Basic Features of a Cell Electroporation Model: Illustrative Behavior for Two Very Different Pulses

Science increasingly involves complex modeling. Here we describe a model for cell electroporation in which membrane properties are dynamically modified by poration. Spatial scales range from cell membrane thickness (5 nm) to a typical mammalian cell radius (10  \(\upmu\) m), and can be used with idealized and experimental pulse waveforms. The model consists of traditional passive components and additional active components representing nonequilibrium processes. Model responses include measurable quantities: transmembrane voltage, membrane electrical conductance, and solute transport rates and amounts for the representative “long” and “short” pulses. The long pulse—1.5 kV/cm, 100  \(\upmu\) s—evolves two pore subpopulations with a valley at \({\sim}\) 5 nm, which separates the subpopulations that have peaks at \({\sim}\) 1.5 and \({\sim}\) 12 nm radius. Such pulses are widely used in biological research, biotechnology, and medicine, including cancer therapy by drug delivery and nonthermal physical tumor ablation by causing necrosis. The short pulse—40 kV/cm, 10 ns—creates 80-fold more pores, all small ( \(<\) 3 nm; \(\sim\) 1 nm peak). These nanosecond pulses ablate tumors by apoptosis. We demonstrate the model’s responses by illustrative electrical and poration behavior, and transport of calcein and propidium. We then identify extensions for expanding modeling capability. Structure-function results from MD can allow extrapolations that bring response specificity to cell membranes based on their lipid composition. After a pulse, changes in pore energy landscape can be included over seconds to minutes, by mechanisms such as cell swelling and pulse-induced chemical reactions that slowly alter pore behavior.     

The challenge for genetic epidemiologists: how to analyze large numbers of SNPs in relation to complex diseases

Background

Molecular genetic approaches have much to offer population biology. Despite recent advances, convenient techniques to develop and screen highly-resolving markers can be limiting for some applications and taxa. We describe an improved PCR-based, cloning-free, nuclear marker development procedure, in which single-stranded conformation polymorphism (SSCP) plays a central role. Sequence-variable alleles at putative nuclear loci are simultaneously identified and isolated from diploid tissues. Based on a multiple allele alignment, locus-specific primers are designed in conserved regions, minimizing 'null' alleles. Using two undescribed endemic Australian Collembola as exemplars, we outline a comprehensive approach to generating and validating suites of codominant, sequence-yielding nuclear loci for previously unstudied invertebrates.

Results

Six markers per species were developed without any baseline genetic information. After evaluating the characteristics of each new locus via SSCP pre-screening, population samples were genotyped on the basis of either DNA sequence, restriction site, or insertion/deletion variation, depending on which assay was deemed most appropriate. Polymorphism was generally high (mean of nine alleles per locus), and the markers were capable of resolving population structuring over very fine spatial scales (<100 km). SSCP coupled with targeted DNA sequencing was used to obtain genotypic, genic and genealogical information from six loci (three per species). Phylogeographic analysis identified introns as being most informative.

Conclusion

The comprehensive approach presented here feasibly overcomes technical hurdles of (i) developing suitably polymorphic nuclear loci for non-model organisms, (ii) physically isolating nuclear allele haplotypes from diploid tissues without cloning, and (iii) genotyping population samples on the basis of nuclear DNA sequence variation.     

Biologically relevant effects of mRNA amplification on gene expression profiles

Background  

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results.     

HeatMapper: powerful combined visualization of gene expression profile correlations, genotypes, phenotypes and sample characteristics

Background  

Accurate interpretation of data obtained by unsupervised analysis of large scale expression profiling studies is currently frequently performed by visually combining sample-gene heatmaps and sample characteristics. This method is not optimal for comparing individual samples or groups of samples. Here, we describe an approach to visually integrate the results of unsupervised and supervised cluster analysis using a correlation plot and additional sample metadata.     

Do distribution volumes and clearances relate to tissue volumes and blood flows? A computer simulation

Background

Thoracotomy is associated with severe pain that may persist for years. Acupuncture is a complementary therapy with a proven role in pain control. A randomized trial showed that acupuncture was effective in controlling pain after abdominal surgery, but the efficacy of this technique for the treatment of thoracotomy pain has not been established. We developed a novel technique for convenient application of acupuncture to patients undergoing thoracotomy, and in a Phase II trial evaluated the safety of this intervention and the feasibility of doing a randomized trial.

Methods

Adult patients scheduled for unilateral thoracotomy with preoperative epidural catheter placement received acupuncture immediately prior to surgery. Eighteen semi-permanent intradermal needles were inserted on either side of the spine, and four were inserted in the legs and auricles. Needles were removed after four weeks. Using a numerical rating scale, pain was measured on the first five postoperative days. After discharge, pain was assessed using the Brief Pain Inventory at 7, 30, 60 and 90 days.

Results

Thirty-six patients were treated with acupuncture. Of these, 25, 23, and 22 patients provided data at 30, 60, and 90 days, respectively. The intervention was well tolerated by patients with only one minor and transient adverse event of skin ulceration.

Conclusion

The rate of data completion met our predefined criterion for determining a randomized trial to be feasible (at least 75% of patients tolerated the intervention and provided evaluable data). This novel intervention is acceptable to patients undergoing thoracotomy and does not interfere with standard preoperative care. There was no evidence of important adverse events. We are now testing the hypothesis that acupuncture significantly adds to standard perioperative pain management in a randomized trial.     

Reversal of profound vecuronium-induced neuromuscular block under sevoflurane anesthesia: sugammadex versus neostigmine

Background

The perioperative period is characterized by an intense inflammatory response. Perioperative inflammation promotes postoperative morbidity and increases mortality. Blunting the inflammatory response to surgical trauma might thus improve perioperative outcomes. We are studying three interventions that potentially modulate perioperative inflammation: corticosteroids, tight glucose control, and light anesthesia.

Methods/Design

The DeLiT Trial is a factorial randomized single-center trial of dexamethasone vs placebo, intraoperative tight vs. conventional glucose control, and light vs deep anesthesia in patients undergoing major non-cardiac surgery. Anesthetic depth will be estimated with Bispectral Index (BIS) monitoring (Aspect medical, Newton, MA). The primary outcome is a composite of major postoperative morbidity including myocardial infarction, stroke, sepsis, and 30-day mortality. C-reactive protein, a measure of the inflammatory response, will be evaluated as a secondary outcome. One-year all-cause mortality as well as post-operative delirium will be additional secondary outcomes. We will enroll up to 970 patients which will provide 90% power to detect a 40% reduction in the primary outcome, including interim analyses for efficacy and futility at 25%, 50% and 75% enrollment.

Discussion

The DeLiT trial started in February 2007. We expect to reach our second interim analysis point in 2010. This large randomized controlled trial will provide a reliable assessment of the effects of corticosteroids, glucose control, and depth-of-anesthesia on perioperative inflammation and morbidity from major non-cardiac surgery. The factorial design will enable us to simultaneously study the effects of the three interventions in the same population, both individually and in different combinations. Such a design is an economically efficient way to study the three interventions in one clinical trial vs three.

Trial registration

This trial is registered at Clinicaltrials.gov #: NTC00433251     

Solubilization and characterization of the anthrax toxin pore in detergent micelles

Proteolytically activated Protective Antigen (PA) moiety of anthrax toxin self‐associates to form a heptameric ring‐shaped oligomer (the prepore). Acidic pH within the endosome converts the prepore to a pore that serves as a passageway for the toxin's enzymatic moieties to cross the endosomal membrane. Prepore is stable in solution under mildly basic conditions, and lowering the pH promotes a conformational transition to an insoluble pore‐like state. N‐tetradecylphosphocholine (FOS14) was the only detergent among 110 tested that prevented aggregation without dissociating the multimer into its constituent subunits. FOS14 maintained the heptamers as monodisperse, insertion‐competent 440‐kDa particles, which formed channels in planar phospholipid bilayers with the same unitary conductance and ability to translocate a model substrate protein as channels formed in the absence of detergent. Electron paramagnetic resonance analysis detected pore‐like conformational changes within PA on solubilization with FOS14, and electron micrograph images of FOS14‐solubilized pore showed an extended, mushroom‐shaped structure. Circular dichroïsm measurements revealed an increase in α helix and a decrease in β structure in pore formation. Spectral changes caused by a deletion mutation support the hypothesis that the 2β2‐2β3 loop transforms into the transmembrane segment of the β‐barrel stem of the pore. Changes caused by selected point mutations indicate that the transition to α structure is dependent on residues of the luminal 2β11‐2β12 loop that are known to affect pore formation. Stabilizing the PA pore in solution with FOS14 may facilitate further structural analysis and a more detailed understanding of the folding pathway by which the pore is formed.     

Entomotoxic action of Sambucus nigra agglutinin i in Acyrthosiphon pisum aphids and Spodoptera exigua caterpillars through caspase‐3‐like‐dependent apoptosis

In this project, the toxicity and mechanism of action of the ricin‐B‐related lectin SNA‐I from elderberry (Sambucus nigra) in the pea aphid (Acyrthosiphon pisum) and the beet armyworm (Spodoptera exigua), two important pest insects in agriculture, were studied. SNA‐I is a chimeric lectin belonging to the class of ribosome‐inactivating proteins and consists of an A‐chain with N‐glycosidase activity and a carbohydrate‐binding B‐chain. Incorporation of 2 mg/ml of SNA‐I in the diet of neonates and adults of A. pisum caused 40–46% mortality within 2 days, while in third instars of S. exigua, the larval biomass was significantly reduced by 12% after feeding for 3 days on a diet containing 5 mg/g of SNA‐I. Interestingly, extracts of the (mid)gut of treated A. pisum and S. exigua demonstrated DNA fragmentation and this was accompanied with an increase in caspase‐3‐like activity. The involvement of cell death or apoptosis in the entomotoxicity of SNA‐I through induction of caspase‐3‐like activity was also confirmed by addition of the permeable caspase‐3 inhibitor III in the diet, leading to a rescue of the treated aphid neonates. Finally, similar to the chimeric lectin SNA‐I, the hololectin SNA‐II, consisting of two carbohydrate‐binding B‐chains caused high mortality to neonate A. pisum aphids with an LC₅₀ of 1.59 mg/ml, suggesting that the entomotoxic action of the lectins under study mainly relies on their carbohydrate‐binding activity. © 2010 Wiley Periodicals, Inc.