Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice

Duchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.     

Electrostatic interactions in ionic homopolypeptides in solutions of moderate ionic strength

At sufficiently high ionic strength, long-range electrostatic interactions in a polyelectrolyte such as poly(L -glutamic acid) might be adequately approximated in matrix calculations by use of statistical weights representing second-order interactions. The validity of this assumption has been investigated making use of experimental observations (CD spectra and titration curves) for poly(L -glutamic acid) as a function of temperature in 0.1–0.5M sodium chloride. Theoretical analysis, using a statistical weight matrix proposed by Warashina and Ikegami, is based on the Zimm-Rice theory. Implementation differs from that of Warashina and Ikegami in one respect. Refinement of the initial estimates is achieved using a form of the configuration partition function which does not assume diagonalization of the statistical weight matrix. This difference is of no consequence for the values of σ and s, but it does produce somewhat different values for the statistical weights used to represent the electrostatic interactions. The method used to treat electrostatic interactions in poly(L -glutamic acid) in 0.1M sodium chloride can be viewed as successful in that it properly reproduces the helix–coil transition and titration curves in this solvent and the molecular-weight dependence of the titration curves yields values for s in harmony with those obtained using a treatment which is independent of model, and gives a reasonable ionic-strength dependence for the electrostatic parameters. Furthermore, the model can account for measured helix–coil transitions and titration curves in homopolypeptides in which the side chain is —(CH₂)_xNHCO(CH₂)_yCOOH. The model, however, is not exact. It does not properly account for the molecular-weight dependence of the helical content for polymers of low degree of polymerization.     

Effects of drought stress on legume symbiotic nitrogen fixation: physiological mechanisms

Drought stress is one of the major factors affecting nitrogen fixation by legume-rhizobium symbiosis. Several mechanisms have been previously reported to be involved in the physiological response of symbiotic nitrogen fixation to drought stress, i.e. carbon shortage and nodule carbon metabolism, oxygen limitation, and feedback regulation by the accumulation of N fixation products. The carbon shortage hypothesis was previously investigated by studying the combined effects of CO2 enrichment and water deficits on nodulation and N2 fixation in soybean. Under drought, in a genotype with drought tolerant N2 fixation, approximately four times the amount of 14C was allocated to nodules compared to a drought sensitive genotype. It was found that an important effect of CO2 enrichment of soybean under drought was an enhancement of photo assimilation, an increased partitioning of carbon to nodules, whose main effect was to sustain nodule growth, which helped sustain N2 rates under soil water deficits. The interaction of nodule permeability to O2 and drought stress with N2 fixation was examined in soybean nodules and led to the overall conclusion that O2 limitation seems to be involved only in the initial stages of water deficit stresses in decreasing nodule activity. The involvement of ureides in the drought response of N2 fixation was initially suspected by an increased ureide concentration in shoots and nodules under drought leading to a negative feedback response between ureides and nodule activity. Direct evidence for inhibition of nitrogenase activity by its products, ureides and amides, supported this hypothesis. The overall conclusion was that all three physiological mechanisms are important in understanding the regulation of N2 fixation and its response of to soil drying.     

On calcium-buffer dynamics within the excess buffer regime

In intracellular calcium signaling, calcium buffers has been recognized for their role in reshaping and localizing the calcium concentration profile in the vicinity of the channel, as well as reducing the effective diffusion of free calcium. In the presence of an excess of endogenous or exogenous buffers, linearization of the reaction-diffusion system describing the calcium-buffer dynamics has been instrumental in understanding the extent of the microdomain formation and in quantifying the apparent diffusion of the free calcium. In these linearized models, the conclusions are usually drawn from the steady-state solutions upon the opening of the channel. In this work, using the joint Laplace-Fourier method, we give an explicit integral transient solution, as well as, the long-time asymptotic behavior of the linearized calcium-buffer dynamics. The results confirm and emphasize the long stated intuitions on the diffusive character of the calcium-buffer dynamics. Numerical validations of our analytical results will be discussed.     

A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256

Biofilm formation of Staphylococcus epidermidis on smooth polymer surfaces has been shown to be mediated by the ica operon. Upon activation of this operon, a polysaccharide intercellular adhesin (PIA) is synthesized that supports bacterial cell-to-cell contacts and triggers the production of thick, multilayered biofilms. Thus, the ica gene cluster represents a genetic determinant that significantly contributes to the virulence of specific Staphylococcus epidermidis strains. PIA synthesis has been reported recently to undergo a phase variation process. In this study, biofilm-forming Staphylococcus epidermidis strains and their PIA-negative phase variants were analysed genetically to investigate the molecular mechanisms of phase variation. We have characterized biofilm-negative variants by Southern hybridization with ica-specific probes, polymerase chain reaction and nucleotide sequencing. The data obtained in these analyses suggested that in approximately 30% of the variants the missing biofilm formation was due to the inactivation of either the icaA or the icaC gene by the insertion of the insertion sequence element IS256. Furthermore, it was shown that the transposition of IS256 into the ica operon is a reversible process. After repeated passages of the PIA-negative insertional mutants, the biofilm-forming phenotype could be restored. Nucleotide sequence analyses of the revertants confirmed the complete excision of IS256, including the initially duplicated 8 bp target sites. These results elucidate, for the first time, a molecular mechanism mediating phase variation in staphylcocci, and they demonstrate that a naturally occurring insertion sequence element is actively involved in the modulation of expression of a Staphylococcus virulence factor.     

A two-stage ultrafiltration process for separating multiple components of Tetraselmis suecica after cell disruption

A two-stage ultrafiltration process was applied to the aqueous phase of Tetraselmis suecica after breaking its cell wall by high-pressure homogenization. Microscopic observation revealed that the cells were completely disrupted from 600 bar and cell fragmentation of the cells was also noticeable after 800 bar. In addition, the highest concentration of all the molecules of interest in the aqueous phase was observed at 1,000 bar and a temperature of 46 °C while preserving the integrity of the molecules of interest in the downstream process. After centrifugation, the aqueous phase was submitted to ultrafiltration through two consecutive membranes of different molecular weight cutoffs. Complete retention of starch was possible with a 100-kDa membrane and separation of sugars from proteins with a 10-kDa membrane on the remaining mixture. After testing the process with model solutions, the transmembrane pressure selected was 2.07 bar, which succeeded in retaining starch and pigments during the first part of the process, and proteins during the second part. A linear correlation between the permeate flux rate and the pressure was observed in both parts of the process.     

Single versus multiple enemies and the impact on biological control of spider mites in cassava fields in West-Africa

To determine whether to use single or multiple predator species for biological pest control requires manipulative field experiments. We performed such tests in Benin (West Africa) in cassava fields infested by the cassava green mite Mononychellus tanajoa, and the cotton red mite Oligonychus gossypii. These fields also harboured the cassava apex-inhabiting predator Typhlodromalus aripo and either the leaf-inhabiting predator Amblydromalus manihoti or Euseius fustis. We manipulated predator species composition on individual plants to determine their effect on prey and predator densities. In fields with T. aripo plus A. manihoti, M. tanajoa densities were reduced by T. aripo alone or together with A. manihoti, but neither of these predators, alone or together, reduced O. gossypii densities. In fields with T. aripo plus E. fustis, T. aripo alone or together with E. fustis exerted significant control over O. gossypii, but weak control over M. tanajoa. Densities of any of the predator species were not affected by co-occurring predator species, suggesting a minor role for intraguild predation in the field, contrary to earlier experiments on small plants in the laboratory. We conclude that (1) T. aripo is the most effective predator species in suppressing M. tanajoa, (2) two predator species, T. aripo and E. fustis, are needed to suppress O. gossypii, and (3) predator species together on the same plant do not negatively affect each other nor the extent to which they control their prey. We argue that intraguild predation is reduced due to partial niche separation among predator species.     

Single pH buffer refolding screen for protein from inclusion bodies

We previously reported the set up of an automated test for screening the refolding of recombinant proteins expressed as inclusion bodies in Escherichia coli[1]. The screen used 96 refolding buffers and was validated with 24 proteins, 70% of which remained soluble in at least one buffer. In the present paper, we have analyzed in more detail these experimental data to see if the refolding process can be driven by general rules. Notably, we found that proteins with an acidic isoelectric point (pI) refolded in buffers the average pH of which was alkaline and conversely. In addition, the number of refolding buffers wherein a protein remained soluble increased with the difference between its pI and the average pH of the buffers in which it refolded. A trend analysis of the other variables (ionic strength, detergents, etc.) was also performed. On the basis of this analysis, we devised and validated a new refolding screen made of a single buffer for acidic proteins and a single buffer for alkaline proteins.     

Mericarp morphology and its systematic implications for the genus Salvia L. section Hymenosphace Benth. (Lamiaceae) in Turkey

Mericarp morphology of 15 taxa of Salvia L. section Hymenosphace Benth. in Turkey were investigated by light microscopy (LM) and scanning electron microscopy (SEM) to evaluate the utility of mericarp characters in systematic studies. Mericarps ranged from 2.50 to 5.38?mm in length and 2.04 to 4.70?mm in width. Mericarp shape was prolate-spheroid or near spherical with a length-to-width ratio of 1.02?C1.48. Transverse sections of the mericarps were rounded or rounded-trigonous. Mericarp surfaces presented colliculate, reticulate, verrucate or foveate sculpturing patterns, mostly as a result of the sculpturing of the exocarp cell walls with the pattern determined by whether the periclinal walls were concave or convex and whether the anticlinal walls were raised or sunken. Colliculate surface sculpturing (periclinal walls convex) was the most common among the taxa examined. The variation in the nature of surface sculpturing, mericarp shape and size, exocarp cell shape, nature of transverse sections and abscission scar diameter proved useful diagnostic characters. Variation was sufficient to distinguish taxa at species level, including morphologically similar species. Data provided here are also relevant to phylogenetic questions at higher levels within Salvia.