Mono-specific algal diets shape microbial networking in the gut of the sea urchin Tripneustes gratilla elatensis.

BACKGROUND: Algivorous sea urchins can obtain energy from a diet of a single algal species, which may result in consequent changes in their gut microbe assemblies and association networks. METHODS: To ascertain whether such changes are led by specific microbes or limited to a specific region in the gut, we compared the microbial assembly in the three major gut regions of the sea urchin Tripneustes gratilla elatensis when fed a mono-specific algal diet of either Ulva fasciata or Gracilaria conferta, or an algal-free diet. DNA extracts from 5 to 7 individuals from each diet treatment were used for Illumina MiSeq based 16S rRNA gene sequencing (V3-V4 region). Niche breadth of each microbe in the assembly was calculated for identification of core, generalist, specialist, or unique microbes. Network analyzers were used to measure the connectivity of the entire assembly and of each of the microbes within it and whether it altered with a given diet or gut region. Lastly, the predicted metabolic functions of key microbes in the gut were analyzed to evaluate their potential contribution to decomposition of dietary algal polysaccharides. RESULTS: Sea urchins fed with U. fasciata grew faster and their gut microbiome network was rich in bacterial associations (edges) and networking clusters. Bacteroidetes was the keystone microbe phylum in the gut, with core, generalist, and specialist representatives. A few microbes of this phylum were central hub nodes that maintained community connectivity, while others were driver microbes that led the rewiring of the assembly network based on diet type through changes in their associations and centrality. Niche breadth agreed with microbes' richness in genes for carbohydrate active enzymes and correlated Bacteroidetes specialists to decomposition of specific polysaccharides in the algal diets. CONCLUSIONS: The dense and well-connected microbial network in the gut of Ulva-fed sea urchins, together with animal's rapid growth, may suggest that this alga was most nutritious among the experimental diets. Our findings expand the knowledge on the gut microbial assembly in T. gratilla elatensis and strengthen the correlation between microbes' generalism or specialism in terms of occurrence in different niches and their metabolic arsenal which may aid host nutrition.

Variable electric fields for high throughput electroporation protocol design in curvilinear coordinates.

The mathematical solution to the electric field equation in cylindrical coordinates, has suggested to us a new experimental methodology and device for reducing experimental effort in designing electroporation protocols. Using a new cylindrical electroporation system, we show, with an Escherichia coli cell model, how key electroporation parameters emerge precisely from single experiments rather than through interpolation from numerous experiments in the conventional Cartesian electroporation system.

Towards electroporation based treatment planning considering electric field induced muscle contractions.

The electric field threshold for muscle contraction is two orders of magnitudes lower than that for electroporation. Current electroporation treatment planning and electrode design studies focus on optimizing the delivery of electroporation electric fields to the targeted tissue. The goal of one part of this study was to investigate the relation between the volumes of tissue that experience electroporation electric fields in a targeted tissue volume and the volumes of tissue that experience muscle contraction inducing electric fields around the electroporated tissue volume, (V(MC)), during standard electroporation procedures and for various electroporation electrodes designs. The numerical analysis shows that conventional electroporation protocols and electrode design can generate muscle contraction inducing electric fields in surprisingly large volumes of non-target tissue, around the electroporation treated tissue. In studying various electrode configurations, we found that electrode placement in a structure we refer to as a "Current Cage" can substantially reduce the volume of non-target tissue exposed to electric fields above the muscle contraction threshold. In an experimental study on a tissue phantom we compare a commercial two parallel needle electroporation system with the Current Cage design. While tissue electroporated volumes were similar, V(MC) of tissue treated using the Current Cage design electrodes was an order of magnitude smaller than that using a commercially available system. An important aspect of the entire study is that it suggests the benefit of including the calculations of V(MC) for planning of electroporation based treatments such as DNA vaccination, electrochemotherapy and irreversible electroporation.

Listeria monocytogenes cell wall constituents exert a charge effect on electroporation threshold.

Genetically engineered cells with mutations of relevance to electroporation, cell membrane permeabilization by electric pulses, can become a promising new tool for fundamental research on this important biotechnology. Listeria monocytogenes mutants lacking DltA or MprF and assayed for sensitivity to the cathelicidin like anti-microbial cationic peptide (mCRAMP), were developed to study the effect of cell wall charge on electroporation. Working in the irreversible electroporation regime (IRE), we found that application of a sequence of 50 pulses, each 50µs duration, 12.5kV/cm field, delivered at 2Hz led to 2.67±0.29 log reduction in wild-type L. monocytogenes, log 2.60±0.19 in the MprF-minus mutant, and log 1.33±0.13 in the DltA-minus mutant. The experimental observation that the DltA-minus mutant was highly susceptible to cationic mCRAMP and resistant to IRE suggests that the charge on the bacterial cell wall affects electroporation and shows that this approach may be promising for fundamental studies on electroporation.

A statistical model for multidimensional irreversible electroporation cell death in tissue.

BACKGROUND: Irreversible electroporation (IRE) is a minimally invasive tissue ablation technique which utilizes electric pulses delivered by electrodes to a targeted area of tissue to produce high amplitude electric fields, thus inducing irreversible damage to the cell membrane lipid bilayer. An important application of this technique is for cancer tissue ablation. Mathematical modelling is considered important in IRE treatment planning. In the past, IRE mathematical modelling used a deterministic single value for the amplitude of the electric field required for causing cell death. However, tissue, particularly cancerous tissue, is comprised of a population of different cells of different sizes and orientations, which in conventional IRE are exposed to complex electric fields; therefore, using a deterministic single value is overly simplistic. METHODS: We introduce and describe a new methodology for evaluating IRE induced cell death in tissue. Our approach employs a statistical Peleg-Fermi model to correlate probability of cell death in heterogeneous tissue to the parameters of electroporation pulses such as the number of pulses, electric field amplitude and pulse length. For treatment planning, the Peleg-Fermi model is combined with a numerical solution of the multidimensional electric field equation cast in a dimensionless form. This is the first time in which this concept is used for evaluating IRE cell death in multidimensional situations. RESULTS: We illustrate the methodology using data reported in literature for prostate cancer cell death by IRE. We show how to fit this data to a Fermi function in order to calculate the critical statistic parameters. To illustrate the use of the methodology, we simulated 2-D irreversible electroporation protocols and produced 2-D maps of the statistical distribution of cell death in the treated region. These plots were compared to plots produced using a deterministic model of cell death by IRE and the differences were noted. CONCLUSIONS: In this work we introduce a new methodology for evaluation of tissue ablation by IRE using statistical models of cell death. We believe that the use of a statistical model rather than a deterministic model for IRE cell death will improve the accuracy of treatment planning for cancer treatment with IRE.

Galvanic apparent internal impedance: an intrinsic tissue property.

Using basic galvanic cell principles, the ability of tissues to generate electrical current through electrolysis was characterized. Studying Zn/Cu electrolysis in animal organs revealed a fundamental and measurable tissue-specific property - the galvanic apparent internal impedance (GAII), that is most likely related to the salt bridge function of tissues delineated by electrodes. Further to the fundamental knowledge acquired, GAII enables a new diagnostic method to distinguish between tissue types and to determine their health status without a need for expensive calibration, as often required when external power source is used. We demonstrated the GAII sensitivity in detecting tissue ablation with microwave heating or irreversible electroporation. The results open the way for a novel, inexpensive self-powered tissue diagnostic system for a wide range of applications such as minimally invasive tissue health status, ischemia, hydration, real time intra-operative control of minimally invasive surgery, medical imaging, virtual biopsy and many others.

Irreversible electroporation for microbial control of drugs in solution.

The purpose of this study was to examine the feasibility of using irreversible electroporation (IRE) as a non-chemical method for eliminating microorganisms of liquid drugs. The studied drug was a topical ophthalmic medication, a pharmaceutical field in which the problem of microbial contamination has not yet been adequately solved, especially in the case of eye drops prescribed for chronic use. Commercially available Hylo-Comod preservative-free eye drop solution was subjected to contamination with Escherichia coli bacteria (10(6) colony forming units/mL). Electroporation parameters for bacterial control were investigated by comparing the effects of electrical fields of 5.4, 7.2, and 10 kV/cm, delivered as 100-micros square pulses at 1 Hz in sequences of 10 pulses, 20 pulses, or 20 pulses delivered as four sets of five pulses with 1-min intervals between each set. Microorganism survival after treatment was determined by pour plate counting. Effects of the treatment parameters on temperature and pH were recorded. Bacterial survival was lowest (0.14% +/- 0.03%) after application of 20 pulses delivered as four separate sets. With that application mode, the solution remained at pH 7.5 and the temperature rose to 35.6 degrees +/- 0.2 degrees C. Because IRE can be efficiently delivered under conditions that avoid the potentially deleterious effects of electrical pulses on temperature and pH, it appears to be a feasible method for bacterial control of drugs in solution. The principles established in this study can be applied to any drug in solution and optimized individually according to the solution's composition.