Valproic acid affects the engraftment of TPO-expanded cord blood cells in NOD/SCID mice

Hematopoietic stem and progenitor cells (HSPC) can improve the long-term outcome of transplanted individuals and reduce the relapse rate. Valproic acid (VPA), an inhibitor of histone deacetylase, when combined with different cytokine cocktails, induces the expansion of CD34+ cell populations derived from cord blood (CB) and other sources. We evaluated the effect of VPA, in combination with thrombopoietin (TPO), on the viability and expansion of CB-HSPCs and on short- and long-term engraftability in the NOD/SCID mouse model. In vitro, VPA+TPO inhibited HSPC differentiation and preserved the CD34+ cell fraction; the self-renewal of the CD34+ TPO+VPA-treated cells was suggested by the increased replating efficiency. In vivo, short- and long-term engraftment was determined after 6 and 20 weeks. After 6 weeks, the median chimerism percentage was 13.0% in mice transplanted with TPO-treated cells and only 1.4% in those transplanted with TPO+VPA-treated cells. By contrast, after 20 weeks, the engraftment induced by the TPO+VPA-treated cells was three times more effective than that induced by TPO alone, and over ten times more effective compared to the short-term engraftment induced by the TPO+VPA-treated cells. The in vivo results are consistent with the higher secondary plating efficiency of the TPO+VPA-treated cells in vitro.     

Evaluation of the occlusion vertical dimension of complete dentures after microwave disinfection

doi: 10.1111/j.1741‐2358.2011.00567.x Evaluation of the occlusion vertical dimension of complete dentures after microwave disinfection Objectives: An increase in occlusal vertical dimension (OVD) after microwave disinfection may result in the need for adjustments in the complete dentures. This in vitro study evaluated the increase in OVD of maxillary complete dentures submitted to microwave disinfection protocols. Material and methods: Thirty sets of complete dentures were evaluated as follows: Group 1–15 sets had the maxillary complete dentures submitted to microwave disinfection (650 W/3 min), once a week, for 4 weeks. Group 2–15 sets had the maxillary complete dentures submitted to microwave disinfection (650 W/3 min), three times a week, for 4 weeks. The vertical dimension was measured with a micrometre (in mm) before disinfection protocols (baseline readings) and after each week of disinfection. Data were analysed using Wilcoxon and Friedman tests (α = 0.05). Results: For Group 1, no significant difference was found between the increases in OVD and zero, and no significant difference was found between the weeks. For Group 2, the increases in OVD were significantly greater than zero, and the Friedman test showed that weeks 3 and 4 had significantly greater changes than week 1 and that week 4 had significantly greater change than week 2. Conclusion: Microwave disinfection only promoted significant increase in OVD in Group 2, in which the values increased progressively.     

Anti-tumor activity of the combination of cetuximab, an anti-EGFR blocking monoclonal antibody and ZD6474, an inhibitor of VEGFR and EGFR tyrosine kinases

PURPOSE: The epidermal growth factor receptor (EGFR) autocrine pathway plays an important role in cancer cell growth. Vascular endothelial growth factor A (VEGF-A) is a key regulator of tumor-induced endothelial cell proliferation and vascular permeability. ZD6474 is an orally available, small molecule inhibitor of VEGF receptor-2 (VEGFR-2), EGFR and RET tyrosine kinase activity. We investigated the activity of ZD6474 in combination with cetuximab, an anti-EGFR blocking monoclonal antibody, to determine the anti-tumor activity of EGFR blockade through the combined use of two agents targeting the receptor at different molecular sites in cancer cells and of VEGFR-2 blockade in endothelial cells. EXPERIMENTAL DESIGN: The anti-tumor activity in vitro and in vivo of ZD6474 and/or cetuximab was tested in human cancer cell lines with a functional EGFR autocrine pathway. RESULTS: The combination of ZD6474 and cetuximab determined synergistic growth inhibition in all cancer cell lines tested as assessed by the Chou and Talalay method. In nude mice bearing established human colon carcinoma (GEO) or lung adenocarcinoma (A549) xenografts and treated with ZD6474 and/or cetuximab for 4 weeks, a reversible tumor growth inhibition was caused by each drug. In contrast, a more significant tumor growth delay resulted from the combination of the two agents with an approximately 100-110 days increase in mice median overall survival as compared to single agent treatment. CONCLUSIONS: This study provides a rationale for evaluating in a clinical setting the double blockade of EGFR in combination with inhibition of VEGFR-2 signaling as cancer therapy.     

Spectroscopic study of the interaction of Ni-5-triethyl ammonium methyl salicylidene ortho-phenylendiiminate with native DNA

The interaction of native calf thymus DNA with the cationic Ni(II) complex of 5-triethyl ammonium methyl salicylidene ortho-phenylendiimine (NiL²⁺), in 1 mM Tris-HCl aqueous solutions at neutral pH, has been monitored as a function of the metal complex-DNA molar ratio by UV absorption spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. The dramatic modification of the DNA CD spectrum, the appearance of a broad induced CD band in the range 350-400 nm, the strong increase of the DNA melting temperature (T_m) and the fluorescence quenching of ethidium bromide-DNA solutions, in the presence of increasing amounts of the NiL²⁺ metal complex, support the existence of a tight intercalative interaction of NiL²⁺ with DNA, analogous to that recently reported for both ZnL²⁺ and CuL²⁺. The intrinsic binding constant (K_b) and the interaction stoichiometry (s), determined by UV spectrophotometric titration, are equal to 4.3 × 10⁶ M⁻¹ and 1.0 base pair per metal complex, respectively. Interestingly, the value of K_b is slightly higher and 10 times higher than that relative to the CuL²⁺-DNA and the ZnL²⁺-DNA systems, respectively. Speculations can be performed to rationalize the observed trend, on the basis of the electronic and geometrical structures of the three complexes of the same ligand. Analogously to what previously observed for CuL²⁺, the shape of the CD of the NiL²⁺-DNA system at NiL²⁺-DNA molar ratios higher than 0.5 is indicative of the formation of supramolecular aggregates in solutions, as a possible consequence of the electrostatic interaction between the cationic complex and the negatively charged phosphate groups of DNA.     

Evaluation of Morphology and Seed Production of Alpine Cat's Tail [Phleum rhaeticum (Humph.) Rauschert] Germplasm in View of its Use for Ecological Restoration at High Altitude

"Site-specific" vegetation, that is germplasm ecologically adapted to the prevailing pedoclimatic conditions and native to the target region, is increasingly required in the revegetation of disturbed areas at high altitude. Some key site-specific species have been reported for the Alpine region. Better knowledge is needed on the existing variability of these species, while high seed yield is required to ensure the availability of commercially adequate seed supply. Natural populations of Alpine cat's tail from the Rhaetian Alps were evaluated for morphology and seed production at a mountain and at a lowland site of northern Italy aiming at (1) identifying possible superior populations for the selection of adapted high-yielding cultivars and (2) verifying whether the seed multiplication of site-specific species could also be undertaken in areas different from those of origin. The germplasm from one valley featured an interesting architecture of traits, representing a possible ideotype for seed production purposes. Individual populations across different valleys also proved to be a valuable source of variation for economically useful traits. A discussion on possible implications of use of nonlocal germplasm sources for restoration purposes is provided. The lowland site gave much lower seed production than the mountain location and experienced outstanding plant mortality after the first summer. The specific site conditions, namely the occurrence of an appreciable level of heat and drought stress in summer and a severe rust infection rather than the altitude per se, limited the suitability of this site for seed production of the alpine germplasm.     

Cremation practices coexisting at the S’Illot des Porros Necropolis during the Second Iron Age in the Balearic Islands (Spain)

The necropolis of S’Illot des Porros, one of the most important prehistoric funerary sites of the Balearic Islands (Spain), was in use from the VIth and Vth century BCE until the Ist century CE. Located in a funerary area which contains two cementeries and one sanctuary, this site is constituted by three funerary chambers named A, B and C, respectively. Investigations on all the human burnt bone remains of the chambers, carried out mainly by the X-ray diffraction and supplemented in some cases by Fourier Transform Infrared spectroscopy pointed to the simultaneous use of inhumation and cremation funerary rites, probably due to existing social differences.In particular, it was argued that the chambers were differentiated, i.e., B was dedicated to inhumations and A to cremations, the cremations found in chamber B very likely being a result of a cleaning-purification of the burial area. Moreover, chamber C, which is the most ancient (IVth century BCE) and with the largest number of inhumed remains, contains the smallest number of remains that were exposed to fire and just in one case it seems possible to attribute a genuine high-temperature cremation.     

Detecting Stress at the Whole-Ecosystem Level: The Case of a Mountain Lake (Lake Santo, Italy)

Detecting the early signs of stress is imperative for the conservation of natural ecosystems. They may, however, go unrecognized because ecosystems, when disturbed, may act as sinks that absorb the external impact without showing any evident changes. This seems to be the case for Lake Santo, a small water body located in a mountainous area of northern Italy. Tourism activity in this area began to develop in the early 1970s and grew continuously over the following 20 years. This activity caused a continually increasing nutrient load into the waters, but surprisingly the lake has remained oligo-mesotrophic, as it was before human pressure became a stressor to the lake. To anticipate possible severe damage to the ecosystem, we searched for early signs of stress by carrying out a retrospective analysis based on a whole-ecosystem approach using trophic flow networks. Ecosystem properties of the lake as calculated from network analysis for the disturbed (year 1991) and unimpacted (year 1973) configurations were compared, with the support of sensitivity analysis and statistical tests. We found evidence that in the period 1970–90 nutrient enrichment did change the course of normal development as the observed increase in system throughput was accompanied by a drop in the level of mutual organization of flows, which instead would be expected to increase during the natural progression of the ecosystem. The scenario that emerged from the comparison of system-level indices, cycling activity, trophic structure, and trophic efficiency indicates that the ecosystem has been subjected to stress. In particular, the type of disturbance corresponds to a quantitative definition of eutrophication.     

Sourdough bread made from wheat and nontoxic flours and started with selected lactobacilli is tolerated in celiac sprue patients

This work was aimed at producing a sourdough bread that is tolerated by celiac sprue (CS) patients. Selected sourdough lactobacilli had specialized peptidases capable of hydrolyzing Pro-rich peptides, including the 33-mer peptide, the most potent inducer of gut-derived human T-cell lines in CS patients. This epitope, the most important in CS, was hydrolyzed completely after treatment with cells and their cytoplasmic extracts (CE). A sourdough made from a mixture of wheat (30%) and nontoxic oat, millet, and buckwheat flours was started with lactobacilli. After 24 h of fermentation, wheat gliadins and low-molecular-mass, alcohol-soluble polypeptides were hydrolyzed almost totally. Proteins were extracted from sourdough and used to produce a peptic-tryptic digest for in vitro agglutination tests on K 562(S) subclone cells of human origin. The minimal agglutinating activity was ca. 250 times higher than that of doughs chemically acidified or started with baker's yeast. Two types of bread, containing ca. 2 g of gluten, were produced with baker's yeast or lactobacilli and CE and used for an in vivo double-blind acute challenge of CS patients. Thirteen of the 17 patients showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread. When fed the sourdough bread, the same 13 patients had values for excreted rhamnose and lactulose that did not differ significantly from the baseline values. The other 4 of the 17 CS patients did not respond to gluten after ingesting the baker's yeast or sourdough bread. These results showed that a bread biotechnology that uses selected lactobacilli, nontoxic flours, and a long fermentation time is a novel tool for decreasing the level of gluten intolerance in humans.     

A nucleophilic catalysis step is involved in the hydrolysis of aryl phosphate monoesters by human CT acylphosphatase

Acylphosphatase, one of the smallest enzymes, is expressed in all organisms. It displays hydrolytic activity on acyl phosphates, nucleoside di- and triphosphates, aryl phosphate monoesters, and polynucleotides, with acyl phosphates being the most specific substrates in vitro. The mechanism of catalysis for human acylphosphatase (the organ-common type isoenzyme) was investigated using both aryl phosphate monoesters and acyl phosphates as substrates. The enzyme is able to catalyze phosphotransfer from p-nitrophenyl phosphate to glycerol (but not from benzoyl phosphate to glycerol), as well as the inorganic phosphate-H(2)18O oxygen exchange reaction in the absence of carboxylic acids or phenols. In short, our findings point to two different catalytic pathways for aryl phosphate monoesters and acyl phosphates. In particular, in the aryl phosphate monoester hydrolysis pathway, an enzyme-phosphate covalent intermediate is formed, whereas the hydrolysis of acyl phosphates seems a more simple process in which the Michaelis complex is attacked directly by a water molecule generating the reaction products. The formation of an enzyme-phosphate covalent complex is consistent with the experiments of isotope exchange and transphosphorylation from substrates to glycerol, as well as with the measurements of the Br?nsted free energy relationships using a panel of aryl phosphates with different structures. His-25 involvement in the formation of the enzyme-phosphate covalent complex during the hydrolysis of aryl phosphate monoesters finds significant confirmation in experiments performed with the H25Q mutated enzyme.     

Sequence-dependent DNA curvature and flexibility from scanning force microscopy images

This paper reports a study of the sequence-dependent DNA curvature and flexibility based on scanning force microscopy (SFM) images. We used a palindromic dimer of a 1878-bp pBR322 fragment and collected a large pool of SFM images. The curvature of each imaged chain was measured in modulus and direction. It was found that the ensemble curvature modulus does not allow the separation of static and dynamic contributions to the curvature, whereas the curvature, when its direction in the two dimensions is taken into account, permits the direct separation of the intrinsic curvature contributions static and dynamic contributions. The palindromic symmetry also acted as an internal gauge of the validity of the SFM images statistical analysis. DNA static curvature resulted in good agreement with the predicted sequence-dependent intrinsic curvature. Furthermore, DNA sequence-dependent flexibility was found to correlate with the occurrence of A.T-rich dinucleotide steps along the chain and, in general, with the normalized basepair stacking energy distribution.