bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibacterial Effect ofLuma apiculata(DC.) Burret Extracts in Clinically Important Bacteria
Authors: Araya-Contreras Tiare , Veas Rhonda , Escobar Carlos A. , Machuca Pamela , Bittner Mauricio
Published: October 2019

Nosocomial infections caused by bacteria are one of the main public health problems. Moreover, the resistance to antibiotics by these bacteria makes it necessary to find new treatments to fight them.Objective. To evaluate the antibacterial activity ofLuma apiculata(DC.) Burret extracts on bacteria of clinical importance.Materials and Methods. In this study, extracts were obtained at room temperature by successive extraction ofL. apiculataleaves, flowers, and branches and treated separately with solvents of ascending polarity (i.e., hexane, methylene dichloride, ethyl acetate, ethanol, methanol, and water) to extract the compounds depending on their polarity. Then, the extract’s antibacterial activity was tested againstStaphylococcus aureus,Staphylococcus epidermidis,Staphylococcus saprophyticus,Enterococcussp,Acinetobacter baumanii,Pseudomonas aeruginosa, andEscherichia coli.Results. The hexane extract ofL. apiculataleaves resulted to be active against all bacteria tested. Among them,S. aureusshowed to be the more susceptible, showing a minimum inhibitory concentration (MIC) of 120 μg/ml. In addition, a growth curve was performed, and colonies were counted. A decrease in bacterial growth was observed when the hexane extract ofL. apiculataleaves was added. Besides, the hexane extracts ofL. apiculataflowers resulted to be active against all Gram-positive tested bacteria. However, at higher concentrations, this extract resulted inactive for the Gram-negative bacteria tested. The hexane extract ofL. apiculatabranches resulted to be inactive in all cases. The extracts obtained treating separately leaves, flowers, or branches with solvents of major polarity than the hexane in a successive extraction of ascending polarity methodology resulted also to be inactive as an antimicrobial against all bacteria tested.Discussion/Conclusion. The hexane extract ofL. apiculataleaves showed the lower MIC againstS. aureuswhen compared with extracts obtained from other parts of the plant. The growth curve and the colonies count suggest a bacteriostatic activity of theL. apiculataleaves extract againstStaphylococcus aureus.
Evolutionary Cooperation in Networked Public Goods Game with Dependency Groups
Authors: Zhu Xuzhen , Su Xin , Ma Jinming , Tian Hui , Liu RunRan
Published: October 2019

Either in microlevel organizations or macrolevel societies, the individuals acquire benefits or payoffs by forming interdependency groups linked by common interests. Conducting research on the effects of interdependency groups on the evolution of cooperation could have a better understanding of the social dilemma problem. In this paper, we studied a spatial public goods game with nonlocal interdependency groups where each of participants is located in a two‐dimensional square lattice or Watts–Strogatz small‐world network with payoffs obtaining from the interactions with nearest neighbors. In terms of the enhancement factor, the effects of group density on the evolutionary cooperation can be quite different. For a low enhancement factor, the cooperation level is a nonmonotonic function with the varying density of interdependency groups in the system, which means a proper density of interdependency groups can best promote the cooperative level. For a moderate enhancement factor, a higher density of interdependency groups can always correspond to a higher cooperative level. However, if the enhancement factor is too high, a high density of interdependency groups can impede the evolutionary cooperation. We give the explanations for the different roles of group density of interdependency by using the transition probabilities of C players into D players as well as the reverse. Our findings are very helpful for the understanding of emergence cooperation as well as the cooperation regulation in the selfish individuals.
Validation of a Simple and Robust Liebermann–Burchard Colorimetric Method for the Assay of Cholesterol in Selected Milk Products in Ghana
Authors: Adu Joseph K. , Amengor Cedric D. K. , Kabiri Naomi , Orman Emmanuel , Patamia Stella Abla Gameli , Okrah Bernice Korkor
Published: October 2019

Cholesterol plays a key role in the synthesis of bile acids and steroid hormones in the human body. However, excessively high levels are usually implicated in cardiovascular diseases. For this reason, it is essential to monitor exposure to high levels of it in products meant for human consumption, and this calls for the need to develop analytical methods to detect them. The use of Liebermann–Burchard reaction in this study has been explored to develop a simple, reliable, and robust quantitative colorimetric method to assay cholesterol, and hence provide a good alternative to chromatographic methods. The developed method was validated and used to determine the contents of cholesterol in selected dairy products on the Kumasi Metropolis market. The method demonstrated a good linearity (R2=0.996) over concentration range of 0.01–0.08 mg/ml. It was also shown to be precise and robust. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 0.00430 mg/ml and 0.01304 mg/ml, respectively. Ten selected brands of canned milk (B1–B5) and fresh yoghurt products (A1–A5) were then assayed using the developed method. The results showed that three products from each category had cholesterol contents above the allowable content of 5 mg/100 g in dairy products. The study thus has proposed a simple colorimetric method that can be adopted by dairy products manufacturing facilities to rapidly determine cholesterol contents during manufacturing in order to monitor the safe consumption of their products, and eliminate or minimize possible future health hazards.
Reproducibility Experimentation among Computer-Aided Inspection Software from a Single Point Cloud
Authors: Jbira Ibtissem , Tahan Antoine , Bonsaint Serge , Mahjoub Mohamed Ali , Louhichi Borhen
Published: October 2019

The ISO GPS and ASME Y14.5 standards have defined dimensional and geometrical tolerance as a way to express the limits of surface part variations with respect to nominal model surfaces. A quality-control process using a measuring device verifies the conformity of the parts to these tolerances. To convert the control measurement points as captured by a device such as a coordinate measurement machine (CMM) or noncontact scan, it is necessary to select the appropriate algorithm (e.g., least square size and maximum inscribed size) and to include the working hypotheses (e.g., treatment of outliers, noise filtering, and missing data). This means that the operator conducting the analysis must decide on which algorithm to use. Through a literature review of current software programs and algorithms, many inaccuracies were found. A benchmark was therefore developed to compare the algorithm performance of three computer-aided inspection (CAI) software programs. From the same point cloud and on the same specifications (requirements and tolerances), three CAI options have been tested with several dimensional and geometrical features.
Linear Quadratic Optimal Control Design: A Novel Approach Based on Krotov Conditions
Authors: Kumar Avinash , Jain Tushar
Published: October 2019

This paper revisits the problem of synthesizing the optimal control law for linear systems with a quadratic cost. For this problem, traditionally, the state feedback gain matrix of the optimal controller is computed by solving the Riccati equation, which is primarily obtained using calculus of variations‐ (CoV‐) based and Hamilton–Jacobi–Bellman (HJB) equation‐based approaches. To obtain the Riccati equation, these approaches require some assumptions in the solution procedure; that is, the former approach requires the notion of costates and then their relationship with states is exploited to obtain the closed‐form expression of the optimal control law, while the latter requires a priori knowledge regarding the optimal cost function. In this paper, we propose a novel method for computing linear quadratic optimal control laws by using the global optimal control framework introduced by V. F. Krotov. As shall be illustrated in this article, this framework does not require the notion of costates and any a priori information regarding the optimal cost function. Nevertheless, using this framework, the optimal control problem gets translated to a nonconvex optimization problem. The novelty of the proposed method lies in transforming this nonconvex optimization problem into a convex problem. The convexity imposition results in a linear matrix inequality (LMI), whose analysis is reported in this work. Furthermore, this LMI reduces to the Riccati equation upon imposing optimality requirements. The insights along with the future directions of the work are presented and gathered at appropriate locations in this article. Finally, numerical results are provided to demonstrate the proposed methodology.
Comparison of Clinical Outcomes of Two-Level PELD and Foraminoplasty PELD for Highly Migrated Disc Herniations: A Comparative Study
Authors: Wu Xinbo , Fan Guoxin , He Shisheng , Gu Xin , Yang Yunfeng
Published: October 2019

Objective. The aim of this study is to compare the clinical outcomes of two-level percutaneous endoscopic lumbar discectomy (PELD) and foraminoplasty PELD in treating highly migrated lumbar disc herniations. Methods. Patients with highly migrated lumbar disc herniations were enrolled from May 2014 to June 2016. Low back pain and leg pain were evaluated by the Visual Analog Scale (VAS), and functional outcomes were assessed with the Oswestry Disability Index (ODI). The satisfaction rate of clinical outcomes was assessed according to the modified MacNab criteria. In addition, the intraoperative duration and postoperative complications were also recorded. Results. Forty patients, 14 cases in two-level PELD group and 26 cases in foraminoplasty PELD group, were included. The VAS scores of low back pain (P=0.67) and leg pain (P=0.86), as well as the ODI scores (P=0.87), were comparative between two-level PELD and foraminoplasty PELD groups. The satisfaction rate of clinical outcomes based on the modified MacNab criteria in the two-level PELD group was equivalent to that in foraminoplasty PELD group (92.9% versus 92.3%, P=0.92). In addition, the intraoperative duration of two-level PELD group was longer than that of foraminoplasty PELD group (80.2 ± 6.6 min versus 64.1 ± 7.3 min, P<0.01). The postoperative complications in the two-level PELD group (postoperative dysesthesia: N = 1) were relatively fewer as compared to those in the foraminoplasty PELD group (postoperative dysesthesia: N = 1; recurrence: N = 1; nucleus pulposus residues: N = 1). Conclusions. Both two-level PELD and foraminoplasty PELD are safe and effective surgical procedures for the patients with highly migrated lumbar disc herniations. Moreover, the two-level PELD technique has merits in reducing the incidence of postoperative nucleus pulposus residue.
Linnaeus tercentenary and invertebrate taxonomy: an introduction*
Authors: ZHANG ZHI-QIANG , SHEAR WILLIAM A.
Published: October 2019

Carl Linnaeus (1707–1778) is the founding father of modern taxonomy. The binominal nomenclatural system for species and the principles of biological classification that he developed in the 18th century provide the basis on which we name and group organisms today.  The tenth edition of his monumental Systema Naturae (Linnaeus 1758) marks the beginning of zoological nomenclature (ICZN 1999) and zoology as a modern branch of the natural sciences.  Linnaeus’ genius was to see and understand what others had not: the requirement for a simple, easily applied, and consistent system of classification.  That the fundamental principles of his work have survived two and a half centuries of repeated challenges stands as a tribute to that genius.
Phylogeny and higher classification of the scale insects (Hemiptera: Sternorrhyncha: Coccoidea)*
Authors: GULLAN P. J. , COOK L. G.
Published: October 2019

The superfamily Coccoidea contains nearly 8000 species of plant-feeding hemipterans comprising up to 32 families divided traditionally into two informal groups, the archaeococcoids and the neococcoids. The neococcoids form a monophyletic group supported by both morphological and genetic data. In contrast, the monophyly of the archaeococcoids is uncertain and the higher level ranks within it have been controversial, particularly since the late Professor Jan Koteja introduced his multi-family classification for scale insects in 1974. Recent phylogenetic studies using molecular and morphological data support the recognition of up to 15 extant families of archaeococcoids, including 11 families for the former Margarodidae sensu lato, vindicating Koteja’s views. Archaeococcoids are represented better in the fossil record than neococcoids, and have an adequate record through the Tertiary and Cretaceous but almost no putative coccoid fossils are known from earlier. In contrast, the sister group of the scale insects (Aphidoidea) has a more informative Jurassic and Triassic record. Relationships among most scale insect families are unresolved in phylogenetic trees based on nuclear DNA sequences, and most nodes in trees based on morphological data, including those from adult males, are poorly supported. Within the neococcoids, the Eriococcidae is not monophyletic and the monophyly of the Coccidae and Diaspididae may be compromised by the current family-level recognition of a few species-poor autapomorphic groups.
Phylogeny and systematics of Diptera: Two decades of progress and prospects*
Authors: YEATES DAVID K. , WIEGMANN BRIAN M. , COURTNEY GREG W. , MEIER RUDOLF , LAMBKIN CHRISTINE , PAPE THOMAS
Published: October 2019

The Diptera, or true flies (mosquitoes, gnats, and house flies) comprise 12-15% of animal species, and are the most ecologically diverse order of insects, spanning ecological roles from detritivory to vertebrate blood feeding and leaf mining.  The earliest known fossil Diptera are from the early Triassic 240 mya, and the order probably arose in the late Permian.  The earliest brachyceran fossils are found in the late Triassic and earliest Jurassic, but the diversification of the extremely diverse Calyptrata (ca. 30% of described species) began in the late Creataceous.  The monophyly of the order is supported by numerous morphological and biological characters and molecular data sets. The major lineages within the order are well established, and we summarize major recent phylogenetic analyses in a supertree for the Diptera.  Most studies concur that the traditional subordinal group Nematocera is paraphyletic, but relationships between the major lineages of these flies are not recovered consistently.  There is particular instability around the placement of the tipulids and their relatives and the families of the Psychodomorpha as traditionally defined.  The other major suborder, Brachycera, is clearly monophyletic, and the relationships between major brachyceran lineages have become clearer in recent decades.  The Eremoneura, Cyclorrhapha, Schizophora and Calyptrata are monophyletic, however the “Orthorrhapha” and “Aschiza” are paraphyletic, and it is likely that the “Acalyptrata” are also.  Ongoing phylogenetic analyses that span the diversity of the order shall establish a robust phylogeny of the group with increased quantitative rigor.  This will enable a more precise understanding of the evolution of the morphology, biogeography, biology, and physiology of flies.
Phylum Echinodermata*
Authors: PAWSON DAVID L.
Published: October 2019

The Phylum Echinodermata, comprising approximately 7,000 living species, and 13,000 fossil species, is epitomized by the familiar sea star, a universal symbol of the marine realm.  This distinctive group of animals may be briefly defined as possessing a skeleton of calcium carbonate in the form of calcite; a unique water-vascular system which mediates feeding, locomotion, and other functions; and a more or less conspicuous five-part radial symmetry.  A closer look at some extant echinoderms will show that some taxa of sea cucumbers lack calcite in their body walls, some taxa of sea stars have “outgrown” five-part symmetry and may have 50 or more arms, and many echinoderms show a more or less conspicuous bilateral symmetry superimposed upon a radial pattern.  Fossil echinoderms can be even more puzzling, for some are decidedly asymmetrical, and others may lack evidence of a water-vascular system.  Perhaps the only truly reliable taxonomic character of the phylum is that its members today are restricted to the marine realm.
Reflections on PEET, the Partnerships for Enhancing Expertise in Taxonomy*
Authors: RODMAN JAMES E.
Published: October 2019

The 12-year old PEET engages a retired program officer in a question-answer session focused on the aspirations and tribulations of the NSF-sponsored research, training, and computer infrastructure program, Partnerships for Enhancing Expertise in Taxonomy.
A modern look at the Animal Tree of Life*
Authors: GIRIBET GONZALO , DUNN CASEY W. , EDGECOMBE GREGORY D. , ROUSE GREG W.
Published: October 2019

The phylogenetic interrelationships of animals (Metazoa) have been elucidated by refined systematic methods and by new techniques, notably from molecular biology. In parallel with the strong molecular focus of contemporary metazoan phylogenetics, morphology has advanced with the introduction of new approaches, such as confocal laser scanning microscopy and cell-labelling in the study of embryology. The discovery of new animal diversity (previously unknown groups like Cycliophora and Micrognathozoa) has invigorated the field as well. At present, broad consensus exists for the monophyly of bilaterian animals, a split of Bilateria into Deuterostomia and Protostomia, a division of protostomes into a clade of mostly spiral cleavers (Lophotrochozoa) and a moulting clade (Ecdysozoa), a ‘restricted’ deuterostome hypothesis that excludes the lophophorate phyla, and a basal position of acoel and nemertodermatid flatworms within Bilateria. However, the position of several protostome phyla, especially Bryozoa and Chaetognatha, remains intractable. Phylogenomic approaches such as Expressed Sequence Tags are showing much promise for resolving ongoing controversies at the base of the animal tree, especially the branching pattern among ctenophores, sponges and cnidarians.
Establishment of the new genus Parvuspina for an undescribed Lithosiini species of Asura/Miltochrista generic complex (Lepidoptera: Erebidae: Arctiinae)
Authors: SINGH NAVNEET , KIRTI JAGBIR SINGH , DATTA HARVINDER SINGH
Published: October 2019

The Asura/Miltochrista generic complex belongs to Lithosiini (Erebidae, Arctiinae). It is one of the most diverse and taxonomically unresolved groups of this tribe. At present, this generic complex includes about 25 valid genera, as well as several generic/specific taxa of unclear taxonomic status (Holloway 2001, Kirti & Singh 2016, Volynkin 2017, Volynkin & Ivanova 2017, Volynkin et al. 2017, Volynkin & Černý 2016).
Leaf-litter thrips of the genus Psalidothrips (Thysanoptera, Phlaeothripidae) from Australia, with fifteen new species
Authors: WANG JUN , MOUND LAURENCE A. , TREE DESLEY J.
Published: October 2019

A key is given to 16 Psalidothrips species known from Australia. In contrast to members of this genus from other parts of the world, most of these species show a strong tendency toward wing loss, with associated character states, also remarkable reduction in sclerotisation of the thoracic sclerites. Character state variation is discussed, and 15 new species are described and illustrated: P. bipictus sp.n., P. brittoni sp.n., P. cecryphalus sp.n., P. daguilari sp.n., P. driesseni sp.n., P. gloriousi sp.n., P. greensladeae sp.n., P. howei sp.n., P. minantennus sp.n., P. platetus sp.n., P. postlei sp.n., P. tritus sp.n., P. trivius sp.n., P. verus sp.n. and P. wellsae sp.n.
Staining the target: CD37 expression in lymphomas
Authors: Bertoni Francesco , Stathis Anastasios
Published: October 2019

In this issue of Blood, Xu-Monette et al show that CD37 protein expression is a strong prognostic marker in diffuse large B-cell lymphoma (DLBCL) treated with R-CHOP.1 CD37 negativity is associated with poor outcome and a high prevalence of adverse genetic and biologic features.1
Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein
Authors: Mouro-Chanteloup Isabelle , D'Ambrosio Anne Marie , Gane Pierre , Le Van Kim Caroline , Raynal Virginie , Dhermy Didier , Cartron Jean-Pierre , Colin Yves
Published: October 2019

In most cases, the lack of Rh in Rhnull red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter–based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.
Dose Intensity and Risk of Relapse for NHL after Allogeneic Transplant.
Authors: Rodriguez Roberto , Nademanee Auayporn , Carter Nora , Smith Eileen , Krishnan Amrita , Popplewell Leslie , Zain Jasmine , Kirschbaum Mark , Sano Allison , Patane Kathy , Nathwani Mudra , Spielberger Ricardo , Sarkodee-Adoo Clarence , Forman Stephen J.
Published: October 2019

Reduced intensity regimens (RIR) have largely replaced conventional myeloablative regimens (CMR) for patients with NHL undergoing allogeneic transplant. However, the impact of dose reduction on relapse has not been extensively studied. We performed a retrospective analysis of 88 patients conditioned with CMR (n=48) and a RIR (n=40) of fludarabine 125mg/m2 and melphalan 140mg/m2. GVHD prophylaxis was with cyclosporine + MTX +/− prednisone for CMR and cyclosporine + MMF +/− MTX for the RIR.

CMR RIR P Value N 48 40 Low grade B-cell 18 16 NS Intermediate grade B-cell 16 12 NS Mantle cell 10 5 NS T cell 4 7 NS Age (years, median, range) 44 (18–54) 51 (20–67) 0.0002 No of prior regimens (median) 3 2 0.02 Previous autologous transplant 5 16 0.002 Chemosensitivity at transplant 24 31 0.007 FTBI regimen 41 0 <0.0001 MUD 8 17 0.009 PBSC 16 36 0.0001 Median follow-up (months,range) 65 (33-95) 20 (6-42)

CMR were significantly associated with a lower rate of relapse (RR) of 15% versus 38% after RIR (p=0.017). The 1-year TRM was 38% for CMR and 24% for RIR (p=NS). Kaplan-Meier 2-year OS/PFS for CMR is 52%/48% versus 57%/48% for RIR (p=NS). When analyzed by diagnosis, CMR were significantly associated with decreased RR (10% vs 60%, p=0.004) for intermediate grade B-cell (figure 1). Univariate analysis of patient and treatment-related prognostic factors showed improved survival with chemosensitive disease; treatment intensity was the single predictor of relapse for the entire group, but, when stratified by diagnosis, for intermediate grade B-cell only. In conclusion, CMR provide better disease control for intermediate grade B-cell NHL.

Figure Figure
SDF-1-CXCR4 and HGF-c-met Axes Regulate Mobilization/Recruitment to Injured Tissue of Human Mesenchymal Stem Cells.
Authors: Son Bo-Ra , Zhao Dongling , Marquez-Curtis Leah A. , Shirvaikar Neeta , Ratajczak Mariusz Z. , Janowska-Wieczorek Anna
Published: October 2019

Human mesenchymal stem cells (MSC) have been shown to egress from the bone marrow (BM), circulate in peripheral blood (PB) and differentiate into many cell types, making them attractive as a potential therapeutic tool for organ/tissue regeneration. However the signals required for their mobilization into PB and their recruitment into injured sites are not fully understood. We previously reported that stromal-derived factor (SDF)-1 and hepatocyte growth factor (HGF) are upregulated at sites of tissue damage (Cancer Research2003; 63:7926; Leukemia2004; 18:29) and in this study we examined whether these factors mediate the migration of MSC. We investigated (i) the expression in MSC of CXCR4 and c-met, the cognate receptors of SDF-1 and HGF, (ii) whether they are functional after early and late passages (using a chemotaxis assay across fibronectin and the reconstituted basement membrane Matrigel), and (iii) whether MSC express matrix metalloproteinases (MMPs) known to facilitate mobilization and homing of stem cells. MSC were derived from human bone marrow (BM) or cord blood (CB) and maintained for up to 18 passages (in IMDM and 10–20% FCS) with monitoring of markers for cardiac (Nkx2.5/Csx, GATA-4 and MEF2-C), skeletal muscle (Myo-D and myogenin) and endothelial cells (VE-cadherin and VEGFR-2). We found that (i) CB and BM MSC strongly express CXCR4 and c-met transcripts for up to 15 passages, (ii) these receptors are functional as the MSC cells were chemotactic and chemoinvasive (across Matrigel) towards gradients of SDF-1 (100 ng/mL) or HGF (40 ng/mL), and (iii) CB and BM MSC express MMP-2 mRNA and secrete both latent and active forms of MMP-2. Moreover, we found that CB and BM MSC expressed mRNA for all three cardiac markers and the endothelial marker VE-cadherin, indicating their potential for heart regeneration. In conclusion, these results indicate that the SDF-1-CXCR4 and HGF-c-met axes are important signaling pathways in MSC mobilization and their trafficking in PB, and could be involved in recruitment of MSC to damaged tissues (e.g., myocardium).
Impaired In Vitro Osteogenesis in Multiple Myeloma Bone Marrow Osteoprogenitor Cells.
Authors: Bose Nandita , Kahler Rachel A. , Li Xiaodong , Bergemann Hope , Rajkumar Vincent , Westendorf Jennifer , Anna Masellis
Published: October 2019

In individuals with the plasma cell malignancy multiple myeloma, there is evidence that bone formation rates are reduced and that increased ostoeclastic bone resorption is associated with impaired osteoblast function. We investigated the osteogenic differentiation capacity of myeloma bone marrow mesenchymal progenitor cells (MPCs). We also examined the expression levels and activity of Runx2, the transcription factor required for osteogenesis.

Bone marrow MPCs were cultured in osteogenic induction medium and assessed for bone alkaline phosphatase (bALP) expression (Sigma kit), induction of osteoblast specific genes like osteocalcin (RT-PCR) and mineralization by von Kossa staining. Immunoblot analysis and electrophoretic mobility shift assays (EMSA) were used to determine Runx2 expression and DNA-binding activity respectively. RT-PCR was used to sequence Runx2 and detect any mutations or deletions present within its domains. Transactivation ability of Runx2 was measured by its ability to activate osteocalcin promoter in transient transfection assays.

Myeloma -derived MPCs showed reduced levels of bALP and osteocalcin transcript and a lower degree of mineralization, in osteogenic induction medium, as compared to that of the healthy donors. Immunoblot analysis and EMSA indicated equivalent Runx2 expression and DNA-binding capacity, respectively, in both healthy donor and myeloma-derived MPCs. Sequence analysis of Runx2 indentified a splice variant of Runx2 lacking exon 8 (Runx2D8) in myeloma patients with reduced transactivation ability. Co-transfection of the splice variant led to reduced transcriptional activity of the full-length Runx2. The reduced transactivation ability of spliced Runx2, as well as its suppressive action on the transactivation function of full-length Runx2, likely contributes to the defective osteogenesis clinically observed in myeloma pateints.
Ceramide-1-Phosphate a Novel Mediator for Phagocytosis.
Authors: Hinkovska-Galcheva Vania Tz , Shayman Jmes A. , Kindzelskii Andrei L. , Hiraoka Miki , Abe Akira , Petty Howard R. , Boxer Laurence A.
Published: October 2019

We identified the generation of ceramide-1-phosphate (C1P) through activation of a ceramide kinase in neutrophils. Our previous studies indicated that C1P enhanced calcium-dependent fusion of liposomes. We hypothesized that human ceramide kinase (hCERK) activity and C1P synthesis leads to enhanced phagocytosis through a mechanism involving modulating membrane fluidity and Ca2+ generation. hCERK was stably transfected into COS-1 cells bearing FcγRIIA. hCERK activity was 2.3 times higher in cells transfected with hCERK than in the FcγRIIA cells or cells transfected with pc-vector. Stably transfected cells showed a 3-fold increase in phagocytosis. Besides increasing phagocytosis, the percentage of ingesting COS-1 cells increased from 43+ 11 in control cells and 50 + 11 in pc-vector control to 70 + 9 (p<0.0001, n=6) in pc-hCERK transfected cells. Cells labeled with [3H]-D-erythro-sphingosine and challenged with particles increased both phagocytosis by three fold and C1P levels by two times compared to resting controls. FcγRIIA, pc-vector and pc-hCERK transfected cells were subjected to cellular fractionation. Utilizing an antibody to c-Myc we confirmed that c-Myc tagged pc-hCERK was localized in the raft fraction, which was identified by a caveolin-1 marker. To assess plasma membrane fluidity we labeled COS–1 cells with 2-dimethylamino-6-lauroylnaphthalene (Laurdan). Cells transfected with pc-hCERK showed higher liquid crystalline order than control and vector transfected cells, a condition favorable to promote membrane fusion. Such ordered structures are reported to be the site of Ca2+ waves ignition. High speed imaging revealed that cells bearing pc-hCERK showed two Ca2+ waves beginning at the leading edge of the cell that propagated in both directions. When the two waves reached the vicinity of the phagosome, a secondary waves split off from each of them, then propagated about the perimeter of the phagosome. That was inhibitable by employing a store-operated Ca2+ channel (SOC) inhibitor. This behavior is unique to the FcγRIIA/pc-hCERK transfected cells. In conclusion transfected COS-1 cells were able to increase their C1P levels during phagocytosis. This changed the structural order parameter of the lipid rafts where hCERK is localized and likely contributed to phagocytosis by promoting phagosome development. Lipid rafts were enriched in Ca2+ signaling machinery and in turn pc-hCERK transfection resulted in a novel means to markedly enhance phagocytosis by generating Ca2+ movement from SOC.
Loss of Heterozygosity on Chromosome 9p24 Is the Most Frequent Chromosomal Aberration in Polycythemia Vera and Idiopathic Myelofibrosis.
Authors: Kralovics Robert , Passamonti Francesco , Liu Kun , Teo Soon-Siong , Bench Anthony , Tichelli Andre , Lazzarino Mario , Green Anthony R. , Cazzola Mario , Skoda Radek C.
Published: October 2019

Myeloproliferative disorders (MPD) represent a heterogeneous group of diseases characterized by excessive myeloid cell production and clonal hematopoiesis. The appearance of the MPD stem cell clone is believed to be a consequence of an as yet unknown somatic mutation. The genetic basis for clonal hematopoiesis in MPD has been extensively studied using cytogenetic analysis, but no invariant chromosomal aberration has been found. Recently, we identified loss of heterozygosity of chromosome 9p (9pLOH) as a clonal defect in a small cohort of polycythemia vera (PV) patients. Here we extended this study to a total of 295 patients with MPD; 171 with PV, 91 with essential thrombocythemia (ET), and 33 with idiopathic myelofibrosis (IMF). The 9pLOH was detected in 62/171 patients with PV (36%) and in 10/33 patients with IMF (30%). Thus, 9pLOH is the most frequent chromosomal aberration in PV and IMF described to date. Interestingly, only 2/91 patients with the diagnosis of ET had 9pLOH. The reason for this difference is currently unknown and may be related to the presence of polyclonal hematopoiesis in a proportion of ET. By increasing the microsatellite marker density used for the LOH mapping in all 68 patients with 9pLOH, we identified a 6.2 Mbp minimal common LOH region. Copy number analysis performed by quantitative PCR revealed that all patients with 9pLOH had two copies of the chromosomal region involved in LOH. This finding excludes deletions as the genetic mechanism underlying 9pLOH and indicates that mitotic recombination is involved. This explains why this chromosomal defect remained hidden to cytogenetic analysis, FISH, or comparative genomic hybridization. 9pLOH results in partial uniparental disomy (UDP) of chromosome 9p. The phenotypic consequences of UPD are often associated with genes undergoing genomic imprinting. However, in two of our patients with 9pLOH, we were able to determine the parental origin of the lost chromosome by analyzing their parents. In one patient, the maternal chromosome was lost, whereas in the other patient, the paternal chromosome was missing. This variability of parental origin of UDP makes the involvement of imprinted genes in the expansion of the 9pLOH clone unlikely and favors a tumor suppressor mechanism. CDKN2A and CDKN2B genes (INK4A, INK4B), localized on 9p21, are among the most frequently mutated tumor suppressors in human malignancies. However, the CDKN2A/B locus is not part of the minimal 9pLOH region in MPD. The 6.2 Mbp common 9pLOH region contains 40 genes and ESTs. We determined which of these genes are expressed in hematopoietic cells and are currently sequencing the cDNAs of these genes to search for mutations.
Increased and Pathological Emperipolesis of Neutrophils within Megakaryocytes Associated with Myelofibrosis in GATA-1LowMice.
Authors: Migliaccio Anna Rita , Centurione Lucia , Di Baldassarre Angela , Zingariello Maria , Bosco Domenico , Gatta Valentina , Rana Rosa Alba , Di Virgilio Antonio , Vannucchi Alessandro M.
Published: October 2019

GATA-1 is a transcription factor involved in the intrinsic control of erythroid, megakaryocytic and mast cell differentiation. Experimentally-induced deletion of the megakaryocytic (Mk)-specific regulatory sequences of GATA-1 (Gata1tm2Sho or GATA-1low mutation) results in severe thrombocytopenia, because of defective thrombocytopoiesis, and myelofibrosis. The relationship, if any, between defective megakaryocytopoiesis and development of the disease is still poorly understood. Electron microscopy studies, coupled with immuno-electron microscopy, have allowed us to determine that the GATA-1low mutation blocks Mk maturation between stage I and II, resulting in accumulation of defective Mk in the tissues of GATA-1low mice. The block in maturation includes failure to properly organize α-granules, since von Willebrand factor is barely detectable in mutant Mk while P-selectin, although expressed at normal level, is found mainly associated with the Demarcation Membrane System (DMS) rather than with granules. Conversely, both von Willebrand factor and P-selectin are barely detectable in the megathrombocytes in the blood from GATA-1low mice. Both in marrow and spleen, mutant Mk are surrounded by numerous myeloperoxidase-positive neutrophils, some of which appear in the process of establishing contact with the Mk by fusing their membrane with those of the DMS. As a result, 16 (in spleen) - 34 (in marrow) percent of GATA-1low Mk contain 1-3 neutrophils embedded in a vacuolated cytoplasm. The neutrophil-embedded GATA-1low Mk have morphological features (high electron density and negativity to TUNEL staining) compatible with those of cells dying from para-apoptosis. We suggest that such an increased and pathological neutrophil emperipolesis represents one of the mechanisms leading to myelofibrosis by releasing Mk cytokines (TGF-β) and neutrophil proteases (myeloperoxidase) in the microenvironment which result, respectively, in marrow fibrosis and abnormal stem/progenitor cell trafficking.
Myeloma Cell-Derived Factors Retard the Differentiation and Function of Dendritic Cells.
Authors: Yi Qing , Wang Siqing , Xie Jin , Qian Jianfei , Yang Jin , Freeman Muta E. , Barlogie Bart
Published: October 2019

Multiple myeloma (MM) is a B-cell malignancy often associated with a suppressed immune system. The mechanisms for the immunosuppression are largely unknown. In this study, we examined, using the murine 5T33 myeloma model, the effects of tumor cell or its-derived factors on the differentiation and function of bone marrow-derived dendritic cells (BMDCs). Our results showed that differentiation of BMDCs was retarded in the presence of 10% of tumor-derived supernatant (TSN). This is evident by, compared with control cells, the downregulated expression of surface molecules including CD40, CD86 and MHC class II; secretion of higher levels of IL-10 and lower levels of IL-12; and a poor T-cell response in an allogeneic mixed lymphocyte reaction induced by TSN-treated cells. The same phenomenon was also observed when the bone marrow progenitor cells were cocultured, either in direct contact or separated by transwell membrane, with myeloma cells. The treatment downregulated the expression of phosphorylated extracellular signal-related kinase (ERK) and mitogen-induced extracellular kinase (MEK), and upregulated the expression of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) and signal transducer and activator of transcription-3 (STAT3) in the cells. As a high level of interleukin (IL)-10, IL-6, vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-b can be detected in TSN, we examined whether these cytokines were responsible. Our results showed that addition of TGF-b, IL-10 and/or IL-6 could largely replace TSN in retarding the differentiation of BMDCs, and neutralizing antibodies against these cytokines, especially in combination, were able to block the effects of TSN or tumor cells on BMDCs. Finally, our results showed that inhibiting p38 MAPK or STAT3 restored the differentiation and function of these cells. Hence, our study not only sheds light on the mechanisms of tumor-induced immune evasion in MM and but also provides one of the solutions to overcome the problems.
Identification and Characterization of an ALK Inhibitor That Attenuates the Proliferation and Survival of Anaplastic Large Cell Lymphoma-Derived Cell Lines and ALK Dependent Cell Lines.
Authors: Cheng Mangeng , Wan Weihua , Albom Mark , Weinberg Linda , Angeles Thelma , Quail Matthew , Lu Lihui , Underiner Ted , Hudkins Robert , Ator Mark , Ruggeri Bruce
Published: October 2019

Anaplastic large cell lymphomas (ALCLs) are a unique subgroup of high grade non-Hodgkin lymphomas. Approximately 60% to 70% of ALCLs contain a t (2:5) (p23; q35) chromosomal rearrangement, generating the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein. NPM-ALK encodes a constitutively activated tyrosine kinase that has been demonstrated to be directly involved in the pathogenesis of ALCLs. Currently there is no optimal therapeutic regimen for ALK positive ALCLs. Even with high dose CHOP (cyclophophamide, doxorubicin, vincristine and prednisone)-based chemotherapy, a substantial number of patients with ALK positive ALCLs have very poor outcome, either failing to enter remission or relapsing within a few months from the start of treatment. This prompted a search for small molecule ALK inhibitors as potential therapeutic agents for these patients. In this report, we describe the identification and characterization of a potent small molecule ALK inhibitor, cmpd-1. Cmpd-1 inhibited the ALK kinase activity in an in vitro enzymatic assay with an IC50 value of 4 nM. In NPM-ALK transfected BaF3 cells and ALCL-derived cell lines, cmpd-1 potently inhibited the tyrosine phosphorylation of NPM-ALK with a cellular IC50 value of 10–30 nM. Treatment with cmpd-1 inhibited IL-3 independent proliferation of BaF3 cells expressing constitutively active NPM-ALK (more than 95% inhibition at 100 nM) and the addition of IL-3 rescued the growth of these cells, while cmpd-1 had little effect on the parental BaF3 cells. Inhibition of NPM-ALK activity by cmpd-1 in ALK positive ALCL-derived cell lines, Karpas-299, Sudhl-1, Sup-M2 and Sr-786, resulted in inhibition of cell proliferation (50–90% inhibition at 100–300 nM) and induction of apoptotic cell death, while having marginal effects on the proliferation and survival of K562, an ALK negative leukemia cell line. These data suggest that the inhibition of cell proliferation and induction of apoptosis by cmpd-1 is an ALK-dependent event. Several putative downstream targets of NPM-ALK, including ERK1/2, Stat3 and Akt, were hypo- or unphosphorylated when the ALK positive cells were treated with cmpd-1. With the potent ALK inhibitory activity, cmpd-1 can be used as a molecular and pharmacological tool to study the biological roles of ALK and to dissect ALK-mediated signaling pathways in ALCLs and possibly other tumor types in which ALK is implicated in their pathogenesis.
Preliminary Safety and Efficacy Report of a Randomized Trial of Alemtuzumab vs Chlorambucil as Front-Line Therapy in 297 Patients with Progressive B-Cell Chronic Lymphocytic Leukemia.
Authors: Hillmen Peter , Skotnicki Aleksander B. , Robak Tadeusz , Mayer Jiri , Jaksic Branimir , Vukovic Vojo , Weitman Steven
Published: October 2019

CAM307, a Phase III, open-label, international, randomized trial comparing the efficacy and safety of alemtuzumab (CAMPATH®, MABCAMPATH®) with chlorambucil in B-CLL. Untreated Rai stage I-IV B-CLL patients with evidence of progressive disease were randomized 1:1 to intravenous alemtuzumab 30 mg three times a week for a maximum of 12 weeks or oral chlorambucil 40 mg/m2 once every 28 days, up to a maximum of 12 cycles. Patients with autoimmune thrombocytopenia, prior bone marrow transplant, active infection, positive CMV by quantitative PCR assay, secondary malignancy or CNS involvement were excluded. All alemtuzumab patients received prophylaxis with cotrimoxazole (trimethoprim/ sulfamethoxazole DS) and famciclovir during treatment and after completion of therapy until CD4+ counts were ≥200 cells/μL. The primary endpoint for the trial is progression free survival; secondary endpoints include safety, overall survival and response rate. Patient recruitment has been completed, and demographic data are available for 297 patients (213 males, 84 females; median age 60 years; 67% Rai I/II; 33% Rai III/IV). AEs occurring in >10% of alemtuzumab patients included pyrexia, rigors, dermatitis, urticaria, headache, hypertension, hypotension (“infusion reactions”), CMV antigen reactivation, nausea and neutropenia. In chlorambucil patients, AEs occurring in >10% of patients included nausea and vomiting. A total of 7 deaths have been reported, 2 in the alemtuzumab arm and 5 in the chlorambucil arm. Two deaths in the chlorambucil arm were related to study drug. Symptomatic CMV reactivations have been tracked by investigator reports. Preliminary analysis indicates that 22 of 149 (15%) patients treated with alemtuzumab developed symptomatic CMV reactivation. In 14 of 22 (64%) cases, mild to moderate fever was the only clinical symptom. All symptomatic CMV reactivations resolved promptly with ganciclovir and most patients were able to complete alemtuzumab therapy per protocol. No symptomatic CMV reactivations have been reported in the chlorambucil arm. No deaths due to CMV have been reported. Preliminary efficacy results on approximately 100 patients that have completed 12 months of follow-up following start of treatment and were reviewed by an independent committee will be presented at the meeting. This randomized Phase III trial demonstrates that alemtuzumab as first line therapy has an acceptable toxicity profile compared to chlorambucil. Therefore, this trial will address whether treatment with alemtuzumab as front line therapy results in greater disease reduction than conventional treatment in B-CLL and whether this strategy confers a survival advantage compared to standard approaches.
MLL Partner Gene Domains Contribute Critical Functional Specificity to Immortalization and Differentiation of Hematopoietic Progenitors.
Authors: Santillan Donna A. , Ryan Amanda S. , Zeleznik-Le Nancy J.
Published: October 2019

The in-frame fusion of MLL (mixed-lineage-leukemia) to CBP (CREB binding protein) has been cloned from several patients with therapy-related AML with a t(11;16)(q23;p13). In a murine retroviral transduction model of MLL-CBP, the mice develop a preleukemic phase which evolves to myeloid leukemia, reminiscent of what is seen in patients with the t(11;16). From this work, it was determined that the amino terminus of MLL fused to the bromodomain and HAT domain of CBP are minimally necessary for transformation. To better understand the role of the CBP bromodomain and HAT domain in determining the characteristics of the leukemia, we have undertaken domain-swapping experiments. In the context of the MLL-CBP (bromodomain + HAT domain) fusion, we have replaced the bromodomain of CBP with either the bromodomain from PCAF or the double bromodomain from TAFII250. The HAT domain of CBP has been replaced with either the HAT domain from PCAF or that from GCN5. We are studying the immortalization and differentiation potential of retrovirally transduced hematopoietic precursors in vitro and in vivo. We have observed differences in the ability of the various domain-swapped constructs to immortalize murine bone marrow progenitors in vitro. The PCAF HAT domain can substitute for the CBP HAT domain such that bone marrow progenitor cells have an enhanced proliferative capacity when grown under myeloid differentiation conditions. In contrast, bromodomain-swapped constructs do not grow under these same conditions. In vivo, mice reconstituted with hematopoietic precursors that were transduced with the bromodomain swapped are developing a disease that is different from that seen in mice reconstituted with MLL-CBP transduced hematopoietic precursors. However, mice reconstituted with cells retrovirally transduced with the HAT domain swapped appear to be developing a disease similar to that seen with MLL-CBP. These results suggest an important role for the bromodomain of CBP in determining the phenotype of MLL-CBP related leukemia. Additionally, MLL-CBP (bromodomain + HAT domain) cell lines undergo a phenotypic change when grown in vitro on stromal cells that promote B cell differentiation. This result implies that immortalized cells are not completely restricted in their differentiation potential.