The role of Wnt/β-catenin antagonists - sclerostin and DKK1 - in bone homeostasis and mechanotransduction

Promising avenues for improving bone mass and fracture resistance include loading-based exercise and agents modulating Wnt/β-catenin signalling. This thesis examines the intersection between these. Murine models of tibial loading and unloading, genetic knockout (KO) models of Wnt antagonists sclerostin and dickkopf-1/DKK1 (encoded by Sost and Dkk1 genes), and neutralising antibodies for sclerostin (Scl-Ab) were applied. Sost KO mice underwent unloading and compressive loading of the tibiae. Sclerostin was vital for the bone response to unloading yet not for loading. Rather, loading-induced anabolism was synergistically augmented in Sost KO mice. It was hypothesised that other Wnt antagonists, such as DKK1, may up-regulate following long-term sclerostin deficiency. Similarly, Dkk1 KO mice exhibited a synergistically augmented anabolic bone response to loading compared to wild type. However, compensation by sclerostin was doubtful as the primary cause for the augmented response with similar sclerostin expression seen in non-loaded tibiae of Dkk1 KO and wild type mice, and similar down-regulation following loading. Dkk1 KO mice treated with Scl-Ab resulted in additive increases in bone volume above either individual DKK1 or sclerostin deficiency. Prominent and synergistic effects were within cancellous bone. Immunohistochemical staining did not support the hypothesis that sclerostin up-regulates to compensate for long-term DKK1 deficiency. Finally, Scl-Ab was co-administered to mice undergoing tibial compressive loading, with additional anabolic increases seen above either monotherapy. RNA sequencing provided insight into mechanisms involved in the augmented response to loading with Scl-Ab use. This thesis supports future clinical use of antibodies targeting Wnt antagonists, whereby load/resistance exercise or dual-agents may increase the efficacy of Scl-Ab or DKK1-Ab therapy. This may have a critical impact for treatment of osteoporosis and other conditions of bone-loss

Medium Aevum, le Moyen-Âge, Middle Ages

Could Shakespeare have had the concept “Middle Ages”? This article argues that the question itself is anachronistic, depending upon ideas of periodization which developed slowly and reached their current form during the nineteenth century. By contrast, Shakespeare’s ideas of time are consistent, and he and his contemporaries understood language-change in time, but his ideas of pasts and pastness belong in a different conceptual space

Chemotherapy-induced genotoxic damage to bone marrow cells: Long-term implications

© The Author(s) 2018. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. Mesenchymal stem/stromal cells (MSCs) within the bone marrow (BM) are vitally important in forming the micro-environment supporting haematopoiesis after myeloablative chemotherapy. MSCs are known to be damaged phenotypically and functionally by chemotherapy; however, to the best our knowledge, the persistence of genotoxic effects of chemotherapy on the BM micro-environment has not been studied. We therefore aimed to evaluate genotoxic effects of chemotherapy on the BM both in vitro and in vivo, using the comet and micronucleus assays, focussing on the persistence of DNA lesions that may contribute to complications in the patient.The MSC cell line (HS-5) and primary cord blood mononuclear cells (CBMNCs: a source of undamaged DNA) were exposed to the chemotherapeutic agent cyclophosphamide (CY) within a physiologically relevant in vitro model. CY treatment resulted in significant increases in CBMNC DNA damage at all time points tested (3-48 h exposure). Similarly, HS-5 cells exposed to CY exhibited significant increases in DNA damage as measured by the comet assay, with increased numbers of abnormal cells visible in the micronucleus assay. In addition, even 48 h after removal of 48-h CY treatment, DNA damage remains significantly increased in treated cells relative to controls. In patients treated with chemotherapy for haematological malignancy, highly significant increases in damaged DNA were seen in BM cells isolated from one individual 1 year after completion of therapy for acute leukaemia compared with pretreatment (P < 0.001). Similarly, two individuals treated 7 and 17 years previously with chemotherapy exhibited significant increases of damaged DNA in MSC compared with untreated age- and sex-matched controls (P < 0.05). Unlike haematopoietic cells, MSCs are not replaced following a stem cell transplant.Therefore, long-term damage to MSC may impact on engraftment of either allogeneic or autologous transplants. In addition, persistence of DNA lesions may lead to genetic instability, correlating with the significant number of chemotherapy-treated individuals who have therapy-related malignancies

SPHINX-based combination therapy as a potential novel treatment strategy for acute myeloid leukaemia

Introduction: Dysregulated alternative splicing is a prominent feature of cancer. The inhibition and knockdown of the SR splice factor kinase SRPK1 reduces tumour growth in vivo. As a result several SPRK1 inhibitors are in development including SPHINX, a 3-(trifluoromethyl)anilide scaffold. The objective of this study was to treat two leukaemic cell lines with SPHINX in combination with the established cancer drugs azacitidine and imatinib. Materials and Methods: We selected two representative cell lines; Kasumi-1, acute myeloid leukaemia, and K562, BCR-ABL positive chronic myeloid leukaemia. Cells were treated with SPHINX concentrations up to 10μM, and in combination with azacitidine (up to 1.5 μg/ml, Kasumi-1 cells) and imatinib (up to 20 μg/ml, K562 cells). Cell viability was determined by counting the proportion of live cells and those undergoing apoptosis through the detection of activated caspase 3/7. SRPK1 was knocked down with siRNA to confirm SPHINX results. Results: The effects of SPHINX were first confirmed by observing reduced levels of phosphorylated SR proteins. SPHINX significantly reduced cell viability and increased apoptosis in Kasumi-1 cells, but less prominently in K562 cells. Knockdown of SRPK1 by RNA interference similarly reduced cell viability. Combining SPHINX with azacitidine augmented the effect of azacitidine in Kasumi-1 cells. In conclusion, SPHINX reduces cell viability and increases apoptosis in the acute myeloid leukaemia cell line Kasumi-1, but less convincingly in the chronic myeloid leukaemia cell line K562. Conclusion: We suggest that specific types of leukaemia may present an opportunity for the development of SRPK1-targeted therapies to be used in combination with established chemotherapeutic drugs

Genotoxicity of cytokines at chemotherapy-induced 'storm' concentrations in a model of the human bone marrow

Donor cell leukaemia (DCL) is a complication of haematopoietic stem cell transplantation where donated cells become malignant within the patient's bone marrow. As DCL predominates as acute myeloid leukaemia, we hypothesised that the cytokine storm following chemotherapy played a role in promoting and supporting leukaemogenesis. Cytokines have also been implicated in genotoxicity, thus we explored a cell line model of the human BM to secrete myeloid cytokines following drug treatment, and their potential to induce micronuclei. HS-5 human stromal cells were exposed to mitoxantrone (MTX) and chlorambucil (CHL) and for the first time, were profiled for 80 cytokines using an array. Fifty-four cytokines were detected in untreated cells, of which 24 were upregulated, and 10 were downregulated by both drugs. FGF-7 was the lowest cytokine to be detected in both untreated and treated cells. Eleven cytokines not detected at baseline were detected following drug exposure. TNFα, IL6, GM-CSF, G-CSF and TGFβ1 were selected for micronuclei induction. TK6 cells were exposed to these cytokines in isolation and in paired combinations. Only TNFα and TGFβ1 induced micronuclei at healthy concentrations, but all five cytokines induced micronuclei at storm levels, which was further increased when combined in pairs. Of particular concern was that some combinations induced micronuclei at levels above the mitomycin C positive control, however most combinations were less than the sum of micronuclei induced following exposure to each cytokine in isolation. These data infer a possible role for cytokines through chemotherapy-induced cytokine storm, in the instigation and support of leukaemogenesis in the BM, and implicate the need to evaluate individuals for variability in cytokine secretion as a potential risk factor for complications such as DCL. [Abstract copyright: © The Author(s) 2023. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.

Signature of the Simplicial Supermetric

We investigate the signature of the Lund-Regge metric on spaces of simplicial three-geometries which are important in some formulations of quantum gravity. Tetrahedra can be joined together to make a three-dimensional piecewise linear manifold. A metric on this manifold is specified by assigning a flat metric to the interior of the tetrahedra and values to their squared edge-lengths. The subset of the space of squared edge-lengths obeying triangle and analogous inequalities is simplicial configuration space. We derive the Lund-Regge metric on simplicial configuration space and show how it provides the shortest distance between simplicial three-geometries among all choices of gauge inside the simplices for defining this metric (Regge gauge freedom). We show analytically that there is always at least one physical timelike direction in simplicial configuration space and provide a lower bound on the number of spacelike directions. We show that in the neighborhood of points in this space corresponding to flat metrics there are spacelike directions corresponding to gauge freedom in assigning the edge-lengths. We evaluate the signature numerically for the simplicial configuration spaces based on some simple triangulations of the three-sphere (S^3) and three-torus (T^3). For the surface of a four-simplex triangulation of S^3 we find one timelike direction and all the rest spacelike over all of the simplicial configuration space. For the triangulation of T^3 around flat space we find degeneracies in the simplicial supermetric as well as a few gauge modes corresponding to a positive eigenvalue. Moreover, we have determined that some of the negative eigenvalues are physical, i.e. the corresponding eigenvectors are not generators of diffeomorphisms. We compare our results with the known properties of continuum superspace.Comment: 24 pages, RevTeX, 4 eps Figures. Submitted to Classical Quantum Gravit

Amylase production from marine sponge Hymeniacidon perlevis; potentials sustainability benefits

The marine sponge Hymeniacidon perlevis is a globally distributed and invasive species with extensive filter-feeding characteristics. The symbiotic relationship fostered between the sea sponge and the inhabiting microorganism is key in the production of metabolic enzymes which is the focus of this study. Sponge bacterial symbionts were grown on starch agar for 48hrs. Colourimetric analyses of amylase were conducted at 540nm using a spectrophotometric plate reader. Using an X-Bridge column (3.5μM, 4.6x150mm), 80/20 acetonitrile/water in 0.1% ammonium were the conditions used for the liquid chromatography-mass spectrometry (LC-MS) analyses. Seven reducing sugars were used to optimise LC-MS to determine the presence of the crude enzyme formed. Not all the bacterial symbionts isolated from H perlevis produced alpha and beta amylases to break down starch. From the statistical mean of crude enzyme concentrations from the hydrolysis of starch by amylase, isolate seven had the highest optical density (OD) at 0.43475 while isolate twelve had the lowest OD at 0.141417. From the LC-MS analysis, out of the seven sugars, Glucose and maltose constituted > 65% of the reducing sugars formed from the hydrolysis of starch by the amylases. Isolates 3,6 and 7 produced 6.906 mg/l, 12.309 mg/l, and 5.909 mg/l of glucose, while isolates 3,4,5,6 and 7 produced 203.391 mg/l, 176.238 mg/l, 139.938 mg/l, 39.030 mg/l, and 18.809 mg/l of maltose, respectively. Isolate two had the highest amount of maltose at a concentration of 267.237 mg/l while isolate four had the highest amount of glucose concentration of 53.084 mg/l. Enzymes from marine sponge bacteria offer greater potential for a green and sustainable production process. Amylase extraction from bacterial symbionts in H perlevis is sustainable and should be supported. They can serve as reliable sources of revenue for enzyme industries, and applications in food industries and biotechnological processes