Design, synthesis, biophysical and biological studies of trisubstituted naphthalimides as G-quadruplex ligands

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)(3) binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UV-melting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.     

Polymerization of hemoglobins in Arctic fish: Lycodes reticulatus and Gadus morhua

In vitro, and possibly in vivo, hemoglobin polymerization and red blood cell sickling appear to be widespread in codfish. In this article, we show that the hemoglobins of the two Arctic fish Lycodes reticulatus and Gadus morhua also have the tendency to polymerize, as monitored by dynamic light scattering experiments. The elucidation of the primary structure of the single hemoglobin of the zoarcid L. reticulatus shows the presence of a large number of cysteyl residues in α and β chains. Their role in eliciting the ability to produce polymers was also addressed by MALDI-TOF and TOF-TOF mass spectrometry. The G.morhua globins are also rich in Cys, but unlike in L. reticulatus, polymerization does not seem to be disulfide driven. The widespread occurrence of the polymerization phenomenon displayed by hemoglobins of Arctic fish supports the hypothesis that this feature may bea response to stressful environmental conditions.     

Interactions between 'Candidatus Phytoplasma mali' and the apple endophyte Epicoccum nigrum in Catharanthus roseus plants

Aims: We investigated the ultrastructural and molecular interactions between ‘Candidatus Phytoplasma mali’ and the apple endophyte Epicoccum nigrum in the experimental host Catharanthus roseus to determine whether inoculation of endophyte could trigger defence reactions in the host. Methods and Results: Apple proliferation (AP) symptom severity was evaluated in AP‐grafted plants that were treated by E. nigrum and compared with untreated controls. Phytoplasma concentration was quantified by real‐time PCR in treated and untreated plants. Ultrastructural observations revealed that in endophyte‐treated periwinkles, modifications to phytoplasmas, such as irregular shape and cytoplasm confined to the periphery of the cell, and plant cytological changes, such as abundant callose depositions and P‐protein aggregations in the sieve elements, occurred. AP‐grafted plants that were treated by the endophyte (E. nigrum) showed a reduction in symptom severity; in particular, flowers appeared normal in shape and size, when compared with uninfected controls. Real‐time PCR indicated that phytoplasma concentration in AP‐grafted plants treated with E. nigrum was about 2·8 times lower than that in untreated ones. Conclusions: These results demonstrated that the inoculation with E. nigrum influenced phytoplasma infection in C. roseus plants; plant ultrastructural modifications allowed us to hypothesize an enhancing host defence response. Significance and Impact of the Study: At present, curative protocols against this phytoplasma are not available. Alternative approaches are thus required to reduce disease spread. Our study might represent a first step in the clarification of plant–phytoplasma–endophyte relationships to find possible strategies for the control of phytoplasma diseases.     

Nucleolipid nanovectors as molecular carriers for potential applications in drug delivery

Novel thymidine- or uridine-based nucleolipids, containing one hydrophilic oligo(ethylene glycol) chain and one or two oleic acid residues (called ToThy, HoThy and DoHu), have been synthesized with the aim to develop bio-compatible nanocarriers for drug delivery and/or produce pro-drugs. Microstructural characterization of their aggregates has been determined in pure water and in pseudo-physiological conditions through DLS and SANS experiments. In all cases stable vesicles, with mean hydrodynamic radii ranging between 120 nm and 250 nm have been revealed. Biological validation of the nucleolipidic nanocarriers was ensured by evaluation of their toxicological profiles, performed by administration of the nanoaggregates to a panel of different cell lines. ToThy exhibited a weak cytotoxicity and, at high concentration, some ability to interfere with cell viability and/or proliferation. In contrast, DoHu and HoThy exhibited no toxicological relevance, behaving similarly to POPC-based liposomes, widely used for systemic drug delivery. Taken together, these results show nucleolipid-based nanocarriers as finely tunable, multi-functional self-assembling materials of interest for the in vivo transport of biomolecules or drugs.     

The occurrence of Corvomeyenia carolinensis (Harrison, 1971) (Porifera: Spongillidae) in Connecticut

Examination of spicules from a living sponge confirmed thepresenceof Corvomeyenia carolinensis in Mohawk Pond, LitchfieldCounty, Connecticut. Until recently this species had only beenreported from one locality in South Carolina. Based uponspiculeobservations in dated sediment samples this species appears tohaveexisted within Mohawk Pond throughout the past 100+years.     

Spatial and temporal distribution of freshwater sponges in Connecticut lakes based upon analysis of siliceous spicules in dated sediment cores

A survey of 28 Connecticut water bodies for spicular remainsoffreshwater sponges preserved in lacustrine sediments revealed14species representing 10 genera. Sediments were examined atfivelevels (ca 1991, ca 1970, ca 1934, ca 1890, ca 1875)todocument changes in species composition, distribution, andabundance. Five species were recorded statewide in all fivegeologic regions: Spongilla lacustris, Heteromeyeniatubisperma, Anheteromeyenia ryderi, Eunapius fragilis, andEphydatia muelleri. Spongilla lacustris was the mostcommonand widely distributed species. Factors that may havecontributedto the observed distribution of the spongillids in Connecticutareregional differences in lake water chemistries, individualspeciestolerances, and differences in extent of dispersal of variousspecies. Most of the more abundant species are widelydistributedwith respect to water quality parameters. Anheteromeyeniaargyrosperma, Duosclera mackayi and Trochospongillapennsylvanica appear to be restricted to waters exhibitingthelower ranges for conductivity, alkalinity and calcium content.Ingeneral, a greater number of spicules was found in bottomsedimentsof the surveyed lakes as compared with the surface sediments.Oftenthis was correlated with increased sediment accumulation ratesinmore recent periods. When this fact is taken into account, itappears that populations of a number of sponge species haveremained relatively constant in many of the lakes over thepastcentury. However, in some lakes exhibiting lower or onlyslightlyhigher sediment accumulation rates at the surface, fewerspiculeswere also found in more recent sediments, suggesting an actualdecline in sponge population sizes. Changes in abundance overthelast 100 years may be related to changes in land use practicesinsurrounding watersheds and the associated changes in waterchemistries.     

Influence of storage conditions on MALDI‐TOF MS profiling of gingival crevicular fluid: Implications on the role of S100A8 and S100A9 for clinical and proteomic based diagnostic investigations

Gingival crevicular fluid (GCF) may be a source of diagnostic biomarkers of periodontitis/gingivitis. However, peptide fingerprints may change, depending on GCF collection, handling and storage. We evaluated how storage conditions affect the quality and the reproducibility of MALDI‐TOF profiles of this fluid. GCF was collected on paper strips from four subjects with healthy gingiva. Our findings demonstrated that sample storage conditions significantly affect GCF peptide pattern over time. Specifically, the storage of GCF immediately extracted from paper strips generates less variations in molecular profiles compared to the extraction performed after the storage. Significant spectral changes were detected for GCF samples stored at –20°C directly on the paper strips and extracted after three months, in comparison to the freshly extracted control. Noteworthy, a significant decrease in the peak area of HNP‐3, S100A8, full‐length S100A9 and its truncated form were detected after 3 months at –80°C. The alterations found in the “stored GCF” profile not only may affect the pattern‐based biomarker discovery but also make its use not adequate for in vitro diagnostic test targeting S100A8, S100A9 proposed as potential diagnostic biomarkers for periodontal disease. In summary, this study shows that the best preserved signatures were obtained for the GCF samples eluted in trifluoroacetic acid and then immediately stored at –80°C for 1 month. The wealth of information gained from our data on protein/patterns stability after storage might be helpful in defining new protocols which enable optimal preservation of GCF specimen.     

Automated Identification and Location Analysis of Marked Stem Cells Colonies in Optical Microscopy Images

Embryonic stem cells (ESCs) are characterized by two remarkable peculiarities: the capacity to propagate as undifferentiated cells (self-renewal) and the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives (pluripotency). Although the majority of ESCs divide without losing the pluripotency, it has become evident that ESC cultures consists of multiple cell populations highlighted by the expression of early germ lineage markers during spontaneous differentiation. Hence, the identification and characterization of ESCs subpopulations represents an efficient approach to improve the comprehension of correlation between gene expression and cell specification status. To study markers of ESCs heterogeneity, we developed an analysis pipeline which can automatically process images of stem cell colonies in optical microscopy. The question we try to address is to find out the statistically significant preferred locations of the marked cells. We tested our algorithm on a set of images of stem cell colonies to analyze the expression pattern of the Zscan4 gene, which was an elite candidate gene to be studied because it is specifically expressed in subpopulation of ESCs. To validate the proposed method we analyzed the behavior of control genes whose pattern had been associated to biological status such as differentiation (EndoA), pluripotency (Pou5f1), and pluripotency fluctuation (Nanog). We found that Zscan4 is not uniformly expressed inside a stem cell colony, and that it tends to be expressed towards the center of the colony, moreover cells expressing Zscan4 cluster each other. This is of significant importance because it allows us to hypothesize a biological status where the cells expressing Zscan4 are preferably associated to the inner of colonies suggesting pluripotent cell status features, and the clustering between themselves suggests either a colony paracrine effect or an early phase of cell specification through proliferation. Also, the analysis on the control genes showed that they behave as expected.     

Fhit Delocalizes Annexin A4 from Plasma Membrane to Cytosol and Sensitizes Lung Cancer Cells to Paclitaxel

Fhit protein is lost or reduced in a large fraction of human tumors, and its restoration triggers apoptosis and suppresses tumor formation or progression in preclinical models. Here, we describe the identification of candidate Fhit-interacting proteins with cytosolic and plasma membrane localization. Among these, Annexin 4 (ANXA4) was validated by co-immunoprecipitation and confocal microscopy as a partner of this novel Fhit protein complex. Here we report that overexpression of Fhit prevents Annexin A4 translocation from cytosol to plasma membrane in A549 lung cancer cells treated with paclitaxel. Moreover, paclitaxel administration in combination with AdFHIT acts synergistically to increase the apoptotic rate of tumor cells both in vitro and in vivo experiments.