Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracellular medium

<p>Abstract</p> <p>Background</p> <p>A bacterial artificial chromosomal library of <it>Planobispora rosea</it>, a genetically intractable actinomycete strain, was constructed using <it>Escherichia coli</it>-<it>Streptomyces </it>artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics.</p> <p>Results</p> <p>One clone with a 40 kb insert showed antimicrobial activity against Gram positive bacteria. Insert sequence analysis and subcloning experiments revealed that the bioactivity was due to a 3.5 kb DNA fragment containing two open reading frames. These <it>orfs </it>encode two proteins with high similarity to a putative membrane protein of <it>Streptomyces coelicolor </it>and to the nogalamycin resistance protein SnorO of <it>Streptomyces nogalater</it>, respectively. The role of these two Orfs is unknown in <it>Planobispora. </it>Disruption and complementation experiments revealed that both proteins are necessary for the antibacterial activity and chemical analysis demonstrated that the antibiotic activity was due to thiostrepton, antibiotic used as recombinant clone selection marker.</p> <p>Conclusion</p> <p>Two <it>Planobispora rosea orfs </it>are responsible for increasing intracellular amounts and storage of thiostrepton in <it>Streptomyces lividans</it>.</p

Canonical Wnt signaling induces skin fibrosis and subcutaneous lipoatrophy: A novel mouse model for scleroderma?

Objective Because aberrant Wnt signaling has been linked with systemic sclerosis (SSc) and pulmonary fibrosis, we sought to investigate the effect of Wnt‐10b on skin homeostasis and differentiation in transgenic mice and in explanted mesenchymal cells. Methods The expression of Wnt‐10b in patients with SSc and in a mouse model of fibrosis was investigated. The skin phenotype and biochemical characteristics of Wnt‐10b–transgenic mice were evaluated. The in vitro effects of ectopic Wnt‐10b were examined in explanted skin fibroblasts and preadipocytes. Results The expression of Wnt‐10b was increased in lesional skin biopsy specimens from patients with SSc and in those obtained from mice with bleomycin‐induced fibrosis. Transgenic mice expressing Wnt‐10b showed progressive loss of subcutaneous adipose tissue accompanied by dermal fibrosis, increased collagen deposition, fibroblast activation, and myofibroblast accumulation. Wnt activity correlated with collagen gene expression in these biopsy specimens. Explanted skin fibroblasts from transgenic mice demonstrated persistent Wnt/β‐catenin signaling and elevated collagen and α‐smooth muscle actin gene expression. Wnt‐10b infection of normal fibroblasts and preadipocytes resulted in blockade of adipogenesis and transforming growth factor β (TGFβ)–independent up‐regulation of fibrotic gene expression. Conclusion SSc is associated with increased Wnt‐10b expression in the skin. Ectopic Wnt‐10b causes loss of subcutaneous adipose tissue and TGFβ‐independent dermal fibrosis in transgenic mice. These findings suggest that Wnt‐10b switches differentiation of mesenchymal cells toward myofibroblasts by inducing a fibrogenic transcriptional program while suppressing adipogenesis. Wnt‐10b–transgenic mice represent a novel animal model for investigating Wnt signaling in the setting of fibrosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86862/1/30312_ftp.pd

CALR-positive myeloproliferative disorder in a patient with Ph-positive chronic myeloid leukemia in durable treatment-free remission: a case report

Current diagnostic criteria for Philadelphia-negative myeloproliferative neoplasia (MPN) have been redefined by the discovery of Janus kinase 2 (JAK2), myeloproliferative leukemia (MPL) and calreticulin (CALR) genetic alterations. Only few cases of coexistence of CALR-mutated MPN and Philadelphia-positive chronic myeloid leukemia (CML) have been described so far. Here we report the case of a patient with CML diagnosed in 2001, treated with imatinib and pegylated interferon (IFN) frontline. She reached complete molecular remission (CMR) and discontinued imatinib, maintaining treatment free remission. Due to persistent thrombocytosis, we repeated bone marrow (BM) analysis and diagnosed CARL-mutated essential thrombocythemia (ET). A CALR-positive clone was found to be present since 2001, and was unaffected by imatinib treatment, possibly representing a molecular abnormality arising at stem cell level

Endothelial cells support osteogenesis in an in vitro vascularized bone model developed by 3D bioprinting

Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) 3D bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold which underwent osteogenic differentiation for two weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during two more weeks of culture. We tested three experimental conditions: Condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM):endothelial medium (EM); Condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; Condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In Conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under Condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just four weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development

Unveiling Adatoms in On-Surface Reactions:Combining Scanning Probe Microscopy with van’t Hoff Plots

[Image: see text] Scanning probe microscopy has become an essential tool to not only study pristine surfaces but also on-surface reactions and molecular self-assembly. Nonetheless, due to inherent limitations, some atoms or (parts of) molecules are either not imaged or cannot be unambiguously identified. Herein, we discuss the arrangement of two different nonplanar molecular assemblies of para-hexaphenyl-dicarbonitrile (Ph(6)(CN)(2)) on Au(111) based on a combined theoretical and experimental approach. For deposition of Ph(6)(CN)(2) on Au(111) kept at room temperature, a rhombic nanoporous network stabilized by a combination of hydrogen bonding and antiparallel dipolar coupling is formed. Annealing at 575 K resulted in an irreversible thermal transformation into a hexagonal nanoporous network stabilized by native gold adatoms. However, the Au adatoms could neither be unequivocally identified by scanning tunneling microscopy nor by noncontact atomic force microscopy. By combining van’t Hoff plots derived from our scanning probe images with our density functional theory calculations, we were able to confirm the presence of the elusive Au adatoms in the hexagonal molecular network

Targeting the Endothelin-1 Receptors Curtails Tumor Growth and Angiogenesis in Multiple Myeloma

The endothelin-1 (ET-1) receptors were recently found to mediate pro-survival functions in multiple myeloma (MM) cells in response to autocrine ET-1. This study investigated the effectiveness of macitentan, a dual ET-1 receptor antagonist, in MM treatment, and the mechanisms underlying its activities. Macitentan affected significantly MM cell (RPMI-8226, U266, KMS-12-PE) survival and pro-angiogenic cytokine release by down-modulating ET-1-activated MAPK/ERK and HIF-1 alpha pathways, respectively. HIF-1 alpha silencing abrogated the ET-1 mediated induction of genes encoding for pro-angiogenic cytokines such as VEGF-A, IL-8, Adrenomedullin, and ET-1 itself. Upon exposure to macitentan, MM cells cultured in the presence of the hypoxia-mimetic agent CoCl2, exogenous ET-1, or CoCl2 plus ET-1, down-regulated HIF-1 alpha and the transcription and release of downstream pro-angiogenic cytokines. Consistently, macitentan limited significantly the basal pro-angiogenic activity of RPMI-8226 cells in chorioallantoic membrane assay. In xenograft mouse models, established by injecting NOG mice either via intra-caudal vein with U266 or subcutaneously with RPMI-8226 cells, macitentan reduced effectively the number of MM cells infiltrating bone marrow, and the size and microvascular density of subcutaneous MM tumors. ET-1 receptors targeting by macitentan represents an effective anti-proliferative and anti-angiogenic therapeutic approach in preclinical settings of MM

Endothelin-1 receptor blockade as new possible therapeutic approach in multiple myeloma.

New effective treatments are needed to improve outcomes for multiple myeloma (MM) patients. Receptors with restricted expression on plasmacells (PCs) represent attractive new therapeutic targets. The endothelin-1(EDN1) axis, consisting of EDN1 acting through EDN-receptor A(EDNRA) and B (EDNRB), was previously shown to be overexpressed inseveral tumours, including MM. However, there is incomplete understand-ing of how EDN1 axis regulates MM growth and response to therapy.Besides EDNRA, the majority of MM cell lines and primary malignant PCsexpress high levels of EDNRB and release EDN1. Similarly, bone-marrowmicroenvironment cells also secrete EDN1. Investigating the extent of epi-genetic dysregulation of EDNRB gene in MM, we found that hypermethyla-tion of EDNRB promoter and subsequent down-regulation of EDNRB genewas observed in PCs or B lymphocytes from healthy donors compared toEDNRB-expressing malignant PCs. Pharm acological blockade with the dualEDN1 receptor antagonist bosentan decreased cell viability and MAPK acti-vation of U266 and RPMI-8226 cells. Interestingly, the combination ofbosentan and the proteasome inhibitor bortezomib, currently approved forMM treatment, resulted in synergistic cytotoxic effects. Overall, our datahas uncovered EDN1-mediated autocrine and paracrine mechanisms thatregulate malignant PCs growth and drug response, and support EDN1receptors as new therapeutic targets in MM

Low-dimensional metal-organic coordination structures on graphene

We report the formation of one- and two-dimensional metal-organic coordination structures from para-hexaphenyl-dicarbonitrile (NC-Ph-6-CN) molecules and Cu atoms on graphene epitaxially grown on Ir(111). By varying the stoichiometry between the NC-Ph-6-CN molecules and Cu atoms, the dimensionality of the metal-organic coordination structures could be tuned: for a 3:2 ratio, a two-dimensional hexagonal porous network based on threefold Cu coordination was observed, while for a 1:1 ratio, one-dimensional chains based on twofold Cu coordination were formed. The formation of metal-ligand bonds was supported by imaging the Cu atoms within the metal-organic coordination structures with scanning tunneling microscopy. Scanning tunneling spectroscopy measurements demonstrated that the electronic properties of NC-Ph-6-CN molecules and Cu atoms were different between the two-dimensional porous network and one-dimensional molecular chains

Force-dependent allostery of the α-catenin actinbinding domain controls adherens junction dynamics and functions

α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis

Effects of initial-state dynamics on collective flow within a coupled transport and viscous hydrodynamic approach

We evaluate the effects of preequilibrium dynamics on observables in ultrarelativistic heavy-ion collisions. We simulate the initial nonequilibrium phase within A MultiPhase Transport (AMPT) model, while the subsequent near-equilibrium evolution is modeled using (2+1)-dimensional relativistic viscous hydrodynamics. We match the two stages of evolution carefully by calculating the full energy-momentum tensor from AMPT and using it as input for the hydrodynamic evolution. We find that when the preequilibrium evolution is taken into account, final-state observables are insensitive to the switching time from AMPT to hydrodynamics. Unlike some earlier treatments of preequilibrium dynamics, we do not find the initial shear viscous tensor to be large. With a shear viscosity to entropy density ratio of $0.12$, our model describes quantitatively a large set of experimental data on Pb+Pb collisions at the Large Hadron Collider(LHC) over a wide range of centrality: differential anisotropic flow $v_n(p_T) ~(n=2-6)$, event-plane correlations, correlation between $v_2$ and $v_3$, and cumulant ratio $v_2\{4\}/v_2\{2\}$.Comment: 10 pages, v2: minor revisio