Carboxy-terminal fragment of osteogenic growth peptide regulates myeloid differentiation through RhoA

The carboxy-terminal fragment of osteogenic growth peptide, OGP(10-14), is a pentapeptide with bone anabolic effects and hematopoietic activity. The latter activity appears to be largely enhanced by specific growth factors. To study the direct activity of OGP(10-14) on myeloid cells, we tested the pentapeptide proliferating/differentiating effects in HL60 cell line. In this cell line, OGP(10-14) significantly inhibited cell proliferation, and enhanced myeloperoxidase (MPO) activity and nitroblue tetrazolium reducing ability. Moreover, it induced cytoskeleton remodeling and small GTP-binding protein RhoA activation. RhoA, which is known to be involved in HL60 differentiation, mediated these effects as shown by using its specific inhibitor, C3. Treatment with GM-CSF had a comparable OGP(10-14) activity on proliferation, MPO expression, and RhoA activation. Further studies on cell proliferation and RhoA activation proved enhanced activity by association of the two factors. These results strongly suggest that OGP(10-14) acts directly on HL60 cells by activating RhoA signaling although other possibilities cannot be ruled out.     

Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging

Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non‐toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label‐free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co‐localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses.      

Inside Cover: Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging (J. Biophotonics 4/2018)

The internalization kinetics and intracellular spatial distribution of functionalized diatomite nanoparticles in human lung epidermoid carcinoma cell line have been investigated by confocal fluorescence and Raman microscopy. In this context, Raman imaging due to its non‐destructive, chemically selective and label‐free working principle provides evidence that the nanovectors are internalized and co‐localize with lipid environments, suggesting an endocytic internalisation route. Nanoparticle uptakes and intracellular persistence are observed up to 72 hours, without damage to cell viability or morphology. Further details can be found in the article by Stefano Managò et al. ( e201700207 )

     

Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2

Basophils circulate in the blood and are able to migrate into tissues at sites of inflammation. Urokinase plasminogen activator (uPA) binds a specific high affinity surface receptor (uPAR). The uPA-uPAR system is crucial for cell adhesion and migration, and tissue repair. We have investigated the presence and function of the uPA-uPAR system in human basophils. The expression of uPAR was found at both mRNA and protein levels. The receptor was expressed on the cell surface of basophils, in the intact and cleaved forms. Basophils did not express uPA at either the protein or mRNA level. uPA (10(-12)-10(-9) M) and its uPAR-binding N-terminal fragment (ATF) were potent chemoattractants for basophils, but did not induce histamine or cytokine release. Inactivation of uPA enzymatic activity by di-isopropyl fluorophosphate did not affect its chemotactic activity. A polyclonal Ab against uPAR inhibited uPA-dependent basophil chemotaxis. The uPAR-derived peptide 84-95 (uPAR84-95) induced basophil chemotaxis. Basophils expressed mRNA for the formyl peptide receptors formyl peptide receptor (FPR), FPR-like 1 (FPRL1), and FPRL2. The FPR antagonist cyclosporin H prevented chemotaxis induced by FMLP, but not that induced by uPA and uPAR84-95. Incubation of basophils with low and high concentrations of FMLP, which desensitize FPR and FPRL1, respectively, but not FPRL2, slightly reduced the chemotactic response to uPA and uPAR84-95. In contrast, desensitization with WKYMVm, which also binds FPRL2, markedly inhibited the response to both molecules. Thus, uPA is a potent chemoattractant for basophils that seems to act through exposure of the chemotactic uPAR epitope uPAR84-95, which is an endogenous ligand for FPRL2 and FPRL1.     

The Synergistic Effect of Everolimus and Chloroquine on Endothelial Cell Number Reduction Is Paralleled by Increased Apoptosis and Reduced Autophagy Occurrence

Endothelial Progenitor Cells (EPCs), a minor subpopulation of the mononuclear cell fraction in peripheral blood, play a critical role in cancer development as they contribute to angiogenesis-mediated pathological neovascularization. In response to tumor cytokines, including VEGF, EPCs mobilize from the bone marrow into the peripheral circulation and move to the tumor bed where they incorporate into sprouting neovessels. In the present study, we evaluated the effects of everolimus (Afinitor, Novartis), a rapamycin analogue, alone or in combination with chloroquine, a 4-alkylamino substituted quinoline family member, one of the autophagy inhibitors, on EPCs biological functions. We found that either everolimus or chloroquine induce growth inhibition on EPCs in a dose-dependent manner after 72 h from the beginning of incubation. The combined administration of the two drugs to EPC was synergistic in inducing growth inhibition; in details, the maximal pharmacological synergism between everolimus and chloroquine in inducing growth inhibition on EPCs cells was recorded when chloroquine was administered 24 h before everolimus. Moreover, we have studied the mechanisms of cell death induced by the two agents alone or in combination on EPCs and we have found that the synergistic effect of combination on EPC growth inhibition was paralleled by increased apoptosis induction and reduced autophagy. These effects occurred together with biochemical features that are typical of reduced autophagic death such as increased co-immunoprecipitation between Beclin 1 and Bcl-2. Chloroquine antagonized the inhibition of the activity of Akt→4EBP1 axis mediated by everolimus and at the same time it blocked the feed-back activation of Erk-1/2 induced by RAD in EPCs. These data suggest a new strategy in order to block angiogenesis in tumours in which this process plays a key role in both the sustainment and spreading of cancer cells.     

MicroRNA-33 and SIRT1 influence the coronary thrombus burden in hyperglycemic STEMI patients

Primary percutaneous coronary intervention (PPCI) is a pivotal treatment in ST-segment elevation myocardial infarction (STEMI) patients. However, in hyperglycemic-STEMI patients, the incidence of death is still significant. Here, the involvement of sirtuin 1 (SIRT1) and miR33 on the pro-inflammatory/pro-coagulable state of the coronary thrombus was investigated. Moreover, 1-year outcomes in hyperglycemic STEMI in patients subjected to thrombus aspiration before PPCI were evaluated. Results showed that hyperglycemic thrombi displayed higher size and increased miR33, reactive oxygen species, and pro-inflammatory/pro-coagulable markers. Conversely, the hyperglycemic thrombi showed a lower endothelial SIRT1 expression. Moreover, in vitro experiments on endothelial cells showed a causal effect of SIRT1 modulation on the pro-inflammatory/pro-coagulative state via hyperglycemia-induced miR33 expression. Finally, SIRT1 expression negatively correlated with STEMI outcomes. These observations demonstrate the involvement of the miR33/SIRT1 pathway in the increased pro-inflammatory and pro-coagulable state of coronary thrombi in hyperglycemic STEMI patients.     

Assessment of DNA damages in lymphocytes of agricultural workers exposed to pesticides by comet assay in a cross-sectional study

Purpose: To assess the predictive power of the comet assay in the context of occupational exposure to pesticides.

Materials and methods: The recruited subjects completed a structured questionnaire and gave a blood sample. Exposure to pesticides was measured by means of an algorithm based on Dosemeci’s work (Agricultural Health Study). Approximately 50 images were analyzed for each sample via fluorescence microscopy. The extent of DNA damage was estimated by tail moment (TM) and is the product of tail DNA (%) and tail Length.

Results: Crude significant risks (odds ratios, ORs) for values higher than the 75th percentile of TM were observed among the exposed subjects (score?>?1). The frequency of some confounding factors (sex, age and smoking) was significantly higher among the exposed workers. A significant dose–effect relationship was observed between TM and exposure score. Significant high-risk estimates (ORs), adjusted by the studied confounding factors, among exposure to pesticides and TM, % tail DNA and tail length were confirmed using unconditional logistic regression models.

Conclusions: The adjusted associations (ORs) between the comet parameters and exposure to pesticides were significant. The sensitivity of the comet test was low (41%), the specificity (89%) and the predictive positive value (0.77) were found acceptable.     

Silicified Biota in High-Altitude,Geothermally Influenced Ignimbrites at El Tatio Geyser Field,Andean Cordillera (Chile)

Biofilms and filamentous communities provided favorable sites for silica precipitation on deeply weathered ignimbrites that make up the substrate at the hydrothermal field of El Tatio (Andean Cordillera, Chile). The amorphous silica encrustation enabled the preservation of a variety of biotic and abiotic features. An integrated study based on optical/scanning electron microscopy and molecular methods of totally to partially silicified microbial communities and biofilms allowed a comparative evaluation of the microfacies and the microbial diversity in the siliceous sinters produced by the digression of a little braided stream departing from a hot spring pool. This study showed useful convergent identifications of certain groups of microbes, such as filamentous cyanobacteria attributed to the genera Phormidium and Rivularia. Together with these microbes, other presumably initial colonizers, such as the halophilic and thermophilic pennate diatoms Nitzschia and Synedra, were widely present and could have contributed to the formation of biofilms and mucus that, as potential home to early silicification, could have contributed to the preservation of microbiologically derived morphologies.     

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

Transformation of potato plastids is limited by low transformation frequencies and low transgene expression in tubers. In order to improve the transformation efficiency, we modified the regeneration procedure and prepared novel vectors containing potato flanking sequences for transgene integration by homologous recombination in the Large Single Copy region of the plastome. Vector delivery was performed by the biolistic approach. By using the improved regeneration procedure and the potato flanking sequences, we regenerated about one shoot every bombardment. This efficiency corresponds to 15–18-fold improvement compared to previous results with potato and is comparable to that usually achieved with tobacco. Further, we tested five promoters and terminators, and four 5′-UTRs, to increase the expression of the gfp transgene in tubers. In leaves, accumulation of GFP to about 4% of total soluble protein (TSP) was obtained with the strong promoter of the rrn operon, a synthetic rbcL-derived 5′-UTR and the bacterial rrnB terminator. GFP protein was detected in tubers of plants transformed with only four constructs out of eleven. Best results (up to approximately 0.02% TSP) were achieved with the rrn promoter and rbcL 5′-UTR construct, described above, and another containing the same terminator, but with the promoter and 5′-UTR from the plastid clpP gene. The results obtained suggest the potential use of clpP as source of novel regulatory sequences in constructs aiming to express transgenes in amyloplasts and other non-green plastids. Furthermore, they represent a significant advancement of the plastid transformation technology in potato, of relevance to its implementation in potato breeding and biotechnology.