Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore

One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-β have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.     

Stratification according to HGG-IMMUNO RPA model predicts outcome in a large group of patients with relapsed malignant glioma treated by adjuvant postoperative dendritic cell vaccination

Purpose

Adult patients with relapsed high-grade glioma are a very heterogenous group with, however, an invariably dismal prognosis. We stratified patients with relapsed high-grade glioma treated with re-operation and postoperative dendritic cell (DC) vaccination according to a simple recursive partitioning analysis (RPA) model to predict outcome.

Patients and methods

Based on age, pathology, Karnofsky performance score, and mental status, 117 adult patients with relapsed malignant glioma, undergoing re-operation, and postoperative adjuvant dendritic cell (DC) vaccination were stratified into 4 classes. Kaplan–Meier survival estimates were generated for each class of this HGG-IMMUNO RPA model. Extent of resection was documented but not included in the prognostic model.

Results

Kaplan–Meier overall survival estimates revealed significant (p?<?0.0001) differences among the 4 HGG-IMMUNO RPA classes. Long-term survivors, surviving more than 24?months after the re-operation and vaccination, are seen in 54.5, 26.7, 11.5, and 0?% for the classes I, II, III, and IV respectively.

Conclusion

This HGG-IMMUNO RPA classification is able to predict overall survival in a large group of adult patients with a relapsed malignant glioma, treated with re-operation and postoperative adjuvant DC vaccination in the HGG-IMMUNO-2003 cohort comparison trial. The model appears useful for prognostic patient counseling for patients participating in DC vaccination trials. A substantial number of long-term survivors after relapse are seen in class I to III, but not in class IV patients.     

The proteome of the human neuroblastoma cell line SH-SY5Y: an enlarged proteome

The human neuroblastoma cell line SH-SY5Y (ATCC: CRL-2266) is widely used as a neural cellular model system. The hitherto existing proteome data (115 proteins) are here extended. A total of 1103 unique proteins of this cell line were identified using 2D-LC combined with MALDI-TOF/TOF-MS, SDS-PAGE with nano-LC-MS/MS, N-terminal COFRADIC analysis with nano-LC-MS/MS and 2D-PAGE with MALDI-TOF/TOF-MS peptide mass fingerprinting. The obtained proteome profile of this cell line is discussed.     

Prolonged gene silencing in hepatoma cells and primary hepatocytes after small interfering RNA delivery with biodegradable poly(beta-amino esters)

Background

Small interfering (si)RNA mediated inhibition of oncogenes or viral genes may offer great opportunities for the treatment of several diseases such as hepatocellular carcinoma and viral hepatitis. However, the development of siRNAs as therapeutic agents strongly depends on the availability of safe and effective intracellular delivery systems. Poly(β‐amino esters) (PbAEs) are, in contrast to many other cationic polymers evaluated in siRNA delivery, biodegradable into smaller, nontoxic molecules.

Methods and Results

We show for the first time that PbAE : siRNA complexes, containing 1,4‐butanediol (PbAE1) or 1,6‐hexanediol (PbAE2) diacrylate‐based polymers, induced efficient gene silencing in both hepatoma cells and primary hepatocytes without causing significant cytotoxicity. Furthermore, carriers that slowly release the siRNA into the cytoplasm and hence induce a prolonged gene silencing are of major clinical interest, especially in fast dividing tumour cells. Therefore, we also studied the duration of gene silencing in the hepatoma cells and found that it was maintained for at least 5 days after siRNA delivery with PbAE2, the polymer with the slowest degradation kinetics.

Conclusions

From the time‐dependent cellular distribution of these PbAE : siRNA complexes, we suggest that the slowly degrading PbAE2 causes a sustained endosomal release of siRNA during a much longer period than PbAE1. This may support the hypothesis that the endosomal release mechanism of PbAE : siRNA complexes is based on an increase of osmotic pressure in the endosomal vesicles after polymer hydrolysis. In conclusion, our results show that both PbAEs, and especially PbAE2, open up new perspectives for the development of efficient biodegradable siRNA carriers suitable for clinical applications. Copyright © 2008 John Wiley & Sons, Ltd.     

External oligonucleotide standards enable cross laboratory comparison and exchange of real-time quantitative PCR data

The quantitative polymerase chain reaction (qPCR) is widely utilized for gene expression analysis. However, the lack of robust strategies for cross laboratory data comparison hinders the ability to collaborate or perform large multicentre studies conducted at different sites. In this study we introduced and validated a workflow that employs universally applicable, quantifiable external oligonucleotide standards to address this question. Using the proposed standards and data-analysis procedure, we obtained a perfect concordance between expression values from eight different genes in 366 patient samples measured on three different qPCR instruments and matching software, reagents, plates and seals, demonstrating the power of this strategy to detect and correct inter-run variation and to enable exchange of data between different laboratories, even when not using the same qPCR platform.     

Production of conjugated linoleic acid and conjugated linolenic acid isomers by Bifidobacterium species

Conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) isomers have attracted great interest because of their potential health benefits. Formation of CLA and CLNA takes place in the rumen during biohydrogenation. Several studies have indicated that certain types of intestinal bacteria, including bifidobacteria, are able to convert linoleic acid (LA) to CLA. The role of intestinal bacteria in the formation of CLNA isomers is largely unknown. In the present study, a screening of 36 different Bifidobacterium strains for their ability to produce CLA and CLNA from free LA and α-linolenic acid (LNA), respectively, was performed. The strains were grown in MRS broth, to which LA or LNA (0.5 mg ml⁻¹) were added after 7 h of bacterial growth. Cultures were further incubated at 37°C for 72 h. Six strains (four Bifidobacterium breve strains, a Bifidobacterium bifidum strain and a Bifidobacterium pseudolongum strain) were able to produce different CLA and CLNA isomers. Conversion percentages varied from 19.5% to 53.5% for CLA production and from 55.6% to 78.4% for CLNA production among these strains. The CLA isomers produced were further identified with Ag⁺-HPLC. LA was mainly converted to t9t11-CLA and c9t11-CLA. The main CLNA isomers were identified with GC-MS as c9t11c15-CLNA and t9t11c15-CLNA.     

Single nucleotide polymorphisms in the FADS gene cluster are associated with delta-5 and delta-6 desaturase activities estimated by serum fatty acid ratios

Genetic variability in the FADS1-FADS2 gene cluster [encoding delta-5 (D5D) and delta-6 (D6D) desaturases] has been associated with plasma long-chain PUFA (LCPUFA) and lipid levels in adults. To better understand these relationships, we further characterized the association between FADS1-FADS2 genetic variability and D5D and D6D activities in adolescents. Thirteen single nucleotide polymorphisms (SNPs) were genotyped in 1,144 European adolescents (mean ± SD age: 14.7 ± 1.4 y). Serum phospholipid fatty acid levels were analyzed using gas chromatography. D5D and D6D activities were estimated from the C20:4n-6/C20:3n-6 and C20:3n-6/C18:2n-6 ratios, respectively. Minor alleles of nine SNPs were associated with higher 18:2n-6 levels (1.9E-18 ≤ P ≤ 6.1E-5), lower C20:4n-6 levels (7.1E-69 ≤ P ≤ 1.2E-12), and lower D5D activity (7.2E-44 ≤ P ≤ 4.4E-5). All haplotypes carrying the rs174546 minor allele were associated with lower D5D activity, suggesting that this SNP is in linkage disequilibrium with a functional SNP within FADS1. In contrast, only the rs968567 minor allele was associated with higher D6D activity (P = 1.5E-6). This finding agrees with an earlier in vitro study showing that the minor allele of rs968567 is associated with a higher FADS2 promoter activity. These results suggest that rare alleles of several SNPs in the FADS gene cluster are associated with higher D6D activity and lower D5D activity in European adolescents.     

Sensitivity of detection of rhinoviruses in spiked clinical samples by nucleic acid sequence-based amplification in the presence of an internal control

The objectives of the study were to determine the sensitivity of Nucleic Acid Sequence-Based Amplification (NASBA) for the detection of rhinovirus (RV) serotype 15 in water and in spiked respiratory specimens in the presence of a newly developed internal control (IC). The sensitivity of NASBA on RNA was 10 molecules per reaction. The sensitivity of RV NASBA on RV-15 in water and in respiratory specimens was equal to that in tissue culture. Addition of 10(4) molecules of rhinovirus internal control (RV IC) did not affect the sensitivity. Viral RNA should be extracted from clinical specimens as rapidly as possible after collection, since loss of detectable RNA occurs after 2 h. NASBA allows a sensitive detection of RV RNA in spiked respiratory specimens in the presence of an internal control.