Attenuation of Eph Receptor Kinase Activation in Cancer Cells by Coexpressed Ephrin Ligands

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting “in trans” with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral “cis” associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.     

Neuromuscular Electrical Stimulation as a Method to Maximize the Beneficial Effects of Muscle Stem Cells Transplanted into Dystrophic Skeletal Muscle

Cellular therapy is a potential approach to improve the regenerative capacity of damaged or diseased skeletal muscle. However, its clinical use has often been limited by impaired donor cell survival, proliferation and differentiation following transplantation. Additionally, functional improvements after transplantation are all-too-often negligible. Because the host microenvironment plays an important role in the fate of transplanted cells, methods to modulate the microenvironment and guide donor cell behavior are warranted. The purpose of this study was to investigate whether the use of neuromuscular electrical stimulation (NMES) for 1 or 4 weeks following muscle-derived stem cell (MDSC) transplantation into dystrophic skeletal muscle can modulate the fate of donor cells and enhance their contribution to muscle regeneration and functional improvements. Animals submitted to 4 weeks of NMES after transplantation demonstrated a 2-fold increase in the number of dystrophin+ myofibers as compared to control transplanted muscles. These findings were concomitant with an increased vascularity in the MDSC+NMES group when compared to non-stimulated counterparts. Additionally, animals subjected to NMES (with or without MDSC transplantation) presented an increased maximal specific tetanic force when compared to controls. Although cell transplantation and/or the use of NMES resulted in no changes in fatigue resistance, the combination of both MDSC transplantation and NMES resulted in a faster recovery from fatigue, when compared to non-injected and non-stimulated counterparts. We conclude that NMES is a viable method to improve MDSC engraftment, enhance dystrophic muscle strength, and, in combination with MDSC transplantation, improve recovery from fatigue. These findings suggest that NMES may be a clinically-relevant adjunct approach for cell transplantation into skeletal muscle.     

MicroRNA-486-3p Regulates γ-Globin Expression in Human Erythroid Cells by Directly Modulating BCL11A

MicroRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNAs target. The functional relevance of microRNAs has been proven in normal and malignant hematopoiesis. While analyzing miRNAs expression profile in unilineage serum-free liquid suspension unilineage cultures of peripheral blood CD34⁺ hematopoietic progenitor cells (HPCs) through the erythroid, megakaryocytic, granulocytic and monocytic pathways, we identified miR-486-3p as mainly expressed within the erythroid lineage. We showed that miR-486-3p regulates BCL11A expression by binding to the extra-long isoform of BCL11A 3′UTR. Overexpression of miR-486-3p in erythroid cells resulted in reduced BCL11A protein levels, associated to increased expression of γ-globin gene, whereas inhibition of physiological miR-486-3p levels increased BCL11A and, consequently, reduced γ-globin expression. Thus, miR-486-3p regulating BCL11A expression might contributes to fetal hemoglobin (HbF) modulation and arise the question as to what extent this miRNA might contribute to different HbF levels observed among β-thalassemia patients. Erythroid cells, differentiated from PB CD34⁺ cells of a small cohort of patients affected by major or intermedia β-thalassemia, showed miR-486-3p levels significantly higher than those observed in normal counterpart. Importantly, in these patients, miR-486-3p expression correlates with increased HbF synthesis. Thus, our data indicate that miR-486-3p might contribute to different HbF levels observed among thalassemic patients and, possibly, to the clinical severity of the disease.     

Sex-sorting of boar spermatozoa does not influence the localization of glucose transporters

Sex-sorting damages spermatozoa function, shortening their lifespan and fertility. This study used an immunofluorescence technique to investigate the effect of sex-sorting on the localization of glucose transporters (GLUTs) in boar spermatozoa. GLUTs are trans-membrane proteins responsible for glucose transport within cells. Distribution of GLUTs on sperm cells was similar in unsorted and sex-sorted semen, suggesting that the flow cytometric sex-sorting process did not affect the sperm energy apparatus.     

Improved BM212 MmpL3 Inhibitor Analogue Shows Efficacy in Acute Murine Model of Tuberculosis Infection

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved in vitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED₉₉ of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery.     

Gene Expression Analysis of PTEN Positive Glioblastoma Stem Cells Identifies DUB3 and Wee1 Modulation in a Cell Differentiation Model

The term astrocytoma defines a quite heterogeneous group of neoplastic diseases that collectively represent the most frequent brain tumors in humans. Among them, glioblastoma multiforme represents the most malignant form and its associated prognosis is one of the poorest among tumors of the central nervous system. It has been demonstrated that a small population of tumor cells, isolated from the brain neoplastic tissue, can reproduce the parental tumor when transplanted in immunodeficient mouse. These tumor initiating cells are supposed to be involved in cancer development and progression and possess stem cell-like features; like their normal counterpart, these cells remain quiescent until they are committed to differentiation. Many studies have shown that the role of the tumor suppressor protein PTEN in cell cycle progression is fundamental for tumor dynamics: in low grade gliomas, PTEN contributes to maintain cells in G1 while the loss of its activity is frequently observed in high grade gliomas. The mechanisms underlying the above described PTEN activity have been studied in many tumors, but those involved in the maintenance of tumor initiating cells quiescence remain to be investigated in more detail. The aim of the present study is to shed light on the role of PTEN pathway on cell cycle regulation in Glioblastoma stem cells, through a cell differentiation model. Our results suggest the existence of a molecular mechanism, that involves DUB3 and WEE1 gene products in the regulation of Cdc25a, as functional effector of the PTEN/Akt pathway.     

Clerocidin-mediated DNA footprinting discriminates among different G-quadruplex conformations and detects tetraplex folding in a duplex environment

Background

G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide.

Methods

Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer.

Results

The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context.

Conclusions

CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions.

General significance

Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions.     

Attachment and Biofilm Forming Capabilities of Staphylococcus epidermidis Strains Isolated from Preterm Infants

Staphylococcus epidermidis, a human commensal, is an important opportunistic, biofilm-forming pathogen and the main cause of late onset sepsis in preterm infants, worldwide. In this study we describe the characteristics of S. epidermidis strains causing late onset (>72 h) bloodstream infection in preterm infants and skin isolates from healthy newborns. Attachment and biofilm formation capability were analyzed in microtiter plates and with transmission electron microscopy (TEM). Clonal relationship among strains was studied with pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was performed, as well as the detection of biofilm-associated genes and of the invasiveness marker IS256 with polymerase chain reaction. Blood and skin isolates had similar attachment and biofilm-forming capabilities and biofilm formation was not related to the presence of specific genes. Filament-like membrane structures were seen by TEM early in the attachment close to the device surface, both in blood and skin strains. Nine of the ten blood isolates contained the IS256 and were also resistant to methicillin and gentamicin in contrast to skin strains. S. epidermidis strains causing bloodstream infection in preterm infants exhibit higher antibiotic resistance and are provided with an invasive genetic equipment compared to skin commensal strains. Adhesion capability to a device surface seems to involve bacterial membrane filaments.     

Direct amplification of new cellulase genes from woodland soil purified DNA

Eight genes encoding cellulolytic enzymes were obtained by direct PCR amplification of genomic DNA recovered from woodland soil samples. The direct amplifications were carried out by using primers designed from available online cellulase nucleotide sequences. The isolated genes were all different from each other and homologous to endo-β-1,4-glucanases of Bacillus subtilis. The cellulases were functionally expressed in Escherichia coli and tested on soluble substrate at 37 and 60 °C, showing different cellulolytic activities. Among these, the enzyme renamed CelWS6 exhibited good activity at higher temperatures. Further analysis of CelWS6 showed a high performance in acid environments (between pH 4.0 and 6.0) and at elevated temperatures with its maximum activity at pH 5.0 and 50 °C. At the optimum pH, it was very stable since more than 80 % of its original activity was maintained after an incubation of 120 min at 60 °C. Because the cellulases had different cellulolytic activities, but similar amino acid sequences, it was possible to assess the relationship between sequence and protein function.