Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM)

Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR).?Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications.     

Temperature preferences of bacteria isolated from seawater collected in Kandalaksha Bay,White Sea,Russia

Fifty-two bacteria were isolated from seawater collected in Kandalaksha Bay, White Sea, Russia, and classified by 16S rDNA sequencing. Most of the strains belonged to ubiquitous microorganisms. Pseudomonas was the most abundant genus (21 strains), including species of P. fluorescens, P. putida and P. syringae. Serratia was also common (10 strains) with species S. plymuthica and S. proteamaculans. Sphingobacterium, Flavobacterium and Pantoea were less represented (5, 3 and 2 strains, respectively). The only typical bacterium of marine Arctic regions was Shewanella baltica. The strains were tested for their optimal growth temperature in the range 0–45°C. The majority appeared to be psychrotolerant (42%) or mesophilic-psychrotolerant (40%). In addition, one strain (Bacillus pumilus) showed a rather narrow mesophilic profile. No true psychrophilic bacteria were found. Most of the strains showed a classical curve with fast growth decrease above the optimum; some others displayed uncommon flat curves with scarce differences between maximum and minimum of growth in a wide range of temperatures. Moreover, few strains presented an unusual profile being, in relation to the optimum, more tolerant to high rather than low temperatures. Preferences of the Kandalaksha Bay strains are generally different from those reported in literature for the same species: optima were at lower temperatures and, sometimes, ranges were broader showing increased eurythermism. This could indicate adaptation to the wide temperature variations recorded in this peculiar environment.     

Arabidopsis AtNek2 Kinase is Essential and Associates with Microtubules

NIMA-related kinases (Neks) are a large family of serine/threonine kinases that have been linked to cell-cycle regulation in fungi and mammals. Large families of NIMA-related kinases are also conserved in plants. We demonstrate that AtNek2, a member of the NIMA-related kinase family in Arabidopsis, is a gene fundamental for plant survival and its down-regulation has a pleiotropic effect on leaf cell morphogenesis and plant development. Intracellular localization of YFP::AtNek2 showed that AtNek2 proteins co-distribute with the microtubular cytoskeleton. As a microtubular-associated protein AtNek2 might influence the dynamics of microtubules and consequently cell morphogenesis. This is supported by the observation that misexpression of AtNek2 in RNAi mutants leads to a distorted organization of cells.     

Effects of doxorubicin cancer therapy on autophagy and the ubiquitin-proteasome system in long-term cultured adult rat cardiomyocytes

The clinical use of anthracyclines in cancer therapy is limited by dose-dependent cardiotoxicity that involves cardiomyocyte injury and death. We have tested the hypothesis that anthracyclines affect protein degradation pathways in adult cardiomyocytes. To this aim, we assessed the effects of doxorubicin (Doxo) on apoptosis, autophagy and the proteasome/ubiquitin system in long-term cultured adult rat cardiomyocytes. Accumulation of poly-ubiquitinated proteins, increase of cathepsin-D-positive lysosomes and myofibrillar degradation were observed in Doxo-treated cardiomyocytes. Chymotrypsin-like activity of the proteasome was initially increased and then inhibited by Doxo over a time-course of 48 h. Proteasome 20S proteins were down-regulated by higher doses of Doxo. The expression of MURF-1, an ubiquitin-ligase specifically targeting myofibrillar proteins, was suppressed by Doxo at all concentrations measured. Microtubule-associated protein?1 light chain 3B (LC3)-positive punctae and both LC3-I and -II proteins were induced by Doxo in a dose-dependent manner, as confirmed by using lentiviral expression of green fluorescence protein bound to LC3 and live imaging. The lysosomotropic drug chloroquine led to autophagosome accumulation, which increased with concomitant Doxo treatment indicating enhanced autophagic flux. We conclude that Doxo causes a downregulation of the protein degradation machinery of cardiomyocytes with a resulting accumulation of poly-ubiquitinated proteins and autophagosomes. Although autophagy is initially stimulated as a compensatory response to cytotoxic stress, it is followed by apoptosis and necrosis at higher doses and longer exposure times. This mechanism might contribute to the late cardiotoxicity of anthracyclines by accelerated aging of the postmitotic adult cardiomyocytes and to the susceptibility of the aging heart to anthracycline cancer therapy.     

Does genetic population structure of Ambrosina bassii L. (Araceae,Ambrosineae) attest a post-Messinian land-bridge between Sicily and Africa?

Aim of the present work is the analysis (through the study of enzyme polymorphism) of Sicilian and African (Tunisian) populations of Ambrosina bassii, a small perennial endemic to the Central-Western Mediterranean basin, in order to verify if the complex geological history of this part of the Mediterranean area left its mark in the present-day genetic structure of this taxon. Starch gel allozyme electrophoresis of seven putative loci of A. bassii was employed to estimate genetic diversity, genetic structure and gene flow. Populations from Sicily, Tunisia and Sardinia (as outgroup) were sampled. Results show that Sicily populations have 4 private alleles, Sardinia 3, Tunisia just one. One allele is private to both Sardinia and Tunisia, another one to Sardinia and Sicily. Even if there are no alleles private to Sicily and Tunisia, “cluster analysis” (based on Nei's genetic distances), “non-metric multidimensional scaling” (computed on the basis of a matrix with F_ST values between populations) and Bayesian analysis point out a clear isolation of Sardinian populations, and a greater similarity between Sicilian and Tunisian populations compared to Sardinian ones. The strong genetic affinity between populations from Sicily and Tunisia, considering the very low dispersal ability of the species, gives evidence of a recent continuity between the populations as well as between the two areas. Considering also the estimates of divergence times, a post-Messinian terrestrial connection between the two landmasses can be hypothesized.     

The enantiomers of epiboxidine and of two related analogs: synthesis and estimation of their binding affinity at α4β2 and α7 neuronal nicotinic acetylcholine receptors

Epiboxidine hydrochlorides (+)-2 and (-)-2, which are the structural analogs of the antipodes of epibatidine (±)-1, as well as the enantiomeric pairs (+)-3/(-)-3 and (+)-4/(-)-4 were synthesized and tested for binding affinity at α4β2 and α7 nicotinic acetylcholine receptor (nAChR) subtypes. Final derivatives were prepared through the condensation of racemic N-Boc-7-azabicyclo[2.2.1]heptane-2-one (±)-5 with the resolving agent (R)-(+)-2-methyl-2-propanesulfinamide. The pharmacological analysis carried out on the three new enantiomeric pairs evidenced an overall negligible degree of enantioselectivity at both nAChRs subtypes, a result similar to that reported for both natural and unnatural epibatidine enantiomers at the same investigated receptor subtypes.     

Oldenlandia diffusa extracts exert antiproliferative and apoptotic effects on human breast cancer cells through ERα/Sp1-mediated p53 activation

Breast cancer is the most frequent tumor and a major cause of death among women. Estrogens play a crucial role in breast tumor growth, which is the rationale for the use of hormonal antiestrogen therapies. Unfortunately, not all therapeutic modalities are efficacious and it is imperative to develop new effective antitumoral drugs. Oldenlandia diffusa (OD) is a well-known medicinal plant used to prevent and treat many disorders, especially cancers. The aim of this study was to investigate the effects of OD extracts on breast cancer cell proliferation. We observed that OD extracts strongly inhibited anchorage-dependent and -independent cell growth and induced apoptosis in estrogen receptor alpha (ERα)-positive breast cancer cells, whereas proliferation and apoptotic responses of MCF-10A normal breast epithelial cells were unaffected. Mechanistically, OD extracts enhance the tumor suppressor p53 expression as a result of an increased binding of ERα/Sp1 complex to the p53 promoter region. Finally, we isolated ursolic and oleanolic acids as the bioactive compounds able to upregulate p53 expression and inhibit breast cancer cell growth. These acids were greatly effective in reducing tamoxifen-resistant growth of a derivative MCF-7 breast cancer cell line resistant to the antiestrogen treatment. Our results evidence how OD, and its bioactive compounds, exert antiproliferative and apoptotic effects selectively in ERα-positive breast cancer cells, highlighting the potential use of these herbal extracts as breast cancer preventive and/or therapeutic agents.     

Wnt/β-catenin signaling pathway and thioredoxin-interacting protein (TXNIP) mediate the "glucose sensor" mechanism in metastatic breast cancer-derived cells MDA-MB-231

In this study we investigated the effect of glucose on GSK3β and β-catenin expression and the involvement of the N-linked glycosylation and hexosamine pathways in the Wnt canonical pathway in response to in vitro conditions resembling normoglycemia (5 mmol) and hyperglycemia (20 mmol) in the metastatic breast cancer-derived cell line MDA-MB-231. We also investigated the relationship between this circuitry and the thioredoxin-interacting protein (TXNIP) regulation that seems to be related. MDA-MB-231 cells were grown either in 5 or 20 mM glucose chronically prior to plating. For glucose shift (5/20), cells were plated in 5 mM glucose and shifted to 20 mM at time 0. Both protein and mRNA levels for GSK3β but only the protein expression for β-catenin, were increased in response to high glucose. Furthermore, we assessed the response of GSK3β, β-catenin, and TXNIP to inhibition of the N-linked glycosylation, hexosamine, and Wnt pathways. Wnt signaling pathway activation was validated by specific reporter assay. We show that high levels of glucose regulate mRNA and protein expression of GSK3β, and consequently higher levels of activated β-catenin protein, which locates to the nucleus and is associated with increased levels of cyclin D1 expression. This event coincides with increased level of N-terminal Ser 9 phosphorylation of GSK3β protein. The inhibition of both the hexosamine pathway and N-linked glycosylation along with Wnt signaling pathway by sFRP1 and DKK1 is associated with significant decrease of the protein levels of GSK3β, β-catenin, and TXNIP RNA. Our work illuminates a novel and never described before function of this signaling pathway that relates glucose metabolism with redox regulation mechanism.