Oligothiophene molecular beacons

Oligomers of thiophene are widely studied compounds for their electronic and optoelectronic properties. Despite their strong fluorescence, their use as markers for biomolecules, especially for oligonucleotides (ONs), is still largely unexplored. Here, we describe the synthesis of a series of ON molecular beacons employing different oligothiophenes as fluorescent probes and discuss their fluorescence emissions in comparative experiments with and without dabcyl as a quencher, in their hairpin and linear conformations, and as duplexes after hybridization with a complementary target.     

New TFO conjugates containing a carminomycinone-derived chromophore.

Conjugates obtained by linking the anthracycline intercalating chromophore to triple helix forming oligonucleotides (TFOs) have been used in a physicochemical study of the stability of triple helices with DNA sequences of pharmacological relevance. The intercalating moiety is represented by carminomycinone derivatives obtained upon O-demethylation and hydrolysis of the glycosidic linkage of daunomycin followed by the introduction of an alkylating residue at two different positions. Results of experiments with a polypurinic region present in the multidrug resistance (MDR) gene indicate that the stability of the triple helix is significantly enhanced by replacement of C's with (5-Me)C's in the TFO sequences tested. The stability is not changed when a 3'-TpT is present in place of a 3'-CpG at the presumed intercalation site of the anthraquinone chromophore. The same carminomycinone derivatives were used for the preparation of conjugates able to form triple helices with the polypurine tract (PPT) present in the human integrated genome of HIV-1 infected cells. Three different TFOs (T(4)(Me)CT(4)(Me)CC, C2; T(4)(Me)CT(4)(Me)CC(Me)CC(Me)CCT, C6; and T(4)(Me)CT(4)G(6), G6) were designed and linked to the anthraquinone moiety. These conjugates showed a significantly enhanced ability to bind the PPT region of HIV with respect to the nonconjugated TFOs.     

Immune Regulatory Neural Stem/Precursor Cells Protect from Central Nervous System Autoimmunity by Restraining Dendritic Cell Function

Background

The systemic injection of neural stem/precursor cells (NPCs) provides remarkable amelioration of the clinico-pathological features of experimental autoimmune encephalomyelitis (EAE). This is dependent on the capacity of transplanted NPCs to engage concurrent mechanisms of action within specific microenvironments in vivo. Among a wide range of therapeutic actions alternative to cell replacement, neuroprotective and immune modulatory capacities of transplanted NPCs have been described. However, lacking is a detailed understanding of the mechanisms by which NPCs exert their therapeutic plasticity. This study was designed to identify the first candidate that exemplifies and sustains the immune modulatory capacity of transplanted NPCs.

Methodology/Principal Findings

To achieve the exclusive targeting of the peripheral immune system, SJL mice with PLP-induced EAE were injected subcutaneously with NPCs and the treatment commenced prior to disease onset. NPC-injected EAE mice showed significant clinical improvement, as compared to controls. Exogenous NPCs lacking the expression of major neural antigens were reliably (and for long-term) found at the level of draining lymph nodes, while establishing sophisticated anatomical interactions with lymph node cells. Importantly, injected NPCs were never found in organs other than lymph nodes, including the brain and the spinal cord. Draining lymph nodes from transplanted mice showed focal up-regulation of major developmental stem cell regulators, such as BMP-4, Noggin and Sonic hedgehog. In lymph nodes, injected NPCs hampered the activation of myeloid dendritic cells (DCs) and steadily restrained the expansion of antigen-specific encephalitogenic T cells. Both ex vivo and in vitro experiments identified a novel highly NPC-specific–BMP-4-dependent–mechanism hindering the DC maturation.

Conclusion/Significance

The study described herein, identifies the first member of the TGF β/BMP family of stem cell regulators as a novel tolerogenic factor released by NPCs. Full exploitation of this pathway as an efficient tool for vaccination therapy in autoimmune inflammatory conditions is underway.     

Evaluation of different types of end-capping modifications on the stability of oligonucleotides toward 3'- and 5'-exonucleases.

Synthetic oligonucleotides are increasingly used because of their potential activity as regulators of gene expression. One of their major drawbacks is instability toward nucleases, in particular exonucleases. In this article, we studied some terminal modifications that can enhance exonuclease resistance, such as end-capping with alkylic chains (1,3-propanediol and 1,6-hexanediol), and with a modified nucleotide (2',3'-secouridine). These compounds were compared with the parent (natural) oligodeoxynucleotide and with different analogs containing a progressive number of phosphorothioate linkages. The resistance toward SVPDE and CSPDE (a 3'- and a 5'-exonuclease) was assessed, in vitro, by two independent techniques, UV and HPLC. Our results showed that the stability of all the modified oligonucleotides was at least 12 times that of the parent compound.     

Human umbilical endothelial cells (HUVECs) have a sex: characterisation of the phenotype of male and female cells

Background

Human umbilical endothelial cells (HUVECs) are widely used to study the endothelial physiology and pathology that might be involved in sex and gender differences detected at the cardiovascular level. This study evaluated whether HUVECs are sexually dimorphic in their morphological, proliferative and migratory properties and in the gene and protein expression of oestrogen and androgen receptors and nitric oxide synthase 3 (NOS3). Moreover, because autophagy is influenced by sex, its degree was analysed in male and female HUVECs (MHUVECs and FHUVECs).

Methods

Umbilical cords from healthy, normal weight male and female neonates born to healthy non-obese and non-smoking women were studied. HUVEC morphology was analysed by electron microscopy, and their function was investigated by proliferation, viability, wound healing and chemotaxis assays. Gene and protein expression for oestrogen and androgen receptors and for NOS3 were evaluated by real-time PCR and Western blotting, respectively, and the expression of the primary molecules involved in autophagy regulation [protein kinase B (Akt), mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] were detected by Western blotting.

Results

Cell proliferation, migration NOS3 mRNA and protein expression were significantly higher in FHUVECs than in MHUVECs. Conversely, beclin-1 and the LC3-II/LC3-I ratio were higher in MHUVECs than in FHUVECs, indicating that male cells are more autophagic than female cells. The expression of oestrogen and androgen receptor genes and proteins, the protein expression of Akt and mTOR and cellular size and shape were not influenced by sex. Body weights of male and female neonates were not significantly different, but the weight of male babies positively correlated with the weight of the mother, suggesting that the mother’s weight may exert a different influence on male and female babies.

Conclusions

The results indicate that sex differences exist in prenatal life and are parameter-specific, suggesting that HUVECs of both sexes should be used as an in vitro model to increase the quality and the translational value of research. The sex differences observed in HUVECs could be relevant in explaining the diseases of adulthood because endothelial dysfunction has a crucial role in the pathogenesis of cardiovascular diseases, diabetes mellitus, neurodegeneration and immune disease.