Exploiting viral cell-targeting abilities in a single polypeptide, non-infectious, recombinant vehicle for integrin-mediated DNA delivery and gene expression

A recombinant, multifunctional protein has been designed for optimized, cell-targeted DNA delivery and gene expression in mammalian cells. This hybrid construct comprises a viral peptide ligand for integrin alpha(V)beta(3) binding, a DNA-condensing poly-L-lysine domain, and a complete, functional beta-galactosidase protein that serves simultaneously as purification tag and DNA-shielding agent. This recombinant protein is stable; it has been produced successfully in Escherichia coli and can be purified in a single step by affinity chromatography. At optimal molar ratios, mixtures of this vector and a luciferase-reporter plasmid form stable complexes that transfect cultured cells. After exposure to these cell-targeted complexes, steady levels of gene expression are observed for more than 3 days after transfection, representing between 20 and 40% of those achieved with untargeted, lipid-based DNA-condensing agents. The principle to include viral motifs for cell infection in single polypeptide recombinant proteins represents a promising approach towards the design of non-viral modular DNA transfer vectors that conserve the cell-target- ing specificity of native viruses and that do not need further processing after bioproduction in a recombinant host.     

Mechanism-based inactivation of VanX, a D-alanyl-D-alanine dipeptidase necessary for vancomycin resistance

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-level resistance to vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu, Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(SPhip-CHF(2))-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K(irr) = 30+/-1 microM; k(inact) = 7.3+/- 0.3 min(-1)) and active site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such as sodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from (19)F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500+/-500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.     

Growth hormone size variants: changes in the pituitary during development of the chicken

There is considerable evidence for the existence of structural variants of growth hormone (GH). The chicken is a useful model for investigating GH heterogeneity as both size and charge immunoreactive-(ir) variants have been observed in the pituitary and plasma. The present study examined the size distribution of ir-GH in the pituitary gland of chicken, from late embryogenesis through adulthood. Pituitaries were homogenized in the presence of protease inhibitor, and the GH size variants were separated by SDS-PAGE, transferred by Western blotting, immunostained with a specific antiserum to chicken GH, and quantitated by chemiluminescence followed by laser densitometry (chemiluminescent assay). Under nonreducing conditions ir-GH bands of 15, 22, 25, 44, 50, 66, 80, 98, 105 and >110 kDa were observed. Both the relative proportion of the GH size variants and the total pituitary content varied with developmental stage and age. The proportion of the 15-kDa fragment was greatest in the embryonic stage, and then it decreased. The proportion of the monomeric 22-kDa form was lowest at 18 days of embryogenesis (dE) and highest at 20 dE. In contrast, the high MW forms (>/=66 kDa) were lowest in embryos, and they increased (P < 0.05) after hatching. The 22-, 44-, 66-, and 80-kDa forms were assayed for activity by radioreceptor assay following isolation by semipreparative SDS-PAGE. Only the 22-kDa GH variant showed radioreceptor activity. Under reducing conditions for SDS-PAGE, ir-GH bands of 13, 15, 18, 23, 26, 36, 39, 44, 48, 59 and 72 kDa were oberved, but most of the high MW form disappeared. There was a concomitant increase in the proportion of the monomeric band and of several submonomeric forms. The present data indicate that the expression, processing, and/or release of some if not all size variants are under some differential control during growth and development of the chicken.     

A cell adhesion peptide from foot-and-mouth disease virus can direct cell targeted delivery of a functional enzyme

The G-H loop of foot-and-mouth disease virus is a disordered protrusion of the VP1 protein exposed on the virion surface. This short stretch includes an arginine-glycine-aspartic acid tripeptide, a recognized integrin-binding motif, which is responsible for cell attachment and infection. Eight copies of a peptide reproducing the amino acid sequence of this FMDV ligand have been displayed in solvent-exposed regions on an enzymatically active recombinant beta-galactosidase. This viral peptide segment enables the chimeric enzyme to bind mammalian cell lines with different efficiencies, probably depending on the number of suitable cell receptors present on each of them. Moreover, it also promotes the internalization of the attached enzyme, which is transiently active inside the cells. These results suggest further exploration of the potential use of short adhesion peptides of viral origin as cell attachment tags to direct the targeted delivery of both genes and enzymes, instead of whole, infectious viruses.     

Regeneration strategies of tree species in the laurel forest of Tenerife (The Canary Islands)

The laurel-forest of the Canary Islands is a montane cloud-forest. In order to gain some knowledge on the processes that maintain tree species diversity, we conducted an analysis of three different laurel-forest plots of the Anaga massif (Tenerife), varying in canopy composition but growing under similar environmental conditions. For each plot we recorded basal area of the canopy trees (h<1.30 m), the density of suckers and seedlings (h>1.30 m), as well as seed-bank composition. The plots have similar regeneration composition, which appears to be independent of differences in canopy composition. Laurus azorica is the most common seedling species, whereas Prunus lusitanica is the most abundant species among suckers and basal shoots. Neither Erica arborea nor Myrica faya, the two main canopy trees in one of the plots, were found in any of the stands as seedlings or suckers, despite their existence as viable seeds in the seed-bank. The regeneration composition and the canopy composition in one of the plots is remarkable different, revealing differents dynamics processes in the three plots. The results suggest the existence of three well-defined ecological groups: pioneer (regeneration primarily by seedlings), non-pioneer (regeneration by seedlings and suckers) and remnant species (regeneration primarily by suckers).These three groups and the effect of small scale disturbances (natural and human-induced), could help to understand the maintenance of tree species richness.     

Sequential Statistical Improvement of the Liquid Cultivation of Helicobacter pylori

Background: Colonization of the gastric mucosa by Helicobacter pylori is one of the most important causes of acute and chronic gastric pathologies in humans. Achieving the growth of H. pylori in liquid media is of great importance in the development of clinical studies. In this study, we developed a sequential optimization strategy based on statistical models to improve the conditions of liquid culture of H. pylori. Materials and Methods: Four statistical models were sequentially used. First, a Box‐Behnken design was used to select the best process conditions (shaking speed, inoculum concentration, and final volume of culture). Secondly, a general factorial design was used to evaluate the influence of adding gel blocks or gel beads (shape and composition). Then a D‐optimal reduce design was carried out to allow the selection of the most influential factors in increasing the cell concentration (culture media components). Finally, another Box‐Behnken design was used to optimize the concentration of the culture media components previously selected. Results: After 12 hours of liquid culture a concentration of 25 × 10⁸ cells per mL (9.4 log₁₀ cells per mL) of H. pylori was obtained, compared with a predicted 32 × 10⁸ (9.5 log₁₀ cells per mL), which means between 1 and 5 log₁₀ units higher than some previous reports. Conclusions: The sequential statistical approach increased the planktonic H. pylori cell culture. The final culture media and conditions were: Brain Heart Infusion, blood agarose (1.5% w/v), lamb’s blood (3.18% v/v), DENT (0.11% v/v), and Vitox (0.52% v/v) at 60 rpm and 37 °C with filtered CO₂ (5% v/v) bubbled directly into the culture media in a final volume of 76.22 mL.     

Polyaniline grafted polyacylonitrile conductive composite fibers for reversible immobilization of enzymes: Stability and catalytic properties of invertase

Polyacylonitrile fibers (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence of potassium dichromate as an oxidizing agent. The effect of aniline concentration on the grafting efficiency and on the electrical surface resistance of PAN/PANI composite fibers was investigated. The surface resistance of the conductive composite fibers in this work was found to be between 8.0 and 0.5 kΩ/cm. As the amount of grafted PANI increased on the PAN fibers the electrical resistance of composite fibers decreased. The PAN/PANI composite fibers were characterized by SEM and FTIR studies. Composite PAN/PANI fibers were used for reversible immobilization of invertase. The immobilization efficiency and the activity of the immobilized invertase (from 1.0 mg/mL invertase solution at pH 5.5) were increased with increasing PANI contents of the composite fibers. The maximum amount of immobilized enzyme onto composite fibers containing 2.0% PANI was about 76.6 mg/g. The optimum pH for the free enzyme was observed at 5.0. On the other hand, immobilized invertase yielded a broad optimum pH profile between pH 5.0 and 7.0. Immobilized invertase exhibited 83% of its original activity even after two months storage at 4 °C while the free enzyme showed only 7% of its initial activity.     

Metazoans of redoxcline sediments in Mediterranean deep-sea hypersaline anoxic basins

Background

The deep-sea hypersaline anoxic basins (DHABs) of the Mediterranean (water depth ~3500 m) are some of the most extreme oceanic habitats known. Brines of DHABs are nearly saturated with salt, leading many to suspect they are uninhabitable for eukaryotes. While diverse bacterial and protistan communities are reported from some DHAB haloclines and brines, loriciferans are the only metazoan reported to inhabit the anoxic DHAB brines. Our goal was to further investigate metazoan communities in DHAB haloclines and brines.

Results

We report observations from sediments of three DHAB (Urania, Discovery, L’Atalante) haloclines, comparing these to observations from sediments underlying normoxic waters of typical Mediterranean salinity. Due to technical difficulties, sampling of the brines was not possible. Morphotype analysis indicates nematodes are the most abundant taxon; crustaceans, loriciferans and bryozoans were also noted. Among nematodes, Daptonema was the most abundant genus; three morphotypes were noted with a degree of endemicity. The majority of rRNA sequences were from planktonic taxa, suggesting that at least some individual metazoans were preserved and inactive. Nematode abundance data, in some cases determined from direct counts of sediments incubated in situ with CellTracker^TM Green, was patchy but generally indicates the highest abundances in either normoxic control samples or in upper halocline samples; nematodes were absent or very rare in lower halocline samples. Ultrastructural analysis indicates the nematodes in L’Atalante normoxic control sediments were fit, while specimens from L’Atalante upper halocline were healthy or had only recently died and those from the lower halocline had no identifiable organelles. Loriciferans, which were only rarely encountered, were found in both normoxic control samples as well as in Discovery and L’Atalante haloclines. It is not clear how a metazoan taxon could remain viable under this wide range of conditions.

Conclusions

We document a community of living nematodes in normoxic, normal saline deep-sea Mediterranean sediments and in the upper halocline portions of the DHABs. Occurrences of nematodes in mid-halocline and lower halocline samples did not provide compelling evidence of a living community in those zones. The possibility of a viable metazoan community in brines of DHABs is not supported by our data at this time.

     

Whole-Genome Analysis of Diversity and SNP-Major Gene Association in Peach Germplasm

Peach was domesticated in China more than four millennia ago and from there it spread world-wide. Since the middle of the last century, peach breeding programs have been very dynamic generating hundreds of new commercial varieties, however, in most cases such varieties derive from a limited collection of parental lines (founders). This is one reason for the observed low levels of variability of the commercial gene pool, implying that knowledge of the extent and distribution of genetic variability in peach is critical to allow the choice of adequate parents to confer enhanced productivity, adaptation and quality to improved varieties. With this aim we genotyped 1,580 peach accessions (including a few closely related Prunus species) maintained and phenotyped in five germplasm collections (four European and one Chinese) with the International Peach SNP Consortium 9K SNP peach array. The study of population structure revealed the subdivision of the panel in three main populations, one mainly made up of Occidental varieties from breeding programs (POP1OC_B), one of Occidental landraces (POP2OC_T) and the third of Oriental accessions (POP3OR). Analysis of linkage disequilibrium (LD) identified differential patterns of genome-wide LD blocks in each of the populations. Phenotypic data for seven monogenic traits were integrated in a genome-wide association study (GWAS). The significantly associated SNPs were always in the regions predicted by linkage analysis, forming haplotypes of markers. These diagnostic haplotypes could be used for marker-assisted selection (MAS) in modern breeding programs.