Synthesis and characterization of tetra-end linked oligonucleotides capable of forming monomolecular G-quadruplexes

The chemical synthesis of two new G-rich Tetra-End-Linked-oligodeoxyribonucleotides (TEL-ODNs) as well as (1)H-NMR and CD spectra of the corresponding monomolecular quadruplexes (IVa and IVb) has been reported. The new TEL-ODNs, characterized by the presence of short branches in the linker moiety, could be very useful for the achievement of monomolecular quadruplexes with predetermined strand orientation.     

Optical tweezers as a probe for oligodeoxyribonucleotide structuration

The aim of this work is to investigate if the optical tweezers (OT) are suitable as a diagnostic tool for monitoring the oligodeoxyribonucleotide (ODN) structural behavior in solution. Preliminary experiments, performed on the quadruplex formed by the ODN sequence TGGGGT, showed that the OT can be used as a probe for ODN structuration by monitoring the medium viscosity changes associated with ODN folding-unfolding processes.     

Reproductive isolation among sympatric cryptic species in marine diatoms

Pseudo-nitzschia is a marine cosmopolitan genus of chain-forming planktonic diatoms. As for the vast majority of phytoplankton organisms, species identification within this genus mostly relies upon morphological features. Taxa were initially identified based on cell shape and gross morphology of their composite silica cell wall, called the frustule. Yet, observations of the frustule in electron microscopy showed many additional characters for species identification and results of molecular studies have demonstrated that genetically distinct groups might exist within morpho-species. However, these studies have not addressed the biological meaning of these genetic differences. Here, we bridge that gap by comparing ultrastructural features and sequence data (three ribosomal and one plastid marker) of 95 strains with results of mating experiments among these strains. Experiments were performed on two morphologically distinct entities: P. delicatissima and P. pseudodelicatissima. Each of the two entities consisted of multiple genetically distinct and reproductively isolated taxa, all occurring in sympatry: P. delicatissima was composed of three phylogenetic and reproductively distinct groups, whereas P. pseudodelicatissima consisted of up to five. Once these taxa had been defined both genetically and biologically, subtle ultrastructural differences could be detected as well. Our findings not only show that cryptic genetic variants abound in sympatry, but also that they are reproductively isolated and, therefore, biologically distinct units.     

GQ-16, a Novel Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand, Promotes Insulin Sensitization without Weight Gain

The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.     

Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells

Recent studies have demonstrated that adipose-derived mesenchymal cells (AMCs) offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage, and fat. Given that cartilage is an avascular tissue and that mesenchymal cells experience hypoxia during prechondrogenic condensation in endochondral ossification, the goal of this study was to understand the influence of oxygen tension on AMC differentiation into bone and cartilage. In vitro chondrogenesis was induced using a three-dimensional micromass culture model supplemented with TGF-1. Collagen II production and extracellular matrix proteoglycans were assessed with immunohistochemistry and Alcian blue staining, respectively. Strikingly, micromasses differentiated in reduced oxygen tension (2% O₂) showed markedly decreased chondrogenesis. Osteogenesis was induced using osteogenic medium supplemented with retinoic acid or vitamin D and was assessed with alkaline phosphatase activity and mineralization. AMCs differentiated in both 21 and 2% O₂ environments. However, osteogenesis was severely diminished in a low-oxygen environment. These data demonstrated that hypoxia strongly inhibits in vitro chondrogenesis and osteogenesis in AMCs. cartilage; bone     

ATM activation and signaling under hypoxic conditions

The ATM kinase has previously been shown to respond to the DNA damage induced by reoxygenation following hypoxia by initiating a Chk 2-dependent cell cycle arrest in the G(2) phase. Here we show that ATM is both phosphorylated and active during exposure to hypoxia in the absence of DNA damage, detectable by either comet assay or 53BP1 focus formation. Hypoxia-induced activation of ATM correlates with oxygen concentrations low enough to cause a replication arrest and is entirely independent of hypoxia-inducible factor 1 status. In contrast to damage-activated ATM, hypoxia-activated ATM does not form nuclear foci but is instead diffuse throughout the nucleus. The hypoxia-induced activity of both ATM and the related kinase ATR is independent of NBS1 and MRE11, indicating that the MRN complex does not mediate the DNA damage response to hypoxia. However, the mediator MDC1 is required for efficient activation of Kap1 by hypoxia-induced ATM, indicating that similarly to the DNA damage response, there is a requirement for MDC1 to amplify the ATM response to hypoxia. However, under hypoxic conditions, MDC1 does not recruit BRCA1/53BP1 or RNF8 activity. Our findings clearly demonstrate that there are alternate mechanisms for activating ATM that are both stress-specific and independent of the presence of DNA breaks.     

Synthesis of quadruplex‐forming tetra‐end‐linked oligonucleotides: Effects of the linker size on quadruplex topology and stability

G‐quadruplexes are characteristic structural arrangements of guanine‐rich DNA sequences that abound in regions with relevant biological significance. These structures are highly polymorphic differing in the number and polarity of the strands, loop composition, and conformation. Furthermore, the cation species present in solution strongly influence the topology of the G‐quadruplexes. Recently, we reported the synthesis and structural studies of new G‐quadruplex forming oligodeoxynucleotides (ODNs) in which the 3′‐ and/or the 5′‐ends of four ODN strands are linked together by a non‐nucleotidic tetra‐end‐linker (TEL). These TEL‐ODN analogs having the sequence TGGGGT are able to form parallel G‐quadruplexes characterized by a remarkable high thermal stability. We report here an investigation about the influence of the reduction of the TEL size on the molecularity, topology, and stability of the resulting TEL‐G‐quadruplexes using a combination of circular dichroism (CD), CD melting, ¹H NMR spectroscopy, gel electrophoresis, and molecular modeling data. We found that all TEL‐(TGGGGT)₄ analogs, regardless the TEL size and the structural orientation of the ODN branches, formed parallel TEL‐G‐quadruplexes. The molecular modeling studies appear to be consistent with the experimental CD and NMR data revealing that the G‐quadruplexes formed by TEL‐ODNs having the longer TEL (L 1 ‐ 4 ) are more stable than the corresponding G‐quadruplexes having the shorter TEL (S 1 ‐ 4 ). The relative stability of S 1 ‐ 4 was also reported. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 466–477, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Early detection of voice diseases via a web-based system

Voice is the result of the coordination of the whole pneumophonoarticulatory apparatus. The analysis of the voice allows the identification of the diseases of the vocal apparatus and currently is carried out from an expert doctor through methods based on the auditory analysis. The paper presents a web-based system for the acquisition and automatic analysis of vocal signals. Vocal signals are submitted by the users through a simple web-interface and are analyzed in real-time by using state-of-the art signal processing techniques, providing first-level information on possible voice alterations. The system offers different analysis functions to the doctors that may analyze suspected cases in detail. The system is currently being tested in the otorhinolaryngologist setting to carry out mass prevention via screening at a regional scale.