On the Mechanism of Cationic Amphiphile-mediated Transfection. To Fuse or not to Fuse: Is that the Question?

Due to charge interaction, cationic lipids spontaneously associate with nucleic acids, resulting in the formation of so-called lipoplexes. Lipoplexes are membranous structures that are capable of transducing genes into cells, eventually leading to expression of the genes (transfection). The mechanism involved in the cellular uptake of lipoplexes is most likely endocytosis, which occurs after nonspecific charge-mediated binding to cellular receptors. An important step in the transfection process following the actual internalization of lipoplexes is the release of the lipoplex and/or its DNA into the cytoplasm in order to evade lysosomal degradation. Here, the membranous nature of the lipoplex seems to be crucial in that it allows the exchange of lipids between the endosomal membrane and the lipoplex, which results in membrane perturbations that are a prerequisite in the endosomal escape of DNA. Interestingly, a hexagonal phase of the lipoplexes has been correlated with efficient transfection and it can be envisaged that such a phase could be instrumental in the creation of membrane perturbations. Subsequent to its release into the cytoplasm, the DNA has to be transferred into the nucleus. The nuclear import of DNA is most likely a protein-mediated process. In addition, the nuclear uptake of DNA may be facilitated at the time of nuclear envelope disassembly during mitosis. Currently, cationic liposomes are widely used as gene carrier system to deliver nucleic acids into cells in culture to study the cell-biological functioning of genes plus accompanying proteins. Ultimately, cationic lipids may be used in gene therapeutic protocols.     

Gene delivery by cationic lipid vectors: overcoming cellular barriers

Non-viral vectors such as cationic lipids are capable of delivering nucleic acids, including genes, siRNA or antisense RNA into cells, thus potentially resulting in their functional expression. These vectors are considered as an attractive alternative for virus-based delivery systems, which may suffer from immunological and mutational hazards. However, the efficiency of cationic-mediated gene delivery, although often sufficient for cell biological purposes, runs seriously short from a therapeutics point of view, as realizing this objective requires a higher level of transfection than attained thus far. To develop strategies for improvement, there is not so much a need for novel delivery systems. Rather, better insight is needed into the mechanism of delivery, including lipoplex–cell surface interaction, route of internalization and concomitant escape of DNA/RNA into the cytosol, and transport into the nucleus. Current work indicates that a major obstacle involves the relative inefficient destabilization of membrane-bounded compartments in which lipoplexes reside after their internalization by the cell. Such an activity requires the capacity of lipoplexes of undergoing polymorphic transitions such as a membrane destabilizing hexagonal phase, while cellular components may aid in this process. A consequence of the latter notion is that for development of a novel generation of delivery devices, entry pathways have to be triggered by specific targeting to select delivery into intracellular compartments which are most susceptible to lipoplex-induced destabilization, thereby allowing the most efficient release of DNA, a minimal requirement for optimizing non-viral vector-mediated transfection. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Statistical modeling of biomedical corpora: mining the Caenorhabditis Genetic Center Bibliography for genes related to life span

Background  

The statistical modeling of biomedical corpora could yield integrated, coarse-to-fine views of biological phenomena that complement discoveries made from analysis of molecular sequence and profiling data. Here, the potential of such modeling is demonstrated by examining the 5,225 free-text items in the Caenorhabditis Genetic Center (CGC) Bibliography using techniques from statistical information retrieval. Items in the CGC biomedical text corpus were modeled using the Latent Dirichlet Allocation (LDA) model. LDA is a hierarchical Bayesian model which represents a document as a random mixture over latent topics; each topic is characterized by a distribution over words.     

Estimating the rate of evolution of the rate of molecular evolution

A simple model for the evolution of the rate of molecular evolution is presented. With a Bayesian approach, this model can serve as the basis for estimating dates of important evolutionary events even in the absence of the assumption of constant rates among evolutionary lineages. The method can be used in conjunction with any of the widely used models for nucleotide substitution or amino acid replacement. It is illustrated by analyzing a data set of rbcL protein sequences.      

Evaluation of a candidate International Standard for Meningococcal Group C polysaccharide

Meningococcal group C (MenC) plain polysaccharide (PS) and conjugate vaccines are primarily evaluated by physicochemical methods to ensure that batches are consistently manufactured. As different assays are employed to quantify the MenC PS content of final formulations and bulk intermediaries, there is a need for an International MenC PS Standard to calibrate internal references used in the different laboratories. Twelve laboratories from nine different countries participated in a collaborative study to determine the MenC PS content of a candidate International Standard MenC PS preparation (08/214) and to assess its suitability. On the basis of the results from this study the candidate standard 08/214 was established as an International Standard for the quantification of MenC PS content in vaccines and components. It has a content of 1.192 ± 0.192 mg MenC PS/ampoule (expanded uncertainty with coverage factor of k = 2.365 corresponding to a 95% level of confidence), as determined by the resorcinol assays carried out by eight of the participating laboratories. The standard is available from The National Institute of Biological Standards and Control who act as guardians and distributors of the material under the auspices of WHO.     

Ensemble attribute profile clustering: discovering and characterizing groups of genes with similar patterns of biological features

Background  

Ensemble attribute profile clustering is a novel, text-based strategy for analyzing a user-defined list of genes and/or proteins. The strategy exploits annotation data present in gene-centered corpora and utilizes ideas from statistical information retrieval to discover and characterize properties shared by subsets of the list. The practical utility of this method is demonstrated by employing it in a retrospective study of two non-overlapping sets of genes defined by a published investigation as markers for normal human breast luminal epithelial cells and myoepithelial cells.     

Interference of serum with lipoplex-cell interaction: modulation of intracellular processing

We have investigated the mechanism of lipoplex-mediated transfection, employing a dialkyl pyridinium surfactant (SAINT-2), and using serum as a modulator of complex stability and processing. Particle size and stability determine lipoplex internalization, the kinetics of intracellular processing, and transfection efficiency. Clustered SAINT-2 lipoplexes are obtained in the absence of serum (-FBS lipoplexes), but not in its presence (+FBS lipoplexes), or when serum was present during lipoplex formation [FBS], conditions that mimic potential penetration of serum proteins. The topology of DNA in [FBS] lipoplexes shifts from a supercoiled, as in -FBS lipoplexes, to a predominantly open-circular conformation, and is more prone to digestion by DNase. Consistently, atomic force microscopy revealed complexes with tubular extensions, reflecting DNA that protrudes from the lipoplex surface. Interestingly, the internalization of [FBS] lipoplexes is approximately three-fold higher than that of -FBS and +FBS lipoplexes, yet their transfection efficiency is approximately five-fold lower. Moreover, in contrast to -FBS and +FBS complexes, [FBS] complexes were rapidly processed into the late endosomal/lysosomal degradation pathway. Intriguingly, transfection by [FBS] complexes is greatly improved by osmotic rupture of endocytic compartments. Our data imply that constraints in size and morphology govern the complex' ability to interact with and perturb cellular membranes, required for gene release. By extrapolation, we propose that serum may regulate these parameters in an amphiphile-dependent manner, by complex 'penetration' and modulation of DNA conformation.     

Influence of Boar and Semen Parameters on Motility and Acrosome Integrity in Liquid Boar Semen Stored for Five Days

Ninety ejaculates from a total of 76 AI boars were extended in Beltsville Thawing Solution (BTS). Boar identity, breed, weight of the ejaculate and sperm concentration were registered. Motility and acrosome integrity were assessed after storage at 16–18°C for 6, 30, 54, 78, and 102 h. Storage time had a significant influence on both motility (p < 0.01) and acrosome integrity (p < 0.001). The Least Square Means for percentage of motility showed a small decline from 79.8% after 6 h of storage to 78.4% at 102 h. Motility at 78 and 102 h was significantly different from motility at 6 h (p < 0.05). The percentage of sperm cells with normal acrosomes declined throughout the experiment. The Least Square Means for 6, 30, 54, 78, and 102 h of storage were 93.9%, 90.6%, 88.0%, 84.8%, and 78.2%, respectively. The decrease in acrosome integrity from one storage time to the next was highly significant throughout the trial (p < 0.001). There was a significant influence of boar (p < 0.001) and sperm concentration (p < 0.01) on motility, while acrosome integrity was affected only by boar (p < 0.001). Breed of the boars and weight of the ejaculate did not influence the dependent variables.     

Lipoplex formation under equilibrium conditions reveals a three-step mechanism

Cellular transfection can be accomplished by the use of synthetic amphiphiles as gene carrier system. To understand the mechanism and hence to improve the efficiency of transfection, insight into the assembly and properties of the amphiphile/gene complex is crucial. Here, we have studied the interaction between a plasmid and cationic amphiphiles, using a monolayer technique, and have examined complex assembly by atomic force microscopy. The data reveal a three-step mechanism for complex formation. In a first step, the plasmids, interacting with the monolayer, display a strong tendency of orientational ordering. Subsequently, individual plasmids enwrap themselves with amphiphile molecules in a multilamellar fashion. The size of the complex formed is determined by the supercoiled size of the plasmid, and calculations reveal that the plasmid can be surrounded by 3 to 5 bilayers of the amphiphile. The eventual size of the transfecting complex is finally governed by fusion events between individually wrapped amphiphile/DNA complexes. In bulk phase, where complex assembly is triggered by mixing amphiphilic vesicles and plasmids, a similar wrapping process is observed. However, in this case, imperfections in this process may give rise to a partial exposure of plasmids, i.e., part of the plasmid is not covered with a layer of amphiphile. We suggest that these exposed sites may act as nucleation sites for massive lipoplex clustering, which in turn may affect transfection efficiency.     

Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape,DNA translocation,and transfection efficiency

Cationic lipids are widely used for gene transfection, but their mechanism of action is still poorly understood. To improve this knowledge, a structure-function study was carried out with two pyridinium-based lipid analogs with identical headgroups but differing in alkyl chain (un)saturation, i.e., SAINT-2 (diC18:1) and SAINT-5 (diC18:0). Although both amphiphiles display transfection activity per se, DOPE strongly promotes SAINT-2-mediated transfection, but not that of SAINT-5, despite the fact that DOPE effectively facilitates plasmid dissociation from either lipoplex. This difference appears to correlate with membrane stiffness, dictated by the cationic lipid packing in the donor liposomes, which governs the kinetics of lipid recruitment by the plasmid upon lipoplex assembly. Because of its interaction with the relatively rigid SAINT-5 membranes, the plasmid becomes inappropriately condensed, which results in formation of structurally deformed lipoplexes. This structural deformation does not affect its cellular uptake but, rather, hampers plasmid translocation across endosomal and/or nuclear membranes. This is inferred from the observation that both lipoplexes effectively translocate much smaller oligonucleotides into cells. In fact, SAINT-5/DOPE-mediated transfection is greatly improved when, before lipoplex assembly, the plasmid is stabilized by condensation with polylysine. The results emphasize a role of the structural shape of the plasmid in gaining cytosolic/nuclear access. Moreover, it has been proposed that such a translocation is promoted when the lipoplex adopts the hexagonal phase, and data are presented that demonstrate that the lamellar SAINT-5/DOPE lipoplex adopts such a phase after its interaction with acidic phospholipid-containing membranes.