Nonviral gene delivery using poly‐D/L aspartate‐diethylenetriamine cationic polymers and polyethylene glycol: A two‐step approach

Cationic polymers have received much attention as promising nonviral vectors for gene transfer. However, development of polymers with low cell toxicities and high transfection efficiencies continue to be a significant problem and a major hurdle to their success. Poly‐D /L aspartate‐diethylenetriamine poly(D /L Asp‐DET) polymers were synthesized and evaluated as nonviral gene delivery agents. Poly(D /L Asp‐DET) polymers display endosome buffering capacity. The polymers condense plasmid DNA above N:P ratios of 1 and form polyplex particles of ～50–100 nm, with zeta potentials between neutral and +40 mV. Transmission electron microscopy shows the polyplexes to be uniform in size and shape. Polyplexes maintain the structural integrity of DNA following incubation in nucleases and also show high transfection efficiencies with minimal toxicity in both HCT‐116 and PC‐3 cell culture. However, it is found that these poly(D /L Asp‐DET)/DNA polyplexes immediately aggregate in salt and serum conditions, making them unsuitable for use in vivo. Therefore, the polyplexes were further modified by covalent addition of polyethylene glycol (PEG). Introduction of this second step produces PEG‐polyplexes of uniform size (below 100 nm), with neutral zeta potentials that are also stable in both salt and serum conditions. These results suggest poly(D /L Asp‐DET) cationic polymers as potentially safe and efficient nonviral gene delivery agents. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Reactivity of Cobalt-Fullerene Complexes towards Deuterium

The adsorption of molecular deuterium (D₂) onto charged cobalt-fullerene-complexes Co_nC₆₀⁺ (n=1–8) is measured experimentally in a few-collision reaction cell. The reactivity is strongly size-dependent, hinting at clustering of the transition metal atoms on the fullerenes. Formation and desorption rate constants are obtained from the pressure-dependent deuterogenation curves. DFT calculations indeed find that this transition metal clustering is energetically more favorable than decorating the fullerene. For n=1, D₂ is predicted to bind molecularly and for n=2 dissociative and molecular configurations are quasi-isoenergetic. For n=3–8, dissociation of D₂ is thermodynamically preferred. However, reaching the ground state configuration with dissociated deuterium on the timescale of the experiment may be hindered by dissociation barriers.     

SDS-induced multi-stage unfolding of a small globular protein through different denatured states revealed by single-molecule fluorescence

Ionic surfactants such as sodium dodecyl sulfate (SDS) unfold proteins in a much more diverse yet effective way than chemical denaturants such as guanidium chloride (GdmCl). But how these unfolding processes compare on a molecular level is poorly understood. Here, we address this question by scrutinising the unfolding pathway of the globular protein S6 in SDS and GdmCl with single-molecule Förster resonance energy transfer (smFRET) spectroscopy. We show that the unfolding mechanism in SDS is strikingly different and convoluted in comparison to denaturation in GdmCl. In contrast to the reversible two-state unfolding behaviour in GdmCl characterised by kinetics on the timescale of seconds, SDS demonstrated not one, but four distinct regimes of interactions with S6, dependent on the surfactant concentration. At ≤1 mM SDS, S6 and surfactant molecules form quasi-micelles on a minute timescale; at millimolar [SDS], the protein denatures through an unfolded/denatured ensemble of highly heterogeneous states on a multi-second timescale; at tens of millimolar of SDS, the protein unfolds into a micelle-packed conformation on the second timescale; and >50 mM SDS, the protein unfolds with millisecond timescale dynamics. We propose a detailed model for multi-stage unfolding of S6 in SDS, which involves at least three different types of denatured states with different level of compactness and dynamics and a continually changing landscape of interactions between protein and surfactant. Our results highlight the great potential of single-molecule fluorescence as a direct probe of nanoscale protein structure and dynamics in chemically complex surfactant environments.

Multi-stage unfolding of S6 in SDS involving various types of denatured states with different levels of compactness and dynamics.     

Expression analyses of human cleft palate tissue suggest a role for osteopontin and immune related factors in palatal development

Cleft lip and/or palate (CL/P) is a common congenital malformation with a complex etiology which is not fully elucidated yet. Epidemiological studies point to different etiologies in the cleft lip and palate subgroups, isolated cleft lip (CL), isolated cleft palate (CP) and combined cleft lip and palate (CLP). In order to understand the biological basis in these cleft lip and palate subgroups better we studied the expression profiles in human tissue from patients with CL/P. In each of the CL/P subgroups, samples were obtained from three patients and gene expression analysis was performed. Moreover, selected differentially expressed genes were analyzed by quantitative RT-PCR, and by immunohistochemical staining of craniofacial tissue from human embryos. Osteopontin (SPP1) and other immune related genes were significantly higher expressed in palate tissue from patients with CLP compared to CP and immunostaining in palatal shelves against SPP1, chemokine receptor 4 (CXCR4) and serglycin (PRG1) in human embryonic craniofacial tissue were positive, supporting a role for these genes in palatal development. However, gene expression profiles are subject to variations during growth and therefore we recommend that future gene expression in CL/P studies should use tissue from the correct embryonic time and place if possible, to overcome the biases in the presented study.     

Upgrading the Nutritional Value of PKC Using a Bacillus subtilis Derived Monocomponent β-Mannanase

Palm kernel cake (PKC) is an abundant side stream that can only be added to non-ruminant feed in small concentrations due to its content of antinutritional factors, mainly galactomannan, which cannot be digested by non-ruminants. β-mannanases can be added to partially hydrolyze galactomannan to form mannose oligosaccharides, which are known to be prebiotic. We here investigate the action of a β-mannanase from B. subtilis on PKC by colorimetry, NMR and fluorescence microscopy. The amount of mannan oligosaccharides in solution was significantly increased by the β-mannanase and their degree of polymerization (DP) was significantly reduced. There was a dose-response behavior in that larger β-mannanase concentrations increased the amount of soluble mannose oligosaccharides while reducing their average DP. Using confocal immunofluorescence microscopy, solubilization of galactomannan in PKC was clearly visualized. Images show a clear disruption of the cellulose and galactomannan structures of the PKC cell walls. We thus show in this study that using commercial dosages of β-mannanase on PKC can lead to formation of prebiotic compounds. Thus, this study suggests that utilization of PKC in poultry feed formulation might be increased by addition of a β-mannanase and would improve the return on investment.