Numerical Modeling of Bi-polar (AC) Pulse Electroporation of Single Cell in Microchannel to Create Nanopores on its Membrane

AC electroporation of a single cell in a microchannel was numerically studied. A \(15\,\upmu\) m diameter cell was considered in a microchannel \(25\,\upmu\) m in height and the influences of AC electric pulse on its membrane were numerically investigated. The cell was assumed to be suspended between two electroporative electrodes embedded on the walls of a microchannel. An amplitude and a time span of applied electric pulse were chosen to be 80 kV/m and \(10\,\upmu\) s, respectively. For different frequency values (50, 100, 200, and 500 kHz), simulations were performed to show how the cell membrane was electroporated and the creation of nanopores. Obtained numerical results show that the most and the largest nanopores are created around poles of cell (nearest points of cell membrane to the electrodes). The numerical simulations also demonstrate that increased frequency will slightly decrease electroporated area of the cell membrane; additionally, growth of the created nanopores will be stabilized. It has also been proven that size and number of the created nanopores will be decreased by moving from the poles to the equator of the cell. There is almost no nanopore created in the vicinity of the equator. Frequency affects the rate of generation of nanopores. In case of AC electroporation, creation of nanopores has two phases that periodically repeat over time. In each period, the pore density sharply increases and then becomes constant. Enhancement of the frequency will result in decrease in time span of the periods. In each period, size of the created nanopores sharply increases and then slightly decreases. However, until the AC electric pulse is present, overall trends of creation and development of nanopores will be ascending. Variation of the size and number of created nanopores can be explained by considering time variation of transmembrane potential (difference of electric potential on two sides of cell membrane) which is clear in the results presented in this study.     

A Numerical Study on Pressure Drop in Microchannel Flow with Different Bionic Micro-Grooved Surfaces

The studies of bionics reveal that some aquatic animals and winged insects have developed an unsmoothed surface possessing good characteristics of drag reduction.In this paper,four types of bionic surfaces,placoid-shaped,V-shaped,riblet-shaped,and ridge-shaped grooved surfaces,are employed as the microchannel surfaces for the purpose of reducing pressure loss.Lattice Boltzmann Method (LBM),a new numerical approach on mescoscopic level,is used to conduct the numerical investigations.The results show that the micro-grooved surfaces possess the drag reduction performance.The existence of the vortices formed within the grooves not only decrease the shear force between fluid and wall but also minimize the contact area between fluid and walls,which can lead to a reduction of pressure loss.The drag reduction coefficient (η) for these four types of　micro-structures could be generalized as follows:ηridge-shaped ＞ ηV-shaped ＞ ηplacoid-shaped ＞ ηriblet-shaped.Besides,the geometrical optimizations for the ridge-shaped grooves,which have the highest drag reduction performance,are performed as well.The results suggest that,for the purpose of drag reduction,the ridge-shaped grooves with smaller width to height ratio are recommended for the lower Reynolds number flow,while the ridge-shaped grooves with larger width to height ratio are be more suitable for the larger Reynolds number flow.     

A Parametric Study Delineating Irreversible Electroporation from Thermal Damage Based on a Minimally Invasive Intracranial Procedure

Background  

Irreversible electroporation (IRE) is a new minimally invasive technique to kill undesirable tissue in a non-thermal manner. In order to maximize the benefits from an IRE procedure, the pulse parameters and electrode configuration must be optimized to achieve complete coverage of the targeted tissue while preventing thermal damage due to excessive Joule heating.     

铜绿假单胞菌最佳电转化条件的研究

以临床分离的一株铜绿假单胞菌 (Pseudomonasaeruginosa)PA68作受体菌 ,将具有卡那霉素抗性标记的质粒pSMC2 8通过电转化导入到受体菌中 ,研究细胞生长状态、电击电压、细胞浓度、感受态细胞的贮备方式对转化效率的影响。结果表明 ,在细胞生长至OD5 40 =0 7～ 0 8时收集菌体 ,在低温 (2℃ )条件下 ,制备浓度为 10 11个细胞 mL的感受态细胞 ,在较高的电压 (2 6kV)电击下 ,能获得较高的转化效率。最高可达 1 68× 10 8个转化子 μgDNA(CFU μgDNA)。用此优化的转化条件 ,在国际上首次成功地将Mu转座复合物导入到P .aeruginosa中 ,并获得 2 4× 10 4 CFU μgDNA的高转化效率。由于Mu转座重组技术具有随机单点插入的优点 ,克服了传统转座子能在染色体上迁移的缺点 ,保证了表型的改变与转座子插入位点所在的基因突变的一一对应关系 ,为进一步研究P .aerugi nosa的基因组功能奠定基础     

A Single-Cell Transcriptomic Atlas of Human Skin Aging

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Expression of Oat-phyA-cDNA in a Suspension Cell Culture of Transgenic Tobacco: A Single-Cell System for the Study of Phytochrome Function

A culture of callus cells has been developed from a transgenicline of tobacco which contains an introduced phyA-cDNA encodingphytochrome A. Suspension cultures of the cells were shown toaccumulate a significant immunodetectable level of the heterologousphytochrome, but not of the native phyA-gene product. The red-irradiatedform (P_fr) of the heterologous phytochrome was specificallydegraded in vivo, and the red-irradiated (P_fr) and far-red-irradiated(P_r) forms demonstrated different patterns of in vitro proteolyticcleavage. These results strongly suggested that the phytochromeapoprotein was associated with a chromophore moiety which mediatedred/far-red sensitive conformational changes of the molecule.Exogenous application of 4-amino-5-hexynoic acid (AHA) to thetransgenic suspension cultures resulted in the accumulationof a population of phytochrome which was stable under red lightand gave identical patterns of in vitro digestion in the redand far-red irradiated forms, i.e. the spectral activity ofphytochrome was inhibited. Application of exogenous 5-aminolevulinicacid (ALA) or biliverdin overcame the inhibitory effects ofAHA to restore spectral sensitivity of the phytochrome pool.These results are consistent with the proposed pathway of phytochromechromophore biosynthesis in intact plant systems. Thus, thetransgenic suspension cultures provided a single-cell systemin which spectrally-active phytochrome, apparently indistinguishablefrom the native phytochrome synthesized in etiolated seedlings,was accumulated. Photoregulation of expression of the genesencoding the small subunit of ribulose-1,5-bisphosphate carboxylaseand chlorophyll a/b binding proteins demonstrated that the heterologousphytochrome population mediated rapid changes in gene expressionin the de-differentiated cells. It is therefore proposed thatsuch a suspension culture of transgenic cells offers a modelsystem for the study of phytochrome function. Key words: Cell cultures, transgenic tobacco, phytochrome, oat-phy A-cDNA, gene expression     

A Single-Cell Genome for Thiovulum sp.

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 μm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.     

Electroporation of cells

By measuring uptake of the membrane impermeable dye. phenosafranine, it can be shown that the plasma membrane of intact cells within cell aggregates can be reversibly permeabilized by electroporation. However, the plant cell wall is a barrier to DNA uptake by intact cells, although under certain circumstances expression of DNA, electroporated into intact cells, can be demonstrated. The level of expression is about 20–50 times lower than that obtained by electroporation of protoplasts, and depends on cell wall properties and pretreatments of cell aggregates. In contrast, efficient transformation of whole cells of bacteria and yeasts can be achieved by electroporation. Factors which influence DNA transfer into whole plant cells and the possibility of stable transformation are discussed.     

Electroporation in dense cell suspension—Theoretical and experimental analysis of ion diffusion and cell permeabilization

Electroporation is a process where increased permeability of cells exposed to an electric field is observed. It is used in many biomedical applications including electrogene transfection and electrochemotherapy. Although the increased permeability of the membrane is believed to be the result of pores due to an induced transmembrane voltage U_m, the exact molecular mechanisms are not fully explained.     

A Quantitative Comparison of Single-Cell Whole Genome Amplification Methods

Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.     

Oligotrophic Bacterioplankton with a Novel Single-Cell Life Strategy

A large fraction of the marine bacterioplankton community is unable to form colonies on agar surfaces, which so far no experimental evidence can explain. Here we describe a previously undescribed growth behavior of three non-colony-forming oligotrophic bacterioplankton, including a SAR11 cluster representative, the world's most abundant organism. We found that these bacteria exhibit a behavior that promotes growth and dispersal instead of colony formation. Although these bacteria do not form colonies on agar, it was possible to monitor growth on the surface of seawater agar slides containing a fluorescent stain, 4′,6′-diamidino-2-phenylindole (DAPI). Agar slides were prepared by pouring a solution containing 0.7% agar and 0.5 μg of DAPI per ml in seawater onto glass slides. Prompt dispersal of newly divided cells explained the inability to form colonies since immobilized cells (cells immersed in agar) formed microcolonies. The behavior observed suggests a life strategy intended to optimize access of individual cells to substrates. Thus, the inability to form colonies or biofilms appears to be part of a K-selected population strategy in which oligotrophic bacteria explore dissolved organic matter in seawater as single cells.     

Electroporation of craniofacial mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse ^1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis ^11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers ¹⁴. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (˜100 million years closer) ¹⁵. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.     

A Single-Cell Model of Phase-Driven Control of Ventricular Fibrillation Frequency

The mechanisms controlling the rotation frequency of functional reentry in ventricular fibrillation (VF) are poorly understood. It has been previously shown that Ba²⁺ at concentrations up to 50 μmol/L slows the rotation frequency in the intact guinea pig (GP) heart, suggesting a role of the inward rectifier current (I_K1) in the mechanism governing the VF response to Ba²⁺. Given that other biological (e.g., sinoatrial node) and artificial systems display phase-locking behavior, we hypothesized that the mechanism for controlling the rotation frequency of a rotor by I_K1 blockade is phase-driven, i.e., the phase shift between transmembrane current and voltage remains constant at varying levels of I_K1 blockade. We measured whole-cell admittance in isolated GP myocytes and in transfected human embryonic kidney (HEK) cells stably expressing Kir 2.1 and 2.3 channels. The admittance phase, i.e., the phase difference between current and voltage, was plotted versus the frequency in control conditions and at 10 or 50 μmol/L Ba²⁺ (in GP heart cells) or 1 mM Ba²⁺ (in HEK cells). The horizontal distance between plots was called the “frequency shift in a single cell” and analyzed. The frequency shift in a single cell was −14.14 ± 5.71 Hz (n = 14) at 10 μM Ba²⁺ and −18.51 ± 4.00 Hz (n = 10) at 50 μM Ba²⁺, p < 0.05. The values perfectly matched the Ba²⁺-induced reduction of VF frequency observed previously in GP heart. A similar relationship was found in the computer simulations. The phase of Ba²⁺-sensitive admittance in GP cells was −2.65 ± 0.32 rad at 10 Hz and −2.79 ± 0.26 rad at 30 Hz. In HEK cells, the phase of Ba²⁺-sensitive admittance was 3.09 ± 0.03 rad at 10 Hz and 3.00 ± 0.17 rad at 30 Hz. We have developed a biological single-cell model of rotation-frequency control. The results show that although rotation frequency changes as a result of I_K1 blockade, the phase difference between transmembrane current and transmembrane voltage remains constant, enabling us to quantitatively predict the change of VF frequency resulting from I_K1 blockade, based on single-cell measurement.     

Estimating Single-Cell Lag Times via a Bayesian Scheme

Network models offer computationally efficient tools for estimating the variability of single-cell lag phases. Currently, optical methods for estimating the variability of single-cell lag phases use single-cell inocula and are technically challenging. A Bayesian network model incorporating small uncertain inocula addresses these limitations.     

Oriented Embedding of Single-Cell Organisms

The dexribed technique facilitates oriented embedding of individual cells in various media for both light and electron microscopy. A fixed Specimen is embedded in a small cube of 2% agar at 40 C and subsequently sealed in the desired orientation to a strip of black paper which then serves as a tab for transferring the specimen during dehydrating and embedding procedures. The beveled ends of the strip indicate the exact location of the specimen in the cube. This technique can be employed for the embedding media used in both light and electron microscopy. It ah permits photomicrographs of the whole specimen to be made which can be compared with photomicrographs of individual sections cut from the specimen in a selected plane.     

质谱流式技术用于单细胞检测

质谱流式技术(mass cytometry)是利用质谱原理对单细胞进行多参数检测的流式技术,能够在单细胞水平实现超过50种标志物的同时测量,显著增强了对细胞生长进程和复杂细胞系统的评估能力。该文简要介绍了质谱流式技术的基本工作原理,并从金属元素标记、质量分析器、高维单细胞数据处理等方面展开论述,阐明设计新型金属元素标签和选择飞行时间质谱的必要性,归纳分析高维单细胞数据的算法并总结各种算法的优点和局限性。     

The Effects of Irreversible Electroporation on the Achilles Tendon: An Experimental Study in a Rabbit Model

Background

To evaluate the potential effects of irreversible electroporation ablation on the Achilles tendon in a rabbit model and to compare the histopathological and biomechanical changes between specimens following electroporation ablation and radiofrequency ablation.

Methods

A total of 140 six-month-old male New Zealand rabbits were used. The animals were randomly divided into two groups, 70 in the radiofrequency ablation group and 70 in the electroporation group. In situ ablations were applied directly to the Achilles tendons of rabbits using typical electroporation (1800 V/cm, 90 pulses) and radiofrequency ablation (power control mode) protocols. Histopathological and biomechanical evaluations were performed to examine the effects of electroporation ablation and radiofrequency ablation over time.

Results

Both electroporation and radiofrequency ablation produced complete cell ablation in the target region. Thermal damage resulted in tendon rupture 3 days post radiofrequency ablation. In contrast, electroporation-ablated Achilles tendons preserved their biomechanical properties and showed no detectable rupture at this time point. The electroporation-ablated tendons exhibited signs of recovery, including tenoblast regeneration and angiogenesis within 2 weeks, and the restoration of their integral structure was evident within 12 weeks.

Conclusions

When applying electroporation to ablate solid tumors, major advantage could be that collateral damage to adjacent tendons or ligaments is minimized due to the unique ability of electroporation ablation to target the cell membrane. This advantage could have a significant impact on the field of tumor ablation near vital tendons or ligaments.