Architecturally induced multiresponsive vesicles from well-defined polypeptides: formation of gene vehicles

A series of novel, partially labeled amphiphilic triblock copolypeptides, PLL-b-PBLG-d7-b-PLL, has been synthesized, where PLL and PBLG-d7 are poly(L-lysine hydrochloride) and poly(gamma-benzyl-d7-L-glutamate), respectively. The synthetic approach involved the sequential ring-opening polymerization (ROP) of gamma-benzyl-L-glutamate and epsilon-Boc-L-lysine N-carboxy anhydrides by a diamino initiator using high-vacuum techniques, followed by the selective deprotection of the Boc groups. Combined characterization results showed that the copolypeptides exhibit high degrees of molecular and compositional homogeneity. The synthesized copolypeptides had similar molecular weights, while the composition of the middle block ranged between 19 and 74% with respect to the monomeric units. Due to the macromolecular architecture of the copolypeptide and the rigid nature of the middle block, the formation of monolayers was favored, and, surprisingly, vesicles were formed in water at neutral pH over the entire compositional range. The vesicular structures were extensively characterized by static and dynamic light scattering, small-angle neutron scattering, atomic force microscopy, cryo-transmission electron microscopy, scanning electron microscopy, UV and Fourier transform infrared spectroscopy, and circular dichroism. In contrast to other vesicular structures derived from conventional polymers, the formed polypeptidic vesicles possess the unique feature of being stimuli-responsive to pH and temperature. When the copolypeptides were mixed with plasmid DNA (pDNA), large vesicular structures were also formed. The molecular characterization of the vectors was performed with most of the methods mentioned above, and indicated that the pDNA is both partially condensed on the PLL phase and partially encapsulated inside the vesicle. Consequently, the synthesized vectors combine the advantages of the polylysine-DNA systems to condense large amounts of genes, as well as those of the liposome-DNA systems to better protect the encapsulated DNA. These vectors are expected to present better gene transfection efficiency to the cell nucleus.     

Diversity of actin architecture in human osteoclasts: network of curved and branched actin supporting cell shape and intercellular micrometer-level tubes

Osteoclasts are multinucleated bone-resorbing cells with a dynamic actin cytoskeleton. Osteoclasts are derived from circulating mononuclear precursors. Confocal and stimulated emission depletion (STED) super-resolution microscopy was used to investigate peripheral blood-derived human osteoclasts cultured on glass surfaces. STED and confocal microscopy demonstrated that the actin was curved and branched, for instance, in the vicinity of membrane ruffles. The overall architecture of the curved actin network extended from the podosomes to the top of the cell. The other novel finding was that a micrometer-level tube containing actin bridged the osteoclasts well above the level of the culture glass. The actin filaments of the tubes originated from the network of curved actin often surrounding a group of nuclei. Furthermore, nuclei were occasionally located inside the tubes. Our findings demonstrated the accumulation of c-Src, cortactin, cofilin, and actin around nuclei suggesting their role in nuclear processes such as the locomotion of nuclei. ARP2/3 labeling was abundant at the substratum level of osteoclasts and in the branched actin network, where it localized to the branching points. We speculate that the actin-containing tubes of osteoclasts may provide a means of transportation of nuclei, e.g., during the fusion of osteoclasts. These novel findings can pave the way for future studies aiming at the elucidation of the differentiation of multinucleated osteoclasts.     

Oblique self-assemblies and order-order transitions in polypeptide complexes with PEGylated triple-tail lipids

We report on highly ordered oblique self-assemblies in ionic complexes of PEGylated triple-tail lipids and cationic polypeptides, as directed by side-chain crystallization, demonstrating also reversible oblique-to-hexagonal order-order transitions upon melting of the side chains. This is achieved in bulk by complexing cationic homopolypeptides, poly-l-lysine (PLys), poly-l-arginine (PArg), and poly-l-histidine (PHis), in stoichiometric amounts with anionic lipids incorporating two hydrophobic alkyl tails and one hydrophilic polyethylene glycol (PEG) tail in a star-shaped A(2)B geometry. Based on Fourier transform infrared spectroscopy (FTIR), the PLys and PArg complexes fold into α-helical conformation. Aiming to periodicities at different length scales, that is, hierarchies, the PEG tails were selected to control the separation of the polypeptide helices in one direction while the alkyl tails determine the distance between the hydrophilic polypeptide/PEG layers, resulting in an oblique arrangement of the helices. We expect that the high overall order, where the self-assembled domains are in 2D registry, is an outcome of a favorable interplay of plasticization due to the hydrophobic and hydrophilic lipid tails combined with the shape persistency of the peptide helices and the crystallization of the lipid alkyl chains. Upon heating the complexes over the melting temperature of the alkyl tails, an order-order transition from oblique to hexagonal columnar morphology was observed. This transition is reversible, that is, the oblique structure with 2D correlation of the helices is fully returned upon cooling, implying that the alkyl tail crystallization guides the structure formation. Also PHis complex forms an oblique self-assembly. However, instead of α-helices, FTIR suggests formation of helical structures lacking intramolecular hydrogen bonds, stabilized by steric crowding of the lipid. The current study exploits competition between the soft and harder domains, which teaches on concepts toward well-defined polypeptide-based materials.     

Data integration workflow for search of disease driving genes and genetic variants

Comprehensive characterization of a gene's impact on phenotypes requires knowledge of the context of the gene. To address this issue we introduce a systematic data integration method Candidate Genes and SNPs (CANGES) that links SNP and linkage disequilibrium data to pathway- and protein-protein interaction information. It can be used as a knowledge discovery tool for the search of disease associated causative variants from genome-wide studies as well as to generate new hypotheses on synergistically functioning genes. We demonstrate the utility of CANGES by integrating pathway and protein-protein interaction data to identify putative functional variants for (i) the p53 gene and (ii) three glioblastoma multiforme (GBM) associated risk genes. For the GBM case, we further integrate the CANGES results with clinical and genome-wide data for 209 GBM patients and identify genes having effects on GBM patient survival. Our results show that selecting a focused set of genes can result in information beyond the traditional genome-wide association approaches. Taken together, holistic approach to identify possible interacting genes and SNPs with CANGES provides a means to rapidly identify networks for any set of genes and generate novel hypotheses. CANGES is available in http://csbi.ltdk.helsinki.fi/CANGES/     

Tight junction proteins ZO-1, occludin, and claudins in developing and adult human perineurium.

In peripheral nerves, groups of Schwann cell-axon units are isolated from the adjacent tissues by the perineurium, which creates a diffusion barrier responsible for the maintenance of endoneurial homeostasis. The perineurium is formed by concentric layers of overlapping, polygonal perineurial cells that form tight junctions at their interdigitating cell borders. In this study, employing indirect immunofluorescence and immunoelectron microscopy, we demonstrate that claudin-1 and -3, ZO-1, and occludin, but not claudin-2, -4, and -5, are expressed in the perineurium of adult human peripheral nerve. We also describe the expression of occludin, ZO-1, claudin-1, -3, and -5 in the developing human perineurium, showing that the expressions of claudin-1 and -3, ZO-1, and occludin follow similar spatial developmental expression patterns but follow different timetables in achieving their respective adult distributions. Specifically, claudin-1 is already largely restricted to perineurium-derived structures at 11 fetal weeks, whereas claudin-3 and occludin are weakly expressed in the perineurial structures at this age and acquire a well-defined perineurial distribution only between 22 and 35 fetal weeks. ZO-1 appears to acquire its mature profile even later during the third trimester. The results of the present and previous studies show that the perineurial diffusion barrier matures relatively late during human peripheral nerve development.     

Identification of genetic polymorphisms of CYP2S1 in a Finnish Caucasian population

CYP2S1 is a recently discovered member of the cytochrome P450 (CYP) gene superfamily. Interestingly, even though the DNA sequence identifies it as the sole member of the new CYP2S family, CYP2S1 exhibits many features typical to CYP1 family members, e.g. dioxin-inducibility mediated by the aryl hydrocarbon receptor (AHR) and the aryl hydrocarbon receptor nuclear translocator (ARNT). In addition, CYP2S1 metabolises some aromatic hydrocarbons as well as cellular substances. These characteristics, together with a wide extrahepatic tissue distribution, suggest that CYP2S1 may have an important role in both exogenous and endogenous metabolism. This is the first study characterising CYP2S1 alleles and naming them with the recommended CYP allele nomenclature. We used denaturing gradient gel electrophoresis (DGGE) and direct sequencing to investigate genetic variation of CYP2S1 in 100 male Finnish Caucasians. Those exons in which variation was found were examined in subsequent 100 subjects. The coding region of all of the nine exons, as well as a 449 bp fragment of the proximal promoter region, was analysed. This systematic investigation revealed eight single nucleotide polymorphisms (SNPs), which comprise nine different variant alleles (haplotypes), in addition to the wild-type allele. Seven of the SNPs occurred in the protein-coding areas and one in the proximal 3' untranslated region (3'UTR). Two of these sequence variations (10347C > T and 13106C > T) result in non-conservative amino acid substitutions, i.e. Arg380Cys and Pro466Leu, respectively. The respective allelic variants, CYP2S1*2 ([10347C > T]) and CYP2S1*3 (13106C > T; 13255A > G]), occurred in our study population at frequencies of 0.50 and 3.75%, respectively. The most common of the variant alleles was CYP2S1*1H (23.8%), harbouring a 13255A > G substitution located in the 3'UTR.     

Hierarchical ionic self-assembly of rod-comb block copolypeptide-surfactant complexes

Novel hierarchical nanostructures based on ionically self-assembled complexes of diblock copolypeptides and surfactants are presented. Rod-coil diblock copolypeptide poly(gamma-benzyl-L-glutamate)-block-poly(L-lysine), PBLG-b-PLL (Mn = 25,000 and 8000 for PBLG and PLL, respectively, polydispersity index 1.08), was complexed with anionic surfactants dodecanesulfonic acid (DSA) or dodecyl benzenesulfonic acid (DBSA), denoted as PBLG-b-PLL(DSA)1.0 and PBLG-b-PLL(DBSA)1.0, respectively. The complexation leading to supramolecular rod-comb architectures was studied by transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), Fourier transform infrared spectroscopy (FTIR), and polarized optical microscopy (POM). PBLG-b-PLL, PBLG-b-PLL(DBSA)1.0, and PBLG-b-PLL(DSA)1.0 self-assemble with alternating PBLG lamellae and PLL-containing lamellae with a periodicity of 27-33 nm. Within the PBLG lamellae, the rod-like PBLG helices pack with a periodicity of ca. 1.3 nm. The internal structure of the PLL-containing lamellae depends on the complexation. For pure PBLG-b-PLL, the PLL chains adopt a random coil conformation and the PLL domains are disordered. For PBLG-b-PLL(DSA)1.0, lamellar self-assembly of periodicity of 3.7 nm within the PLL(DSA)1.0 domains is observed due to crystalline packing of the linear n-dodecyl tails. For PBLG-b-PLL(DBSA)1.0 with branched dodecyl tails, a distinct SAXS reflection is observed, suggesting self-assembly within the PLL(DBSA)1.0 domains with a periodicity of 2.9 nm. However, due to the absence of higher order reflections, the internal structure cannot be conclusively assigned. The efficient plasticization which leads to fluid-like liquid crystallinity in PBLG-b-PLL(DBSA)1.0 and an alpha-helical conformation according to FTIR allows us to suggest that the PLL(DBSA)1.0 domains have a hexagonal internal structure. The interplay of self-assembly at different length scales combined with rod-like liquid crystallinity can open new routes to design functional materials.