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. In this study we analyze transient conductivity changes during the electric pulses and increased membrane permeability for ions and molecules after the pulses in order to determine which parameters affect stabilization of pores, and to analyze the relation between transient pores and long-lived transport pores. By quantifying ion diffusion, fraction of transport pores f(per) was obtained. A simple model, which assumes a quadratic dependence of f(per) on E in the area where U(m)>U(c) very accurately describes experimental values, suggesting that f(per) increases with higher electric field due to larger permeabilized area and due to higher energy available for pore formation. The fraction of transport pores increases also with the number of pulses N, which suggest that each pulse contributes to formation of more and/or larger stable transport pores, whereas the number of transient pores does not depend on N.     

Increase in specific density of levobupivacaine and fentanyl solution ensures lower incidence of inadequate block

The clinical presentation of a subarachnoid block (SAB) is dependent upon the intrathecal spread of local anesthetic (LA). Intrathecal distribution depends on the chemical and physical characteristics of LA, puncture site, technique used, patient anatomical characteristics and hydrodynamic properties of cerebrospinal fluid. We tried to determine whether a combined glucose/LA solution can render a clinically significant difference in sensory block distribution and motor block intensity.This was a controlled, randomized and double blinded study. The surgical procedures were stripping of the great or small saphenous vein and extirpation of remaining varicose veins. The study included 110 patients distributed into two groups: Hyperbaric (7.5 mg levobupivacaine (1.5 ml 0.5% Chirocaine) + 50 microg Fentanyl (0.5 ml Fentanil) and 1 ml 10% glucose (Pliva)) vs. Hypobaric (7.5 mg levobupivacaine (1.5 ml 0.5% Chirocaine) + 50 microg Fentanyl (0.5 ml Fentanil) and 1 ml 0.9% NaCl (Pliva, Zagreb)) adding to a total volume of 3.5 ml per solution. Spinal puncture was at L3-L4 level. Spinal block distribution was assessed in five minute intervals and intensity of motor block was assessed according to the modified Bromage scale. Pain was assessed with the Visual Analogue Scale. A statistically significant difference in sensory block distribution, motor block intensity and recovery time was established between hyperbaric and hypobaric solutions. By increasing the specific density of anesthetic solution, a higher sensory block, with lesser variability, a diminished influence of Body Mass Index, decreased motor block intensity and faster recovery time may be achieved.     

Analytical and numerical quantification and comparison of the local electric field in the tissue for different electrode configurations

Background  

Electrochemotherapy and gene electrotransfer are novel promising treatments employing locally applied high electric pulses to introduce chemotherapeutic drugs into tumor cells or genes into target cells based on the cell membrane electroporation. The main focus of this paper was to calculate analytically and numerically local electric field distribution inside the treated tissue in two dimensional (2D) models for different plate and needle electrode configurations and to compare the local electric field distribution to parameter U/d, which is widely used in electrochemotherapy and gene electrotransfer studies. We demonstrate the importance of evaluating the local electric field distribution in electrochemotherapy and gene electrotransfer.     

Insect cells for antibody production: evaluation of an efficient alternative

In recent years there has been an increase in both availability and demand for therapeutic monoclonal antibodies. Currently, most of these antibodies are produced by stably transfected mammalian cells. In this study we evaluated the use of different baculoviral insect cell systems as an alternative for commonly used production schemes. We expressed the human anti-gp41 antibody 3D6 in Spodoptera frugiperda Sf9, Trichoplusia ni BTI-TN5B1-4 "High Five", and Spodoptera frugiperda SfSWT-1 "Mimic?" insect cells and compared product yield, specificity and glycosylation patterns with a 3D6 antibody expressed in Chinese hamster ovary cells. Using "High Five" cells we achieved amounts of secreted antibody comparable to those resulting from transient expression in mammalian cells. We determined the N-linked oligosaccharide structures present on asparagine-297 in IgG? heavy chains and tested the functionality in terms of antigen binding and the ability to elicit effector functions. Antibodies expressed in all insect cell lines displayed highly specific antigen binding. In general, the insect-produced antibodies carried, as the CHO-produced form, fucosylated N-glycans, including, in the case of "High Five" cells, high levels of core α1,3-fucose. This indicates that in all systems glycoengineering may be required in order to produce optimal glycoforms of this antibody.     

Concentration and purification of rubella virus using monolithic chromatographic support

The production of economically acceptable viral vaccines of high quality requires simple and efficient methods for purification and concentration of viral particles. Ion-exchange chromatography (IEC) has become one of commonly used methods for large-scale downstream purification of viruses. Viruses possess different biological and/or biochemical properties and therefore IEC conditions must be established specifically for each virus. Live attenuated rubella virus vaccines have been manufactured and successfully used widely to protect people from rubella and congenital rubella syndrome for almost 40 years. The aim of this study was to search for an efficient method for concentration and purification of rubella virus using IEC. The selected operating conditions using quaternary amine monolithic supports enabled highly efficient binding, purification and concentration of rubella virus from complex biological suspension without additional procedures. Eluted viral particles maintained their infectivity and viral recovery was almost 100%. At the same time, viral preparation was successfully depleted from host cell protein and DNA. This work indicates the possibility of using monoliths to improve the rubella virus yields in productions where high virus titers during cultivation can hardly be achieved.     

Medical Surveillance for Biological Terrorism Agents

The first recognition of a bioterrorist attack may be sick patients. Recognition and subsequent notification of an attack at the earliest possible moment will assist in rapidly instituting protective measures. New methods of medical surveillance can assist with rapid detection. Syndromic surveillance is the use of newly created or pre-existing databases to track the health of communities. The data can include medical and non-medical information, but typically contains non-specific indicators of health status. These systems are currently being tested and fielded in multiple locations, and nationwide systems are being developed in the United States. Retrospective and prospective analyses have demonstrated the ability of the data to detect disease outbreaks, but the systems have yet to be proven to lead to more effective interventions.     

The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing

Most processed, e.g. fucosylated, N-glycans on insect glycoproteins terminate in mannose, yet the relevant modifying enzymes require the prior action of N-acetylglucosaminyltransferase I. This led to the hypothesis that a hexosaminidase acts during the course of N-glycan maturation. To determine whether the Drosophila melanogaster genome indeed encodes such an enzyme, a cDNA corresponding to fused lobes (fdl), a putative beta-N-acetylglucosaminidase with a potential transmembrane domain, was cloned. When expressed in Pichia pastoris, the enzyme exhibited a substrate specificity similar to that previously described for a hexosaminidase activity from Sf-9 cells, i.e. it hydrolyzed exclusively the GlcNAc residue attached to the alpha1,3-linked mannose of the core pentasaccharide of N-glycans. It also hydrolyzed p-nitrophenyl-N-acetyl-beta-glucosaminide, but not chitooligosaccharides; in contrast, Drosophila HEXO1 and HEXO2 expressed in Pichia cleaved both these substrates but not N-glycans. The localization of recombinant FDL tagged with green fluorescent protein in Drosophila S2 cells by immunoelectron microscopy showed that this enzyme transits through the Golgi, is present on the plasma membrane and in multivesicular bodies, and is secreted. Finally, the N-glycans of two lines of fdl mutant flies were analyzed by mass spectrometry and reversed-phase high-performance liquid chromatography. The ratio of structures with terminal GlcNAc over those without (i.e. paucimannosidic N-glycans) was drastically increased in the fdl-deficient flies. Therefore, we conclude that the fdl gene encodes a novel hexosaminidase responsible for the occurrence of paucimannosidic N-glycans in Drosophila.     

Morphological and proteomic analyses of sugar beet cultures and identifying putative markers for cell differentiation

Normal (N), habituated nonorganogenic (HNO), and tumour (T) sugar beet cell lines were analysed in order to establish specific patterns of extracellular proteins and identify protein markers that might explain the distinct phenotypical characteristics. Electron microscopy showed that the ultrastructure of N cells corresponds to that of parenchyma cells, and that these cells contain plastids with large starch grains. HNO and T cells had enlarged, lobed nuclei with an increased number of nucleoli; the number of nuclei in HNO cells was greater than in T cells. The T plastids were elongated, with reduced thylakoids and abundant phytoferritin deposits, while HNO plastids were small and vacuolated, with an irregular, underdeveloped thylakoid system. The extracellular proteome of the cells was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Greater differences in protein expression were observed between the HNO and N lines than between the T and N lines. Sixteen of the most prominent bands differentially expressed among the cell lines were cut out from the gel and analyzed by mass spectrometry. Cell wall-modifying enzymes were identified, including a peroxidase whose expression was twofold higher in N and T tissue than in HNO tissue; pectinesterase, which was expressed at a level threefold lower in the T line than in the other cell lines; and xyloglucan endotransglucosylase, which was expressed at a level sixfold higher in HNO and T tissue. Three proteins belonged to the chitinase gene family and their expression was higher in HNO and T tissue than in N tissue. The differential expression of these proteins suggests that these play a role in cell line-specific cell wall composition and cell-to-cell adhesion.     

Esomeprazole versus pantoprazole for healing erosive oesophagitis

The aim of this study was to compare the efficacy of esomeprazole and pantoprazole with regard to healing and relief from gastroesophageal reflux disease-related symptoms. I this multicentre, randomized, single-blind study 180 patients (ITT population) diagnosed with endoscopically proven GERD grade A,B,C received esomeprazole (40 mg once daily (o.d.), n = 90) orpantoprazole (40 mg o.d., n = 90). Healing and relief from GERD-related symptoms were assessed at first and final visit (after 4 or 8 weeks of treatment). Esomeprazole 40 mg provided significantly greater healing than pantoprazole 40 mg after 4 weeks of treatment in patients with EE (77.8% vs. 72.2%). Esomeprazole-treated patients were healed after up to 8 weeks of treatment similar those treated with pantoprazole (92.2% vs. 91.1%). The proportion of heartburn-free days was similar in patients treated with esomeprazole and to those treated with pantoprazole.