CRISPR-assisted
Protection from endogenous perforin: glycans and the C terminus regulate exocytic trafficking in cytotoxic lymphocytes.
Publisher: Elsevier BV
Date: 06-2011
DOI: 10.1016/J.IMMUNI.2011.04.007
Abstract: Cytotoxic lymphocyte-mediated apoptosis is dependent on the delivery of perforin to secretory granules and its ability to form calcium-dependent pores in the target cell after granule exocytosis. It is unclear how cytotoxic lymphocytes synthesize and store perforin without incurring damage or death. We discovered that the extreme C terminus of perforin was essential for rapid trafficking from the endoplasmic reticulum to the Golgi compartment. Substitution of the C-terminal tryptophan residue resulted in retention of perforin in the ER followed by calcium-dependent toxic activity that eliminated host cells. We also found that N-linked glycosylation of perforin was critical for transport from the Golgi to secretory granules. Overall, an intact C terminus and N-linked glycosylation provide accurate and efficient export of perforin from the endoplasmic reticulum to the secretory granules and are critical for cytotoxic lymphocyte survival.
The Biodistribution and Immune Suppressive Effects of Breast Cancer–Derived Exosomes
Publisher: American Association for Cancer Research (AACR)
Date: 30-11-2016
DOI: 10.1158/0008-5472.CAN-16-0868
Abstract: Small membranous secretions from tumor cells, termed exosomes, contribute significantly to intercellular communication and subsequent reprogramming of the tumor microenvironment. Here, we use optical imaging to determine that exogenously administered fluorescently labeled exosomes derived from highly metastatic murine breast cancer cells distributed predominantly to the lung of syngeneic mice, a frequent site of breast cancer metastasis. At the sites of accumulation, exosomes were taken up by CD45+ bone marrow–derived cells. Subsequent long-term conditioning of naïve mice with exosomes from highly metastatic breast cancer cells revealed the accumulation of myeloid-derived suppressor cells in the lung and liver. This favorable immune suppressive microenvironment was capable of promoting metastatic colonization in the lung and liver, an effect not observed from exosomes derived from nonmetastatic cells and liposome control vesicles. Furthermore, we determined that breast cancer exosomes directly suppressed T-cell proliferation and inhibited NK cell cytotoxicity, and hence likely suppressed the anticancer immune response in premetastatic organs. Together, our findings provide novel insight into the tissue-specific outcomes of breast cancer–derived exosome accumulation and their contribution to immune suppression and promotion of metastases. Cancer Res 76(23) 6816–27. ©2016 AACR.
The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production
Publisher: American Society of Hematology
Date: 03-03-2022
Abstract: 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.
c-Myb is critical for murine colon development
Publisher: Springer Science and Business Media LLC
Date: 13-10-1999
Abstract: The mammalian colon develops from a simple tube of undifferentiated cells into a complex, highly ordered organ, with a continuously self-renewing epithelial layer. We have previously described c-Myb expression in the epithelia of murine and human colon crypts and documented increased expression in colorectal adenocarcinoma cells. To investigate the role of c-Myb in colonic epithelium development, we have used embryos with a disrupted c-myb gene. Prior to the in utero death of these embryos at E15, we excised colon tissue and transplanted it under the kidney capsule of recipient mice to allow further development and cyto-differentiation. Compared to the colons of wildtype and heterozygous littermates, the c-myb homozygous knockout colon is highly irregular with a disordered epithelium and abnormal crypts. In addition, the expression of Bcl-2, a known target of c-Myb, is reduced and apoptosis is increased, indicating a critical requirement for c-Myb in normal colon development.
SCRIBBLE IMPACTS ON THYMOCYTE NUMBERS IN VIVO
Publisher: Elsevier BV
Date: 09-2016
Hsp70 Architecture: The Formation of Novel Polymeric Structures of Hsp70.1 and Hsc70 after Proteotoxic Stress
Publisher: Public Library of Science (PLoS)
Date: 19-12-2012
Natural Compounds from the Marine Brown Alga Caulocystis cephalornithos with Potent In Vitro-Activity against the Parasitic Nematode Haemonchus contortus
Publisher: MDPI AG
Date: 09-07-2020
Abstract: Eight secondary metabolites (1 to 8) were isolated from a marine sponge, a marine alga and three terrestrial plants collected in Australia and subsequently chemically characterised. Here, these natural product-derived compounds were screened for in vitro-anthelmintic activity against the larvae and adult stages of Haemonchus contortus (barber’s pole worm)—a highly pathogenic parasitic nematode of ruminants. Using an optimised, whole-organism screening system, compounds were tested on exsheathed third-stage larvae (xL3s) and fourth-stage larvae (L4s). Anthelmintic activity was initially evaluated on these stages based on the inhibition of motility, development and/or changes in morphology (phenotype). We identified two compounds, 6-undecylsalicylic acid (3) and 6-tridecylsalicylic acid (4) isolated from the marine brown alga, Caulocystis cephalornithos, with inhibitory effects on xL3 and L4 motility and larval development, and the induction of a “skinny-straight” phenotype. Subsequent testing showed that these two compounds had an acute nematocidal effect (within 1–12 h) on adult males and females of H. contortus. Ultrastructural analysis of adult worms treated with compound 4 revealed significant damage to subcuticular musculature and associated tissues and cellular organelles including mitochondria. In conclusion, the present study has discovered two algal compounds possessing acute anthelmintic effects and with potential for hit-to-lead progression. Future work should focus on undertaking a structure-activity relationship study and on elucidating the mode(s) of action of optimised compounds.
An innovative triple immunogold labeling method to investigate the hemopoietic stem cell niche in situ.
Publisher: Oxford University Press (OUP)
Date: 16-09-2009
DOI: 10.1017/S1431927609990924
Abstract: The ultrastructural study of rare cells within their niche in situ is very difficult. We have developed a method for locating in idual transplanted cells and simultaneously identifying and analyzing the molecules and cellular phenotypes surrounding them in situ using transmission electron microscopy. This innovative method involves triple immunogold labeling combined with serial ultrathin sectioning. We demonstrate the validity of this approach by examining the niche of in idual transplanted cells from a population highly enriched for hemopoietic stem cells and the ultrastructural expression of two key stem cell regulatory molecules, hyaluronic acid and osteopontin. In addition, we describe the phenotypes of the surrounding cells.
Sec15 is an effector for the Rab11 GTPase in mammalian cells
Publisher: Elsevier BV
Date: 10-2004
The location and cellular composition of the hemopoietic stem cell niche
Publisher: Elsevier BV
Date: 02-2012
Programmed Anuclear Cell Death Delimits Platelet Life Span
Publisher: Elsevier BV
Date: 03-2007
DOI: 10.1016/J.CELL.2007.01.037
Abstract: Platelets are anuclear cytoplasmic fragments essential for blood clotting and wound healing. Despite much speculation, the factors determining their life span in the circulation are unknown. We show here that an intrinsic program for apoptosis controls platelet survival and dictates their life span. Pro-survival Bcl-x(L) constrains the pro-apoptotic activity of Bak to maintain platelet survival, but as Bcl-x(L) degrades, aged platelets are primed for cell death. Genetic ablation or pharmacological inactivation of Bcl-x(L) reduces platelet half-life and causes thrombocytopenia in a dose-dependent manner. Deletion of Bak corrects these defects, and platelets from Bak-deficient mice live longer than normal. Thus, platelets are, by default, genetically programmed to die by apoptosis. The antagonistic balance between Bcl-x(L) and Bak constitutes a molecular clock that determines platelet life span: this represents an important paradigm for cellular homeostasis, and has profound implications for the diagnosis and treatment of disorders that affect platelet number and function.
The role of Tenascin C in the lymphoid progenitor cell niche.
Publisher: Elsevier BV
Date: 12-2013
DOI: 10.1016/J.EXPHEM.2013.09.009
Abstract: Hemopoietic stem cells (HSCs) are extrinsically controlled by the bone marrow (BM) microenvironment. Mice devoid of the extracellular matrix molecule Tenascin-C (TNC) were reported to develop normally. The current study explores the relationship between TNC and hemopoiesis, from HSCs within their niche to maturing progenitors in alternate niches. Although the absence of TNC did not alter the size of the BM stem cell pool, we report decreased thymic T cell progenitors with redistribution to other lymphoid organs, suggesting an anchoring role for TNC. TNC did not play an essential role in stem and progenitor cell homing to BM, but significantly altered lymphoid primed progenitor cell homing. These cells express the TNC receptor, integrin α9β1, with the same reduced homing evident in the absence of this integrin. The absence of TNC also resulted in an increased proportion and number of mature circulating T cells. In addition, the absence of TNC significantly impaired hemopoietic reconstitution after transplant and increased stem and progenitor cell mobilization. In summary, our analysis revealed unidentified roles for TNC in hemopoiesis: in lineage commitment of thymic T cell progenitors, peripheral T cell migration, and hemopoietic reconstitution.
The Asymmetric Cell Division Regulators Par3, Scribble and Pins/Gpsm2 Are Not Essential for Erythroid Development or Enucleation
Publisher: Public Library of Science (PLoS)
Date: 17-01-2017
ROLE OF THE POLARITY PROTEIN, SCRIBBLE, IN HEMATOPOIESIS AND LEUKEMIA
Publisher: Elsevier BV
Date: 08-2014
BMPs regulate differentiation of a putative visceral endoderm layer within human embryonic stem-cell-derived embryoid bodies
Publisher: Canadian Science Publishing
Date: 02-2007
DOI: 10.1139/O06-145
Abstract: Human embryonic stem cells (HESCs), pluripotent cells derived from the inner cell mass (ICM) of human blastocysts, represent a novel tool for the study of early human developmental events. When cultured in suspension with serum, HESCs form spherical structures resembling embryoid bodies (EBs). We show that differentiation of HESCs within EBs occurs radially, with central cells then undergoing apoptosis in association with EB cavitation. Cells within the outer layer of cavitating EBs display stage-specific immunoreactivity to pan-keratin, cytokeratin-8, GATA6, α-fetoprotein, and transthyretin specific antibodies, and hybridization to disabled-2, GATA4, and GATA6 specific riboprobes. Transmission electron microscopy of these cells reveals clathrin-coated micropinocytotic vesicles, microvilli, and many vacuoles, a phenotype consistent with mouse visceral endoderm (VE) rather than mouse definitive or parietal endoderm. When cultured in media supplemented with the BMP inhibitor noggin, or in the absence of serum, HESC derivatives do not develop the mouse VE-like phenotype. The addition of BMP-4 to noggin-treated HESCs cultured in serum or in serum-free conditions reconstituted development of the VE-like phenotype. These data demonstrate that human EBs undergo developmental events similar to those of mouse EBs and that in vitro BMP signalling induces derivatives of the human ICM to express a phenotype similar to mouse VE.
Extended Platelet In Vivo Survival Results in Exhausted Platelets
Publisher: American Society of Hematology
Date: 03-12-2015
DOI: 10.1182/BLOOD.V126.23.416.416
Abstract: Platelet lifespan is limited to 10 days in humans and 5 days in mice. The intrinsic apoptosis pathway regulates the survival of platelets, where the pro-survival protein Bcl-xLrestrains the essential death mediators Bak and Bax (Mason et al., Cell 2007). Hence, platelet lifespan and platelet counts in mice are increased in the absence of Bak and Bak/Bax. While platelet production in mice is normal in the absence of intrinsic apoptosis (Josefsson et al., J Exp Med 2011), the function of these long-lived platelets has not been investigated. In the current study we examined the functional outcome of extended platelet survival. We found that washed platelets from mice with a constitutive deletion of Bak and a platelet-specific deletion of Bax (Bak-/-BaxPf4Δ/Pf4Δ) were fully resistant to apoptosis induced by the BH3-mimetic ABT-737, as demonstrated by lack of phosphatidylserine exposure (binding of AnnexinV) and unaltered mitochondrial membrane potential. Tail bleeding times into 37°C saline, were extended in the absence of either Bak alone or both Bak and Bax. Furthermore, the electrolytic thrombosis model showed that despite normal time to arterial occlusion, the thrombi formed in Bak-/- BaxPf4Δ/Pf4Δ mice were unstable, a trend also observed in Bak-/-Baxfl/fl mice. The formation of stable thrombi is dependent on the release of secondary agonists, such as ADP and Thromboxane, from activated platelets. To investigate potential defects in platelet signaling pathways in the absence of Bax and/or Bak, we performed in vitro platelet activation assays. Flow cytometric measurements revealed that activation of the PAR4 receptor (by PAR4-AP) or GPVI (by convulxin) led to reduced integrin activation (JON/A) and degranulation (P-selectin exposure) in the absence of Bak and Bak/Bax, while loss of Bax alone had no effect. In contrast, the response to activation with ADP, which does not induce granule release, was similar in platelets from all genotypes. Similarly, platelet aggregation in response to intermediate concentrations of PAR4-AP was severely reduced in the absence of Bak and Bak/Bax, but normal in response to ADP. We next investigated if abnormal degranulation in response to agonists could explain the aggregation defect. Platelet aggregation was performed with PAR4-AP and the platelet supernatants were collected after centrifugation. Dense granule release (ATP and serotonin) and alpha granule release (PF4) were significantly reduced from platelets deficient in Bak, Bak/Bax, but not Bax alone. Untreated resting platelets of all genotypes contained similar amount of granular proteins (ATP, serotonin and PF4). Hence, altered granule content was not the reason behind the abnormality. We next explored if platelet age was a factor behind the observed functional differences. To be able to directly compare platelet function in Bak/Bax deficient mice and wild-type controls, we synchronized platelet age to ~3 days in all genotypes. Platelets were depleted in vivo by injection of anti-platelet serum (APS). Newly generated platelets were collected at 72 h post injection, a time-point were platelet counts had returned to normal. Remarkably, synchronized platelet age normalized PAR4-AP and convulxin dependent integrin activation (JON/A) and degranulation (P-selectin exposure) in the absence of Bak and Bak/Bax to control levels. Similarly, the platelet aggregation and release defects were rescued. Lastly we investigated if synchronizing platelet age would revert the hemostatic defect of Bak/Bax mice in vivo. We determined tail bleeding times using mice, which were either untreated or depleted of platelets 72 h prior to the experiment. Strikingly, synchronization of platelet age to 3 days rescued the hemostatic defect in Bak-/-BaxPf4Δ/Pf4Δ mice. We conclude that extended platelet survival leads to platelet exhaustion, with reduced ability to mobilize granular release. Our studies suggest that, in the context of blood bank storage, extending platelet survival times by pharmacologically inhibiting apoptosis may result in a hemostatically compromised product. No relevant conflicts of interest to declare.
A Homolog of Drosophila grainy head Is Essential for Epidermal Integrity in Mice
Publisher: American Association for the Advancement of Science (AAAS)
Date: 15-04-2005
Abstract: The Drosophila cuticle is essential for maintaining the surface barrier defenses of the fly. Integral to cuticle resilience is the transcription factor grainy head , which regulates production of the enzyme required for covalent cross-linking of the cuticular structural components. We report that formation and maintenance of the epidermal barrier in mice are dependent on a mammalian homolog of grainy head, Grainy head-like 3 . Mice lacking this factor display defective skin barrier function and deficient wound repair, accompanied by reduced expression of transglutaminase 1, the key enzyme involved in cross-linking the structural components of the superficial epidermis. These findings suggest that the functional mechanisms involving protein cross-linking that maintain the epidermal barrier and induce tissue repair are conserved across 700 million years of evolution.
Mammalian inositol polyphosphate 5-phosphatase II can compensate for the absence of all three yeast Sac1-like-domain-containing 5-phosphatases
Publisher: Portland Press Ltd.
Date: 24-04-2001
DOI: 10.1042/BJ3550805
Abstract: Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] plays a complex role in generating intracellular signalling molecules, and also in regulating actin-binding proteins, vesicular trafficking and vacuolar fusion. Four inositol polyphosphate 5-phosphatases (hereafter called 5-phosphatases) have been identified in Saccharomyces cerevisiae: Inp51p, Inp52p, Inp53p and Inp54p. Each enzyme contains a 5-phosphatase domain which hydrolyses PtdIns(4,5)P2, forming PtdIns4P, while Inp52p and Inp53p also express a polyphosphoinositide phosphatase domain within the Sac1-like domain. Disruption of any two yeast 5-phosphatases containing a Sac1-like domain results in abnormalities in actin polymerization, plasma membrane, vacuolar morphology and bud-site selection. Triple null mutant 5-phosphatase strains are non-viable. To investigate the role of PtdIns(4,5)P2 in mediating the phenotype of double and triple 5-phosphatase null mutant yeast, we determined whether a mammalian PtdIns(4,5)P2 5-phosphatase, 5-phosphatase II, which lacks polyphosphoinositide phosphatase activity, could correct the phenotype of triple 5-phosphatase null mutant yeast and restore cellular PtdIns(4,5)P2 levels to near basal values. Mammalian 5-phosphatase II expressed under an inducible promoter corrected the growth, cell wall, vacuolar and actin polymerization defects of the triple 5-phosphatase null mutant yeast strains. Cellular PtdIns(4,5)P2 levels in various 5-phosphatase double null mutant strains demonstrated significant accumulation (4.5-, 3- and 2-fold for ∆inp51∆inp53, ∆inp51∆inp52 and ∆inp52∆inp53 double null mutants respectively), which was corrected significantly following 5-phosphatase II expression. Collectively, these studies demonstrate the functional and cellular consequences of PtdIns(4,5)P2 accumulation and the evolutionary conservation of function between mammalian and yeast PtdIns(4,5)P2 5-phosphatases.
The testis and epididymis are productively infected by SIV and SHIV in juvenile macaques during the post-acute stage of infection
Publisher: Springer Science and Business Media LLC
Date: 2007
A role for Abca12 in regulating terminal differentiation and lipid balance in the developing epidermis
Publisher: Elsevier BV
Date: 08-2009
The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells
Publisher: Springer Science and Business Media LLC
Date: 25-05-2008
DOI: 10.1038/NI.1617
Abstract: Ets-related gene (ERG), which encodes a member of the Ets family of transcription factors, is a potent oncogene. Chromosomal rearrangements involving ERG are found in acute myeloid leukemia, acute lymphoblastic leukemia, Ewing's sarcoma and more than half of all prostate cancers however, the normal physiological function of Erg is unknown. We did a sensitized genetic screen of the mouse for regulators of hematopoietic stem cell function and report here a germline mutation of Erg. We show that Erg is required for definitive hematopoiesis, adult hematopoietic stem cell function and the maintenance of normal peripheral blood platelet numbers.
THE POLARITY PROTEIN, PAR3, IS A KEY REGULATOR OF HAEMATOPOIETIC PROGENITORS
Publisher: Elsevier BV
Date: 09-2015
The class II PI 3-kinase, PI3KC2α, links platelet internal membrane structure to shear-dependent adhesive function
Publisher: Springer Science and Business Media LLC
Date: 17-03-2015
DOI: 10.1038/NCOMMS7535
Abstract: PI3KC2α is a broadly expressed lipid kinase with critical functions during embryonic development but poorly defined roles in adult physiology. Here we utilize multiple mouse genetic models to uncover a role for PI3KC2α in regulating the internal membrane reserve structure of megakaryocytes (demarcation membrane system) and platelets (open canalicular system) that results in dysregulated platelet adhesion under haemodynamic shear stress. Structural alterations in the platelet internal membrane lead to enhanced membrane tether formation that is associated with accelerated, yet highly unstable, thrombus formation in vitro and in vivo. Notably, agonist-induced 3-phosphorylated phosphoinositide production and cellular activation are normal in PI3KC2α-deficient platelets. These findings demonstrate an important role for PI3KC2α in regulating shear-dependent platelet adhesion via regulation of membrane structure, rather than acute signalling. These studies provide a link between the open canalicular system and platelet adhesive function that has relevance to the primary haemostatic and prothrombotic function of platelets.
RAF1 rearrangements are common in pancreatic acinar cell carcinomas.
Publisher: Elsevier BV
Date: 09-2020
Ultrastructure of primitive hematopoietic stem cells isolated using probes of functional status
Publisher: Elsevier BV
Date: 02-1999
DOI: 10.1016/S0301-472X(98)00017-4
Abstract: The most primitive hematopoietic stem cells capable of longterm reconstitution of the entire hematopoietic system following transplantation are characterized by their ability to exclude both Rhodamine 123 and Hoechst 33342 dyes (Rh/Ho(dull)), and are an appropriate target population for the determination of stem cell ultrastructure. We have used a fluorescence-activated cell sorter to enrich to near purity these rare, highly quiescent cells. Analysis of the in vitro growth characteristics of Rh/Ho(dull) cells demonstrated an obligatory requirement for multiple growth factors, with 62% of the sorted population having the capacity to form colonies in the presence of CSF-1 + IL-1alpha + IL-3 + SCF. The Rh/Ho(dull) cells were small, with profiles having a mean diameter of 4.6 microm. Ultrastructural examination showed numerous ribosomes and several mitochondria in the thin rim of cytoplasm surrounding the nucleus, with other cytoplasmic organelles revealed in serial sections. The cells were generally homogeneous in appearance apart from the nucleus, which had an irregular shape with a single deep indentation. The heterochromatin around the margin was distinctly more pronounced in about 50% of nuclei. The findings provide a basis for studying the structural changes that occur with progressive differentiation of early hematopoietic cells.
The Response of Megakaryocytes with Processes to Thrombin
Publisher: Georg Thieme Verlag KG
Date: 1987
Abstract: The response of megakaryocytes to thrombin (1-10 U/ml) has been examined by time-lapse cinemicrography and electron microscopy. The study was confined to mature megakaryocytes which had developed processes following incubation in vitro. The initial response of all cells was to undergo retraction of processes, behaviour thought to be linked with the depolymerization of microtubules which extend longitudinally through the processes. The majority of cells completely withdrew their processes, but about 30% responded differently and underwent only limited retraction, followed by secretion. Analysis of time-lapse film showed that processes from the latter group of cells had formed attachments with the coverslip prior to exposure to thrombin. Within the partially retracted processes of these cells, secretory granules were found to be clustered centripetally and enveloped by a rnicrofilamentous structure in the form of a cylinder. Vacuoles appeared, some of which were located outside the rnicrofilamentous structure. Microtubules were present, but many appeared disorientated. The shape of the rnicrofilamentous structure suggests that the cytoplasm is not organized into putative platelets at the time of process formation.
A Functional Interaction between CD46 and DLG4
Publisher: Elsevier BV
Date: 02-2002
CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer
Publisher: Springer Science and Business Media LLC
Date: 26-05-2020
DOI: 10.1038/S41467-020-16393-4
Abstract: Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.
The histo-morphometric relationship between bone, blood vessels and hemopoietic stem cells
Publisher: Oxford University Press (OUP)
Date: 08-2008
DOI: 10.1017/S1431927608083104
Abstract: Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008
Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets
Publisher: Rockefeller University Press
Date: 12-09-2011
DOI: 10.1084/JEM.20110750
Abstract: It is believed that megakaryocytes undergo a specialized form of apoptosis to shed platelets. Conversely, a range of pathophysiological insults, including chemotherapy, are thought to cause thrombocytopenia by inducing the apoptotic death of megakaryocytes and their progenitors. To resolve this paradox, we generated mice with hematopoietic- or megakaryocyte-specific deletions of the essential mediators of apoptosis, Bak and Bax. We found that platelet production was unperturbed. In stark contrast, deletion of the prosurvival protein Bcl-xL resulted in megakaryocyte apoptosis and a failure of platelet shedding. This could be rescued by deletion of Bak and Bax. We examined the effect on megakaryocytes of three agents that activate the intrinsic apoptosis pathway in other cell types: etoposide, staurosporine, and the BH3 mimetic ABT-737. All three triggered mitochondrial damage, caspase activation, and cell death. Deletion of Bak and Bax rendered megakaryocytes resistant to etoposide and ABT-737. In vivo, mice with a Bak−/− Bax−/− hematopoietic system were protected against thrombocytopenia induced by the chemotherapeutic agent carboplatin. Thus, megakaryocytes do not activate the intrinsic pathway to generate platelets rather, the opposite is true: they must restrain it to survive and progress safely through proplatelet formation and platelet shedding.
High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production
Publisher: Springer Science and Business Media LLC
Date: 13-04-2023
DOI: 10.1038/S41467-023-37780-7
Abstract: 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-Srsf3 Δ/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.
Diverging impact of cell fate determinants Scrib and Llgl1 on adhesion and migration of hematopoietic stem cells
Publisher: Springer Science and Business Media LLC
Date: 06-08-2018
DOI: 10.1007/S00432-018-2724-3
Abstract: Cell fate determinants Scrib and Llgl1 influence self-renewal capacity of hematopoietic stem cells (HSCs). Scrib-deficient HSCs are functionally impaired and lack sufficient repopulation capacity during serial transplantation and stress. In contrast, loss of Llgl1 leads to increased HSC fitness, gain of self-renewal capacity and expansion of the stem cell pool. Here, we sought to assess for shared and unique molecular functions of Llgl1 and Scrib by analyzing their interactome in hematopoietic cells. Interactome analysis was performed by affinity purification followed by mass spectrometry. Motility, migration and adhesion were assessed on primary murine HSCs, which were isolated by FACS sorting following conditional deletion of Scrib or Llgl1, respectively. Imaging of Scrib-deficient HSCs was performed by intravital 2-photon microscopy. Comparison of Scrib and Llgl1 interactome analyses revealed involvement in common and unique cellular functions. Migration and adhesion were among the cellular functions connected to Scrib but not to Llgl1. Functional validation of these findings confirmed alterations in cell adhesion and migration of Scrib-deficient HSCs in vitro and in vivo. In contrast, genetic inactivation of Llgl1 did not affect adhesion or migratory capacity of hematopoietic stem cells. Our data provide first evidence for an evolutionarily conserved role of the cell fate determinant Scrib in HSC adhesion and migration in vitro and in vivo, a unique function that is not shared with its putative complex partner Llgl1.
A Network of PDZ-Containing Proteins Regulates T Cell Polarity and Morphology during Migration and Immunological Synapse Formation
Publisher: Elsevier BV
Date: 06-2005
DOI: 10.1016/J.IMMUNI.2005.04.009
Abstract: T cell shape is dictated by the selective recruitment of molecules to different regions of the cell (polarity) and is integral to every aspect of T cell function, from migration to cytotoxicity. This study describes a mechanism for the regulation of T cell polarity. We show that T cells contain a network of asymmetrically distributed proteins with the capacity to dictate the subcellular localization of both cell surface receptors and morphological determinants in T cells. Proteins from the Scribble, Crumbs3, and Par3 complexes, previously shown to regulate epithelial polarity, were polarized in T cells containing either uropods or immunological synapses. Reduction in Scribble expression prevented the polarization of cell surface receptors and prevented morphological changes associated with uropod formation, migration, and antigen presentation. By dynamically coordinating molecular distribution throughout the T cell, this network provides a mechanism by which T cell function and polarity are linked.
The polarity protein PARD3 and cancer
Publisher: Springer Science and Business Media LLC
Date: 07-06-2021
DOI: 10.1038/S41388-021-01813-6
Abstract: Tissue disorganisation is one of the main hallmarks of cancer. Polarity proteins are responsible for the arrangement of cells within epithelial tissues through the asymmetric organisation of cellular components. Partition defective 3 (PARD3) is a master regulator of the Par polarity complex primarily due to its ability to form large complexes via its self-homologous binding domain. In addition to its role in polarity, PARD3 is a scaffolding protein that binds to intracellular signalling molecules, many of which are frequently deregulated in cancer. The role of PARD3 has been implicated in multiple solid cancers as either a tumour suppressor or promoter. This dual functionality is both physiologically and cell context dependent. In this review, we will discuss PARD3's role in tumourigenesis in both laboratory and clinical settings. We will also review several of the mechanisms underpinning PARD3's function including its association with intracellular signalling pathways and its role in the regulation of asymmetric cell ision.
Optimization of a transplant model to assess skin reconstitution from stem cell‐enriched primary human keratinocyte populations
Publisher: Wiley
Date: 2005
DOI: 10.1111/J.0906-6705.2005.00252.X
Abstract: Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.
The TAM receptor TYRO3 is a critical regulator of myelin thickness in the central nervous system
Publisher: Wiley
Date: 12-09-2018
DOI: 10.1002/GLIA.23481
Abstract: Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system (CNS). Major deficits arise in MS patients due to an inability to repair damaged myelin sheaths following CNS insult, resulting in prolonged axonal exposure and neurodegeneration. The TAM receptors (Tyro3, Axl, and Mertk) have been implicated in MS susceptibility, demyelination and remyelination. Previously, we have shown that Tyro3 regulates developmental myelination and myelin thickness within the optic nerve and rostral region of the corpus callosum (CC) of adult mice. In this study we have verified and extended our previous findings via a comprehensive analysis of axonal ensheathment and myelin thickness in the CC of unchallenged mice, following demyelination and during myelin repair. We show that the loss of the Tyro3 receptor correlates with significantly thinner myelin sheaths in both unchallenged mice and during remyelination, particularly in larger caliber axons. The hypomyelinated phenotype observed in the absence of Tyro3 occurs independently of any influence upon oligodendrocyte precursor cell (OPC) maturation, or density of oligodendrocytes (OLs) or microglia. Rather, the primary effect of Tyro3 is upon the radial expansion of myelin. The loss of Tyro3 leads to a reduction in the number of myelin lamellae on axons, and is therefore most likely a key component of the regulatory mechanism by which oligodendrocytes match myelin production to axonal diameter.
The relationship between bone,,hemopoietic stem cells,and vasculature
Publisher: American Society of Hematology
Date: 11-08-2011
DOI: 10.1182/BLOOD-2010-08-303800
Abstract: A large body of evidence suggests hemopoietic stem cells (HSCs) exist in an endosteal niche close to bone, whereas others suggest that the HSC niche is intimately associated with vasculature. In this study, we show that transplanted hemopoietic stem and progenitor cells (HSPCs) home preferentially to the trabecular-rich metaphysis of the femurs in nonablated mice at all time points from 15 minutes to 15 hours after transplantation. Within this region, they exist in an endosteal niche in close association with blood vessels. The preferential homing of HSPCs to the metaphysis occurs rapidly after transplantation, suggesting that blood vessels within this region may express a unique repertoire of endothelial adhesive molecules. One candidate is hyaluronan (HA), which is highly expressed on the blood vessel endothelium in the metaphysis. Analysis of the early stages of homing and the spatial dis-tribution of transplanted HSPCs at the single-cell level in mice devoid of Has3-synthesized HA, provides evidence for a previously undescribed role for HA expressed on endothelial cells in directing the homing of HSPCs to the metaphysis.
Thrombocytopenia and kidney disease in mice with a mutation in the C1galt1 gene
Publisher: Proceedings of the National Academy of Sciences
Date: 31-10-2006
Abstract: An N -ethyl- N -nitrosourea mutagenesis screen in mice was performed to isolate regulators of circulating platelet number. We report here recessive thrombocytopenia and kidney disease in plt1 mice, which is the result of a severe but partial loss-of-function mutation in the gene encoding glycoprotein- N -acetylgalactosamine-3-β-galactosyltransferase (C1GalT1), an enzyme essential for the synthesis of extended mucin-type O-glycans. Platelet half-life and basic hemostatic parameters were unaffected in plt1 lt1 mice, and the thrombocytopenia and kidney disease were not attenuated on a lymphocyte-deficient rag1 -null background. gpIbα and podocalyxin were found to be major underglycosylated proteins in plt1 lt1 platelets and the kidney, respectively, implying that these are key targets for C1GalT1, appropriate glycosylation of which is essential for platelet production and kidney function. Compromised C1GalT1 activity has been associated with immune-mediated diseases in humans, most notably Tn syndrome and IgA nephropathy. The disease in plt1 lt1 mice suggests that, in addition to immune-mediated effects, intrinsic C1Gal-T1 deficiency in megakaryocytes and the kidney may contribute to pathology.
A role for pericytes as microenvironmental regulators of human skin tissue regeneration
Publisher: American Society for Clinical Investigation
Date: 03-08-2009
DOI: 10.1172/JCI38535
Tetrahydroquinoxalines induce a lethal evisceration phenotype in Haemonchus contortus in vitro
Publisher: Elsevier BV
Date: 04-2019
An ultrastructural investigation of tumors undergoing regression mediated by immunotherapy
Publisher: Impact Journals, LLC
Date: 14-12-2017
Radiation response of mouse lymphoid and myeloid cell lines. Part II. Apoptotic death is shown by all lines examined
Publisher: Informa UK Limited
Date: 1994
DOI: 10.1080/09553009414550251
Abstract: The mode of death induced by gamma-irradiation in a panel of 10 mouse lymphoid or myeloid cell lines was examined. Four of these lines were known to lose viability (membrane integrity) rapidly after irradiation, whilst the others were known to lose viability considerably more slowly. However, based on the criteria of morphology and DNA degradation pattern, all 10 lines showed apoptotic death. The occurrence of apoptosis after irradiation in rapid-dying lymphoid cell lines was consistent with published results, whilst the demonstration of apoptosis in slow-dying lines was unexpected. Cells of the slow-dying lymphoid lines underwent one or more mitoses prior to death, a feature also reported for fibroblastoid cell lines. However, the occurrence of radiation-induced necrosis in fibroblasts suggests that the pathways leading to 'mitotic death' differ between fibroblastoid and lymphoid cell lines.
A Mouse Model of Harlequin Ichthyosis Delineates a Key Role for Abca12 in Lipid Homeostasis
Publisher: Public Library of Science (PLoS)
Date: 19-09-2008
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View Funded ActivityLinkage Infrastructure, Equipment And Facilities - Grant ID: LE0347356
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