The role of plasmid constructs containing the SV40 DNA nuclear-targeting sequence in cationic lipid-mediated DNA delivery

One of the steps that limit transfection efficiency in non-viral gene delivery is inefficient nuclear import of plasmid DNA, once it has been delivered into the cytoplasm. Recently, via microinjection into the cytoplasm and in situ hybridizations into a few cell types, it was shown that a region of Simian virus 40(SV40), specifically a c. 372-bp fragment of SV40 genomic DNA encompassing the SV40 promoter-enhancer-origin of replication (SV40 DTS), could enable the nuclear import of a plasmid carrying these sequences (Dean D.A. Exp. Cell Res. 230 (1997) 293). In this report, we address the issue of the suitability of the SV40 DTS for cationic lipid-mediated gene delivery, and its capacity to improve the efficiency of the transfection process. For this study, we used transient reporter gene expression assays on various cell types. The gene expression from the plasmid constructs carrying the SV40 DTS varied with cell type and plasmid construct used. Such cell-type and plasmid-construct dependency on gene expression from plasmids containing the SV40 DTS suggests that the gene expression from plasmids is not entirely dependent on its ability to enhance the nuclear import of said plasmids.     

Anchor-dependent lipofection with non-glycerol based cytofectins containing single 2-hydroxyethyl head groups

Detailed structure-activity investigations aimed at probing the anchor chain length dependency for glycerol-based lipofectins have been reported previously. Herein, we report on the first detailed investigation on the anchor-dependent transfection biology of non-glycerol based simple monocationic cytofectins containing single 2-hydroxyethyl head group functionality using 11 new structural analogs of our previously published first generation of non-glycerol based transfection lipids (lipids 1-11). The C-14 and C-16 analogs of DOMHAC (lipids 4 and 5, respectively) were found to be remarkably efficient in transfecting COS-1 cells. In addition, the present anchor-dependency investigation also revealed that the C-14 analog of DOHEMAB (lipid 10) is significantly efficient in transfecting both COS-1 and NIH3T3 cells. Our results also indicate that too strong lipid-DNA interactions might result in weaker transfection for non-glycerol based cationic lipids. In summary, the anchor-dependence investigations presented here convincingly demonstrate that non-glycerol based cationic lipids containing a single hydroxyethyl head group and hydrophobic C-14 or C-16 anchors are promising non-toxic cationic transfection lipids for future use in liposomal gene delivery.     

Quantitative aspects of endocytic activity in lipid-mediated transfections.

Variation in transfection efficiency observed in different cell-types is poorly understood. To investigate the influence of endocytic activity on lipid-mediated transfections, we have monitored both the processes in 12 different cell-types. The endocytic activity shows a strong positive correlation (P < 0.01), with transfection efficiency. Treatment with wortmannin resulted in cell-type-dependent inhibition of transfection. Studies on M-phase cells by confocal microscopy show that compared to interphase cells, uptake of cationic liposomes was substantially reduced. In addition, transfection efficiency of cells in mitotic phase was inhibited by >70% compared to controls. Our study based on several cell-types demonstrates for the first time that quantitative aspects of endocytosis have decisive influence on the overall process of lipid-mediated transgene expression.     

Identification of quantitative trait loci for resistance to shoot fly in sorghum [<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench]

The shoot fly is one of the most destructive insect pests of sorghum at the seedling stage. Deployment of cultivars with improved shoot fly resistance would be facilitated by the use of molecular markers linked to QTL. The objective of this study was to dissect the genetic basis of resistance into QTL, using replicated phenotypic data sets obtained from four test environments, and a 162 microsatellite marker-based linkage map constructed using 168 RILs of the cross 296B (susceptible) × IS18551 (resistant). Considering five component traits and four environments, a total of 29 QTL were detected by multiple QTL mapping (MQM) viz., four each for leaf glossiness and seedling vigor, seven for oviposition, six for deadhearts, two for adaxial trichome density and six for abaxial trichome density. The LOD and R ² (%) values of QTL ranged from 2.6 to 15.0 and 5.0 to 33%, respectively. For most of the QTL, IS18551 contributed resistance alleles; however, at six QTL, alleles from 296B also contributed to resistance. QTL of the related component traits were co-localized, suggesting pleiotropy or tight linkage of genes. The new morphological marker Trit for trichome type was associated with the major QTL for component traits of resistance. Interestingly, QTL identified in this study correspond to QTL/genes for insect resistance at the syntenic maize genomic regions, suggesting the conservation of insect resistance loci between these crops. For majority of the QTL, possible candidate genes lie within or very near the ascribed confidence intervals in sorghum. Finally, the QTL identified in the study should provide a foundation for marker-assisted selection (MAS) programs for improving shoot fly resistance in sorghum.     

Quantitative proteomics for identifying biomarkers for Rabies

Introduction

Rabies is a fatal acute viral disease of the central nervous system, which is a serious public health problem in Asian and African countries. Based on the clinical presentation, rabies can be classified into encephalitic (furious) or paralytic (numb) rabies. Early diagnosis of this disease is particularly important as rabies is invariably fatal if adequate post exposure prophylaxis is not administered immediately following the bite.

Methods

In this study, we carried out a quantitative proteomic analysis of the human brain tissue from cases of encephalitic and paralytic rabies along with normal human brain tissues using an 8-plex isobaric tags for relative and absolute quantification (iTRAQ) strategy.

Results and conclusion

We identified 402 proteins, of which a number of proteins were differentially expressed between encephalitic and paralytic rabies, including several novel proteins. The differentially expressed molecules included karyopherin alpha 4 (KPNA4), which was overexpressed only in paralytic rabies, calcium calmodulin dependent kinase 2 alpha (CAMK2A), which was upregulated in paralytic rabies group and glutamate ammonia ligase (GLUL), which was overexpressed in paralytic as well as encephalitic rabies. We validated two of the upregulated molecules, GLUL and CAMK2A, by dot blot assays and further validated CAMK2A by immunohistochemistry. These molecules need to be further investigated in body fluids such as cerebrospinal fluid in a larger cohort of rabies cases to determine their potential use as antemortem diagnostic biomarkers in rabies. This is the first study to systematically profile clinical subtypes of human rabies using an iTRAQ quantitative proteomics approach.     

Haloperidol-associated stealth liposomes: a potent carrier for delivering genes to human breast cancer cells

Sigma receptors are membrane-bound proteins that are overexpressed in certain human malignancies including breast cancer. These receptors show very high affinity for various sigma ligands including neuroleptics like haloperidol. We hypothesized that in associating haloperidol-linked lipid into the cationic lipid-DNA complex, we can specifically target and deliver genes to breast cancer cells that overexpress sigma receptors. In the present study, haloperidol was chemically modified to conjugate at the distal end of the polyethylene glycollinked phospholipid, which was then incorporated into the cationic liposome known to condense and deliver genes inside cells. The resulting haloperidol-conjugated targeted lipoplex showed at least 10-fold higher (p < 0.001) reporter gene expression in MCF-7 cells than control lipoplex. The reporter gene expression of the targeted lipoplex was significantly blocked by haloperidol (p < 0.001) and by another sigma ligand, 1,3-ditolylguanidine (p < 0.001) in the majority of cationic lipid to DNA charge ratios (+/-). Spironolactone-mediated sigma receptor down-regulation enabled MCF-7 to show 10-fold lower transgene expression with targeted lipoplex compared with that obtained in spironolactone-untreated cells. The targeted lipoplex generated nonspecific gene expression in sigma receptor-nonexpressing human cancer cells such as Hela, KB, HepG2, and Chinese hamster ovary cells. Moreover, the transgene expression remained unabated in physiologically relevant serum concentrations. This is the first study to demonstrate that haloperidol-targeted gene delivery systems can mediate efficient targeting of genes to sigma receptor-overexpressing breast cancer cells, thereby becoming a novel class of therapeutics for the treatment of human cancers.     

Lipase in aqueous‐polar organic solvents: Activity,structure, and stability

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ～20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]‐HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active‐site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased K_m); however significantly enhanced the k_cat. Melting temperature (T_m) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.