Frederick R. Haselton

MITOSIS ENHANCES TRANSGENE EXPRESSION OF PLASMID DELIVERED BY CATIONIC LIPOSOMES

A critical requirement of gene therapy is expression of the delivered transgene. Transgene expression is facilitated by access to the transcription mechanism found primarily in the nucleus. Factors modulating the interactions between intracellular plasmid and nuclear access are not well understood. In this study, the effect of mitosis on transgene expression was examined by quantitative flow cytometry. Transfection of HeLa cells synchronized at late G1 phase or G2/M phase was performed using a liposomal vector containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dioleoyl-phosphatidylethanolamine (DOPE) (1:1 mol/mol). Cell samples were transfected and subsequently maintained in G1 phase for various durations to modulate the time between plasmid entry and mitosis. The plasmid contains the sequence for a mutated green fluorescent protein (GFP(S65T)) that was used to examine transgene expression. Ethidium monoazide-labeled plasmid was employed to examine the association of plasmid with the cell membrane. The percentage of cells expressing GFP(S65T) increased sharply as the synchronized cell population passed through M phase, suggesting that an event associated with mitosis is essential for transgene expression. Expression levels of the transgene then declined 18 h after mitosis irrespective of transfection strategy. All transfection strategies resulted in the same maximum percentage of GFP(S65T) positive cells (40%) and average GFP(S65T) expression level (3.14x106 molecules per positive cell). Association of plasmid with the cell membrane at late G1 phase was 1.5-fold of that at G2/M phase. These data are evidence for control of transgene expression triggered by events associated with cell cycle.

Transfection by Cationic Liposomes Using Simultaneous Single Cell Measurements of Plasmid Delivery and Transgene Expression

Cationic liposomes are potentially important gene transfer vehicles, although their application has been limited by relatively low efficiency of transgene expression. Single cell quantitative methods, such as those used in this study, should permit a more detailed understanding of the relationships between delivered plasmid and transgene expression. Intracellular plasmid delivery and transgene expression were measured simultaneously using photoconjugated ethidium monoazide as an intracellular plasmid delivery marker and green fluorescent protein (GFP(S65T)) as a transgene expression marker. Quantitative flow cytometry was used to estimate plasmid copy number and GFP(S65T) molecules in single cells. The plasmid was delivered to HeLa cells with a cationic liposome vehicle containing 1,2-dioleoyloxy-3-trimethylammonium-propane and dioleoylphosphatidylethanolamine (1:1 mol/mol). Treatment was carried out continuously for 24 h. Flow cytometry measurements on 20,000 cells were performed during treatment and for 48 h post-treatment. On a single cell basis, transgene expression efficiency and average GFP(S65T) expression level increased with intracellular plasmid copy number. After 3-h exposure to the liposomal vector, more than 95% of the cells were positive for plasmid entry, but none had detectable transgene expression. Maximum transgene expression was achieved at 24 h and remained unchanged at the 72-h measurement. At 24 h, the average positive cell contained 1.6 × 105plasmid copies and 2.3 × 106 GFP(S65T) molecules. Importantly, the measurement strategies revealed that transgene expression varied widely within the entire cell population. Although only 30% of all cells expressed transgene, the subpopulation of cells that rapidly incorporated the vector demonstrated 100% efficiency in transgene expression. This study identifies parameters that modulate highly efficient transgene expression from plasmid delivery by cationic liposomes.

TARGETING EXPRESSION WITH LIGHT USING CAGED DNA

In this report, we describe the inactivation and site-specific light induction of plasmid expression using a photosensitive caging compound. Plasmids coding for luciferase were caged with 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) and transfected into ∼1-cm diameter sites of the skin of rats with particle bombardment. Skin sites transfected with caged plasmids did not express luciferase. However, subsequent exposure of transfected skin sites to 355-nm laser light induced luciferase expression in proportion to the amount of light. Liposome transfection of HeLa cells with DMNPE-caged green fluorescent protein (GFP) plasmids showed similar results. Caging DNA with DMNPE blocks expression at the level of transcription, since in vitro production of mRNA from linearized GFP plasmid was also blocked by caging and subsequently restored by exposure to light. Under the reaction conditions of these experiments, our absorbance data indicate that each DMNPE-caged GFP plasmid contains ∼270 caging groups. In addition to inhibition and subsequent restoration of plasmid bioactivity, the presence and photocleavage of this relatively small number of cage groups also alters electrophoretic mobility of plasmids and optical absorption characteristics. This light-induced expression strategy provides a new means to target the expression of genetic material with spatial and temporal specificity.

Designing for chaos: applications of chaotic advection at the microscale

Chaotic advection can play an important role in efficient microfluidic mixers. We discuss a design paradigm that exploits chaotic advection and illustrate by two recent examples, namely enhancing gene expression profiling and constructing an in–line microfluidic mixing channel, how application of this paradigm has led to successful micromixers. We suggest that ‘designing for chaos’, that is, basing practical mixer design on chaotic advection analysis, is a promising approach to adopt in this developing field which otherwise has little to guide it and is constrained by issues of scale and manufacturability.

The Role of Cadherin Endocytosis in Endothelial Barrier Regulation: Involvement of Protein Kinase C and Actin-Cadherin Interactions

We have previously reported that exposure of endothelial monolayers to low (0.12 mM) extracellular calcium significantly decreased the endothelial solute barrier, and that this effect was reversed by restoring ‘normal’ (1.2 mM) calcium (1). This effect was shown to be dependent on cadherins, however the molecular mechanisms through which barrier was altered by low calcium were not characterized. Here we investigated the mechanism of increased endothelial permeability produced by low calcium exposure. Endothelial permeability was significantly increased by exposure to low (0.12 mM) calcium; this effect was attenuated by pre-treatment with the protein kinase C (PKC) inhibitor, staurosporine (2 × 10−7 M) for 30 min. Cell border retraction and gap formation produced by low calcium was also prevented by staurosporine. Treatment of monolayers with 0.12 mM calcium also stimulated the endocytosis of endothelial cadherins. This low calcium mediated cadherin endocytosis was also prevented by pretreatment with staurosporine. Low calcium mediated endocytosis was also prevented by the actin filament toxin, cytochalasin D (1 ug/ml, 30 min). We conclude that the mechanism of low calcium mediated loss of endothelial barrier function is mediated in part by a PKC dependent endocytosis of endothelial cadherins, which may involve interactions with the actin cytoskeleton. Physiological regulation of the in vivo endothelial barrier may also involve PKC dependent-actin mediated endocytosis of cadherin junctional elements.

Flow visualization of steady streaming in oscillatory flow through a bifurcating tube

A steady streaming displacement of fluid elements is observed to occur during oscillatory flow through a Y-shaped tube bifurcation model at Womersley and Reynolds numbers that can exist in the human bronchial tree. The cause of the displacement is the effect of the asymmetric geometry on the oscillating velocity vector field. The steady streaming displacement is greatest for fluid elements that experience the highest velocity through the bifurcation junction. The maximum displacement observed increases with Re and a up to a Re of about 100 and a of about 5, after which a levelling off and gradual decline occur. Photographs of low-Re and low-a: cxpcrimcnts show the effects of secondary components of the steady-streaming displacement field which contribute to a complex circulation.

Development of a low-resource RNA extraction cassette based on surface tension valves.

Nucleic acid-based diagnostics are highly sensitive and specific, but are easily disrupted by the presence of interferents in biological samples. In a laboratory or hospital setting, the influence of these interferents can be minimized using an RNA or DNA extraction procedure prior to analysis. However, in low-resource settings, limited access to specialized instrumentation and trained personnel presents challenges that impede sample preparation. We have developed a self-contained nucleic acid extraction cassette suitable for operation in a low-resource setting. This simple design contains processing solutions preloaded within a continuous length of 1.6 mm inner diameter Tygon tubing. Processing solutions are separated by air gaps and held in place during processing by the surface tension forces at the liquid-air interface, viz. surface tension valves. Nucleic acids preferentially adsorbed to silica-coated magnetic particles are separated from sample interferents using an external magnet to transfer the nucleic acid biomarker through successive solutions to precipitate, wash and elute in the final cassette solution. The efficiency of the extraction cassette was evaluated using quantitative reverse transcriptase PCR (qRT-PCR) following extraction of respiratory syncytial virus (RSV) RNA. RNA was recovered from TE buffer or from lysates of RSV infected HEp-2 cells with 55 and 33% efficiency, respectively, of the Qiagen RNeasy kit. Recovery of RSV RNA from RSV infected HEp-2 cells was similar at 30% of the RNeasy kit. An overall limit of detection after extraction was determined to be nearly identical (97.5%) to a laboratory-based commercially available kit. These results indicate that this extraction cassette design has the potential to be an effective sample preparation device suitable for use in a low-resource setting.

Platelet-derived lysophosphatidic acid decreases endothelial permeability in vitro

We previously reported that platelets release a soluble factor that decreases the solute permeability of cultured bovine aortic endothelial monolayers. This factor was characterized as heat stable, tryspsin sensitive, and not serotonin, adenosine, ADP, or ATP [F. R. Haselton and J. S. Alexander. Am. J. Physiol. 263 (Lung Cell Mol. Physiol. 7): L670-L678, 1992]. We now report its identity as lysophosphatidic acid (LPA). Endothelial permeability decreases rapidly, reversibly, and repeatedly when exposed to platelet supernatants. Continuous exposure produces a sustained decrease in permeability. Methanol extracts of platelet supernatants also decrease endothelial permeability. Treatment of methanol extracts of platelet supernatants with phospholipase B or alkaline phosphatase, which modify the structure of LPA, abolishes the permeability-decreasing activity. However, activity is unaffected by treatment with phospholipase A2. This pattern of enzyme inactivation is consistent with the structure of LPA. Furthermore, synthetic 1-oleoyl-LPA rapidly and significantly decreases endothelial permeability in a concentration-dependent manner. Platelet activation does not appear to be required to produce activity in supernatants from platelet isolations, since P-selectin expression is not increased and thromboxane B2 is < 14 pg/6,000 platelets. Our data show that platelets release a methanol-extractable compound with an enzyme degradation profile consistent with LPA, which decreases the permeability of endothelial monolayers in vitro. In vivo, LPA derived from platelets may be an important mediator of the transport barrier formed by the vascular endothelium.We previously reported that platelets release a soluble factor that decreases the solute permeability of cultured bovine aortic endothelial monolayers. This factor was characterized as heat stable, trypsin sensitive, and not serotonin, adenosine, ADP, or ATP [F. R. Haselton and J. S. Alexander. Am. J. Physiol. 263 ( Lung Cell Mol. Physiol. 7): L670-L678, 1992]. We now report its identity as lysophosphatidic acid (LPA). Endothelial permeability decreases rapidly, reversibly, and repeatedly when exposed to platelet supernatants. Continuous exposure produces a sustained decrease in permeability. Methanol extracts of platelet supernatants also decrease endothelial permeability. Treatment of methanol extracts of platelet supernatants with phospholipase B or alkaline phosphatase, which modify the structure of LPA, abolishes the permeability-decreasing activity. However, activity is unaffected by treatment with phospholipase A2. This pattern of enzyme inactivation is consistent with the structure of LPA. Furthermore, synthetic 1-oleoyl-LPA rapidly and significantly decreases endothelial permeability in a concentration-dependent manner. Platelet activation does not appear to be required to produce activity in supernatants from platelet isolations, since P-selectin expression is not increased and thromboxane B2 is <14 pg/6,000 platelets. Our data show that platelets release a methanol-extractable compound with an enzyme degradation profile consistent with LPA, which decreases the permeability of endothelial monolayers in vitro. In vivo, LPA derived from platelets may be an important mediator of the transport barrier formed by the vascular endothelium.

Control of DNA Hybridization with Photocleavable Adducts

Previous reports have shown that 1‐(4,5‐dimethoxy‐2‐nitro‐phenyl)ethyl ester (DMNPE) adducts coupled to DNA plasmids block transcription in vitro and in vivo until removed with light. In this report, we explore the use of DMNPE to control DNA hybridization. We found that DMNPE‐caged oligonucleotides have changed spectrophotometric and elec‐trophoretic properties that can be restored with light exposure. Caged oligonucleotides have slower electrophoretic mobility than noncaged oligonucleotides and caged oligonucleotides exposed to light. Effects of caging on hybridization were assessed in a fluorescence‐based assay using a 20mer caged DNA oligonucleotide complementary to a 30mer molecular beacon. Fluorescence results indicate that hybridization is reduced and subsequently restored by light. Subsequent gel shift assays confirmed these results. Hybridization activity of caged oligonucleotides with an average of 14–16 DMNPE adducts per oligonucleotide was 14% of noncaged control oligonucleotides and after 365 nm photolysis, increased to nearly 80% of controls. Spectrophotometric characterization of caged oligonucleotides exposed to light and then filtered to remove the released DMNPE adducts indicates two to four attached cage groups remaining following photoactivation. These results suggest that this light‐based technology can be used as a tool for the spatial and temporal regulation of hybridization‐based DNA bioactivity.

Effects of relative humidity and buffer additives on the contact printing of microarrays by quill pins.

DNA microarrays printed with quill pins exhibit significant variation in probe DNA spots. Interspot variations and nonuniform distribution of probe within spots are major sources of experimental uncertainty in microarray analysis. To gain better insight into the sources of variation, we analyzed 450 consecutive depositions printed at relative humidities between 40 and 80% using three print buffers. Increasing relative humidity improved printing performance by delaying pin failure but did not reduce the variability in spot characteristics. Adding either betaine or dimethyl sulfoxide (DMSO) to the print buffer also improved quill pin performance. Least interspot variation was observed with the DMSO additive printed at 80% relative humidity, but this additive also resulted in the greatest intraspot variation. Least intraspot variation was observed with 1.5M betaine printed at 60% relative humidity, but these conditions produced microarrays with high interspot variability. Evaporation of printing solution from the quill reservoir appeared to be the primary cause of interspot and intraspot variations. Our studies indicate that relative humidity and printing solution additives reduce evaporation. Based on the spot variability requirements for a particular application, humidity and additives may be chosen to optimize either inter- or intraspot variability.