Crystal structure of monoclinic calcium pyrophosphate dihydrate (m-CPPD) involved in inflammatory reactions and osteoarthritis

Pure monoclinic calcium pyrophosphate dihydrate (m-CPPD) has been synthesized and characterized by synchrotron powder X-ray diffraction and neutron diffraction. Rietveld refinement of complementary diffraction data has, for the first time, allowed the crystal structure of m-CPPD to be solved. The monoclinic system P21/n was confirmed and unit-cell parameters determined: a = 12.60842 (4), b = 9.24278 (4), c = 6.74885 (2) Å and β = 104.9916 (3)°. Neutron diffraction data especially have allowed the precise determination of the position of H atoms in the structure. The relationship between the m-CPPD crystal structure and that of the triclinic calcium pyrophosphate dihydrate (t-CPPD) phase as well as other pyrophosphate phases involving other divalent cations are discussed by considering the inflammatory potential of these phases and/or their involvement in different diseases. These original structural data represent a key step in the understanding of the mechanisms of crystal formation involved in different types of arthritis and to improve early detection of calcium pyrophosphate (CPP) phases in vivo

Crystallisation of a highly metastable hydrated calcium pyrophosphate phase

A simple and fast synthesis method was set up to obtain pure hydrated calcium pyrophosphate (CPP)phases of biological interest. This work focused on a specific phase synthesised at 25 uC and pH 4.5 in a stirred tank reactor. Powder X-ray diffraction, FTIR spectroscopy, scanning electron microscopy and thermogravimetric analyses revealed that the phase is unknown but presents similarities with a monoclinic tetrahydrated CPP phase (Ca2P2O7?4H2O, m-CPPT b phase) synthesised under the same conditions of pH and temperature. Characterisation of the unreferenced phase (u-CPP) has been performed, especially to better identify its composition, structure and stability, as well as its possible relation to the m-CPPT b phase or to other hydrated CPP phases

Prolonged Complete Response after GEMOX Chemotherapy in a Patient with Advanced Fibrolamellar Hepatocellular Carcinoma

The only currently validated treatment for advanced hepatocellular carcinoma (HCC) is sorafenib. However, sorafenib has been mainly studied in patients with HCC developed in cirrhotic liver. Chemotherapy might be more suitable for patients with HCC in non-cirrhotic liver. We report the case of a young woman with fibrolamellar HCC in a non-cirrhotic liver, with histologically proven metastatic ganglionary relapse after surgical resection of the primary tumour. Chemotherapy with gemcitabine and oxaliplatin (GEMOX regimen) achieved a complete response without relapse five years after discontinuation of chemotherapy. This exceptional case raises the question of clinical trials specifically designed for patients with HCC in non-cirrhotic liver

Structure of the calcium pyrophosphate monohydrate phase (Ca2P2O7·H2O): towards understanding the dehydration process in calcium pyrophosphate hydrates

Calcium pyrophosphate hydrate (CPP, Ca2P2O7·nH2O) and calcium orthophosphate compounds (including apatite, octa­calcium phosphate etc.) are among the most prevalent pathological calcifications in joints. Even though only two dihydrated forms of CPP (CPPD) have been detected in vivo (monoclinic and triclinic CPPD), investigations of other hydrated forms such as tetra­hydrated or amorphous CPP are relevant to a further understanding of the physicochemistry of those phases of biological inter­est. The synthesis of single crystals of calcium pyrophosphate monohydrate (CPPM; Ca2P2O7·H2O) by diffusion in silica gel at ambient temperature and the structural analysis of this phase are reported in this paper. Complementarily, data from synchrotron X-ray diffraction on a CPPM powder sample have been fitted to the crystal parameters. Finally, the relationship between the resolved structure for the CPPM phase and the structure of the tetra­hydrated calcium pyrophosphate [beta] phase (CPPT-[beta]) is discussed

Synthesis and Characterisation of Hydrated Calcium Pyrophosphate Phases of Biological Interest

The details of a synthesis method for biologically relevant hydrated calcium pyrophosphates (CPPs, Ca2P2O7·nH2O) has been elucidated. Control of the pH (from 3.6 to 5.8) and the temperature (from 25 to 90 °C) during the synthesis enabled the preparation of four pure CPP phases within one hour without intermediates: monoclinic and triclinic calcium pyrophosphate dihydrate (CPPD, Ca2P2O7·2H2O), which are the two CPP phases detected in vivo in joints of arthritic patients, monoclinic tetrahydrate β (CPPT, Ca2P2O7·4H2O) and an amorphous phase (a-CPP, Ca2P2O7·nH2O). Four domains corresponding to the four different phases of hydrated calcium pyrophosphate were identified; a-CPP was synthesised over a very wide pH and temperature range (up to 90 °C) within the domain of synthesis conditions explored, including physiological conditions (pH 7.4 and 37 °C). The as-synthesised hydrated CPP phases were characterised by complementary techniques (powder X-ray diffraction, FTIR and Raman spectroscopy, scanning electron microscopy and thermogravimetry) and chemical analyses. Rietveld refinement analyses of the as-synthesised crystalline phases were performed, andthere were significant differences between the m-CPPD Xray diffraction pattern observed and previously published cell parameters. Vibrational spectroscopy allowed the crystalline and amorphous phases synthesised to be clearly distinguished and identified owing to the high flexibility of the pyrophosphate anion. Chemical analyses showed that the synthesis conditions used in this study did not allow significant hydrolysis of the pyrophosphate ions into phosphate ions, and the number of water molecules associated with each synthesised CPP phase was determined by thermogravimetric analysis. Different mechanisms of dehydration were also identified. The study of the formation of synthetic and well-characterised hydrated calcium pyrophosphate phases and their availability in large amounts in vitro could allow progress to be made on the biological role of these phases and their possible transformations. This could also aid their detection in patients suffering from disease caused by calcium salt crystals and could clarify the mechanism by which CPP crystals form and evolve in vivo

From crystalline to amorphous calcium pyrophosphates:a solid state Nuclear Magnetic Resonance perspective

Hydrated calcium pyrophosphates (CPP, Ca2P2O7·nH2O) are a fundamental family of materials among osteoarticular pathologic calcifications. In this contribution, a comprehensive multinuclear NMR (Nuclear Magnetic Resonance) study of four crystalline and two amorphous phases of this family is presented. 1H, 31P and 43Ca MAS (Magic Angle Spinning) NMR spectra were recorded, leading to informative fingerprints characterizing each compound. In particular, different 1H and 43Ca solid state NMR signatures were observed for the amorphous phases, depending on the synthetic procedure used. The NMR parameters of the crystalline phases were determined using the GIPAW (Gauge Including Projected Augmented Wave) DFT approach, based on first-principles calculations. In some cases, relaxed structures were found to improve the agreement between experimental and calculated values, demonstrating the importance of proton positions and pyrophosphate local geometry in this particular NMR crystallography approach. Such calculations serve as a basis for the future ab initio modeling of the amorphous CPP phases. Statement of significance The general concept of NMR crystallography is applied to the detailed study of calcium pyrophosphates (CPP), whether hydrated or not, and whether crystalline or amorphous. CPP are a fundamental family of materials among osteoarticular pathologic calcifications. Their prevalence increases with age, impacting on 17.5% of the population after the age of 80. They are frequently involved or associated with acute articular arthritis such as pseudogout. Current treatments are mainly directed at relieving the symptoms of joint inflammation but not at inhibiting CPP formation nor at dissolving these crystals. The combination of advanced NMR techniques, modeling and DFT based calculation of NMR parameters allows new original insights in the detailed structural description of this important class of biomaterials

Genome-Wide Mapping of Collier In Vivo Binding Sites Highlights Its Hierarchical Position in Different Transcription Regulatory Networks

Collier, the single Drosophila COE (Collier/EBF/Olf-1) transcription factor, is required in several developmental processes, including head patterning and specification of muscle and neuron identity during embryogenesis. To identify direct Collier (Col) targets in different cell types, we used ChIP-seq to map Col binding sites throughout the genome, at mid-embryogenesis. In vivo Col binding peaks were associated to 415 potential direct target genes. Gene Ontology analysis revealed a strong enrichment in proteins with DNA binding and/or transcription-regulatory properties. Characterization of a selection of candidates, using transgenic CRM-reporter assays, identified direct Col targets in dorso-lateral somatic muscles and specific neuron types in the central nervous system. These data brought new evidence that Col direct control of the expression of the transcription regulators apterous and eyes-absent (eya) is critical to specifying neuronal identities. They also showed that cross-regulation between col and eya in muscle progenitor cells is required for specification of muscle identity, revealing a new parallel between the myogenic regulatory networks operating in Drosophila and vertebrates. Col regulation of eya, both in specific muscle and neuronal lineages, may illustrate one mechanism behind the evolutionary diversification of Col biological roles

Inflammatory Potential of Four Different Phases of Calcium Pyrophosphate Relies on NF-κB Activation and MAPK Pathways

Background: Calcium pyrophosphate (CPP) microcrystal deposition is associated with wide clinical phenotypes, including acute and chronic arthritis, that are interleukin 1β (IL-1β)-driven. Two CPP microcrystals, namely monoclinic and triclinic CPP dihydrates (m- and t-CPPD), have been identified in human tissues in different proportions according to clinical features. m-CPP tetrahydrate beta (m-CPPTβ) and amorphous CPP (a-CPP) phases are considered as m- and t-CPPD crystal precursors in vitro.Objectives: We aimed to decipher the inflammatory properties of the three crystalline phases and one amorphous CPP phase and the intracellular pathways involved.Methods: The four synthesized CPP phases and monosodium urate crystals (MSU, as a control) were used in vitro to stimulate the human monocytic leukemia THP-1 cell line or bone marrow-derived macrophages (BMDM) isolated from WT or NLRP3 KO mice. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR; IL-1β, IL-6 and IL-8 production by ELISA; and mitogen-activated protein kinase (MAPK) activation by immunoblot analysis. NF-κB activation was determined in THP-1 cells containing a reporter plasmid. In vivo, the inflammatory potential of CPP phases was assessed with the murine air pouch model via cell analysis and production of IL-1β and CXCL1 in the exudate. The role of NF-κB was determined by a pharmacological approach, both in vivo and in vitro.Results:In vitro, IL-1β production induced by m- and t-CPPD and m-CPPTβ crystals was NLRP3 inflammasome dependent. m-CPPD crystals were the most inflammatory by inducing a faster and higher production and gene expression of IL-1β, IL-6, and IL-8 than t-CPPD, m-CPPTβ and MSU crystals. The a-CPP phase did not show an inflammatory property. Accordingly, m-CPPD crystals led to stronger activation of NF-κB, p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) MAPKs. Inhibition of NF-κB completely abrogated IL-1β and IL-8 synthesis and secretion induced by all CPP crystals. Also, inhibition of JNK and ERK1/2 MAPKs decreased both IL-1β secretion and NF-κB activation induced by CPP crystals. In vivo, IL-1β and CXCL1 production and neutrophil infiltration induced by m-CPPD crystals were greatly decreased by NF-κB inhibitor treatment.Conclusion: Our results suggest that the inflammatory potential of different CPP crystals relies on their ability to activate the MAPK-dependent NF-κB pathway. Studies are ongoing to investigate the underlying mechanisms

Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement

EMT Inducers Catalyze Malignant Transformation of Mammary Epithelial Cells and Drive Tumorigenesis towards Claudin-Low Tumors in Transgenic Mice

The epithelial-mesenchymal transition (EMT) is an embryonic transdifferentiation process consisting of conversion of polarized epithelial cells to motile mesenchymal ones. EMT–inducing transcription factors are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. Supporting oncogenic activity within primary lesions, the TWIST and ZEB proteins can prevent cells from undergoing oncogene-induced senescence and apoptosis by abolishing both p53- and RB-dependent pathways. Here we show that they also downregulate PP2A phosphatase activity and efficiently cooperate with an oncogenic version of H-RAS in malignant transformation of human mammary epithelial cells. Thus, by down-regulating crucial tumor suppressor functions, EMT inducers make cells particularly prone to malignant conversion. Importantly, by analyzing transformed cells generated in vitro and by characterizing novel transgenic mouse models, we further demonstrate that cooperation between an EMT inducer and an active form of RAS is sufficient to trigger transformation of mammary epithelial cells into malignant cells exhibiting all the characteristic features of claudin-low tumors, including low expression of tight and adherens junction genes, EMT traits, and stem cell–like characteristics. Claudin-low tumors are believed to be the most primitive breast malignancies, having arisen through transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Challenging this prevailing view, we propose that these aggressive tumors arise from cells committed to luminal differentiation, through a process driven by EMT inducers and combining malignant transformation and transdifferentiation