Stečki so srednjeveški kamniti nagrobniki, ki jih najdemo še vedno in situ v pokrajinah držav Zahodnega Balkana. Okoli 8% stečkov ima raznovrstne dekoracije v obliki nizkega reliefa, medtem, ko manj ...kot 400 nagrobnikov nosi tudi napise. Zaradi klimatskih vplivov vrezani napisi in ornamenti izginjajo s površine stečkov, zaradi česar jih je vse težje odkriti in prepoznati. V sklopu arheoloških raziskav stečkov na območju srednjeveške župe Dabar (današnji občini Berkovići in Bileća), je bil izveden pilotski projekt 3D skeniranja stečkov z ročnim skenerjem. Osem stečkov, dva z napisom in šest z dekoracijami, je bilo skeniranih v celoti, z namenom da bi lahko ovrednotili vidnost in prepoznavanje umetnostnih izrazov, ter samo tehniko detajlnega dokumentiranja materialne kulture. Članek predstavlja potek skeniranja in analizira rezultate.
Electroporation is a method which uses an adequate number of electric pulses of enough amplitude, duration and number applied to cells, thus inducing transient permeabilization of the cell membrane. ...Due to possibility that microenvironment in applications of in vivo electroporation is slightly acidic, we studied the effects of slightly acidic electroporation and recovery medium. We observed no difference in the permeabilization threshold, detected by propidium iodide, of cells which were electroporated and allowed to recover in growth (pH 7.8) or acidic (pH 6.5) medium. In contrast, statistically significant difference was observed in survival of cells that were exposed to pulse amplitudes greater than permeabilization threshold. Survival of cells was greater if acidic electroporation and recovery medium were used, but acidic extracellular pH decreased gene electrotransfer efficiency. We also observed differences in morphology between cells that were electroporated and left to recover in growth medium and cells that were electroporated and left to recover in acidic medium. Our results imply that slightly acidic extracellular pH allows more efficient repair of damage that is induced on cell membrane during electroporation with high pulse amplitudes.
A high-intensity pulsed electromagnetic field (HI-PEMF) is a non-invasive and non-contact delivery method and may, as such, have an advantage over gene electrotransfer mediated by conventional ...electroporation using contact electrodes. Due to the limited number of in vitro studies in the field of gene electrotransfection by HI-PEMF, we designed experiments to investigate and demonstrate the feasibility of such a technique for the non-viral delivery of genetic material into cells in vitro. We first showed that HI-PEMF causes DNA adsorption to the membrane, a generally accepted prerequisite step for successful gene electrotransfection. We also showed that HI-PEMF can induce gene electrotransfection as the application of HI-PEMF increased the percentage of GFP-positive cells for two different combinations of pDNA size and concentration. Furthermore, by measuring the uptake of larger molecules, i.e., fluorescently labelled dextrans of three different sizes, we showed endocytosis to be a possible mechanism for introducing large molecules into cells by HI-PEMF.
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Gene therapies are revolutionizing medicine by providing a way to cure hitherto incurable diseases. The scientific and technological advances have enabled the first gene therapies to ...become clinically approved. In addition, with the ongoing COVID-19 pandemic, we are witnessing record speeds in the development and distribution of gene-based vaccines. For gene therapy to take effect, the therapeutic nucleic acids (RNA or DNA) need to overcome several barriers before they can execute their function of producing a protein or silencing a defective or overexpressing gene. This includes the barriers of the interstitium, the cell membrane, the cytoplasmic barriers and (in case of DNA) the nuclear envelope. Gene electrotransfer (GET), i.e., transfection by means of pulsed electric fields, is a non-viral technique that can overcome these barriers in a safe and effective manner. GET has reached the clinical stage of investigations where it is currently being evaluated for its therapeutic benefits across a wide variety of indications. In this review, we formalize our current understanding of GET from a biophysical perspective and critically discuss the mechanisms by which electric field can aid in overcoming the barriers. We also identify the gaps in knowledge that are hindering optimization of GET in vivo.
Gene electrotransfer (GET) is a widely used method for nucleic acids’ delivery into cells. We explored, evaluated, and demonstrated the potential use of different pulse durations for introducing ...plasmid DNA (pDNA) into cells in vitro and compared the efficiency and dynamics of transgene expression after GET. We performed experiments on cell suspensions of 1306 fibroblasts and C2C12 myoblasts with four ranges of pulse durations (nanosecond, high frequency bipolar (HF-BP), and micro- and millisecond). Six different concentrations of pDNA encoding green fluorescent protein were used. We show that GET can be achieved with nanosecond pulses with a low pulse repetition rate (10 Hz). The GET’s efficiency depends on the pDNA concentration and cell line. Time dynamics of transgene expression are comparable between millisecond, microsecond, HF-BP, and nanosecond pulses but depend greatly on cell line. Lastly, based on the data obtained in the experiments of pDNA concentration effect on GET the model of the probability of pDNA and cell membrane contact during GET was developed. The model shows that pDNA migration is dominated by diffusion for nanosecond and HF-BP pulses and by electrophoresis for micro- and millisecond pulses. Modeling results can provide valuable guidance for further experiments and interpretations of the results obtained by various pulse protocols.
•Gene electrotransfer can be achieved with high frequency bipolar microsecond pulses.•Gene electrotransfer efficiency depends on plasmid concentration.•Number of transferred plasmid copies is higher ...with longer monopolar pulses.•No correlation between size of plasmid aggregates and transfection efficiency.
High-frequency bipolar pulses (HF-BP) have been demonstrated to be efficient for membrane permeabilization and irreversible electroporation. Since membrane permeabilization has been achieved using HF-BP pulses we hypothesized that with these pulses we can also achieve successful gene electrotransfer (GET). Three variations of bursts of 2 µs bipolar pulses with 2 µs interphase delay were applied in HF-BP protocols. We compared transfection efficiency of monopolar micro and millisecond pulses and HF-BP protocols at various plasmid DNA (pDNA) concentrations on CHO – K1 cells. GET efficiency increased with increasing pDNA concentration. Overall GET obtained by HF-BP pulse protocols was comparable to overall GET obtained by longer monopolar pulse protocols. Our results, however, suggest that although we were able to achieve similar percent of transfected cells, the number of pDNA copies that were successfully transferred into cells seemed to be higher when longer monopolar pulses were used. Interestingly, we did not observe any direct correlation between fluorescence intensity of pDNA aggregates formed on cell membrane and transfection efficiency. The results of our study confirmed that we can achieve successful GET with bipolar microsecond i. e. HF-BP pulses, although at the expense of higher pDNA concentrations.
The exact mechanisms of nucleic acid (NA) delivery with gene electrotransfer (GET) are still unknown, which represents a limitation for its broader use. Further, not knowing the effects that ...different experimental electrical and biological parameters have on GET additionally hinders GET optimization, resulting in the majority of research being performed using a trial-and-error approach. To explore the current state of knowledge, we conducted a systematic literature review of GET papers in in vitro conditions and performed meta-analyses of the reported GET efficiency. For now, there is no universal GET strategy that would be appropriate for all experimental aims. Apart from the availability of the required electroporation device and electrodes, the choice of an optimal GET approach depends on parameters such as the electroporation medium; type and origin of cells; and the size, concentration, promoter, and type of the NA to be transfected. Equally important are appropriate controls and the measurement or evaluation of the output pulses to allow a fair and unbiased evaluation of the experimental results. Since many experimental electrical and biological parameters can affect GET, it is important that all used parameters are adequately reported to enable the comparison of results, as well as potentially faster and more efficient experiment planning and optimization.
Electrochemotherapy treats tumors by combining specific chemotherapeutic drugs with an intracellular target and electric pulses, which increases drug uptake into the tumor cells. Electrochemotherapy ...has been successfully used for treatment of easily accessible superficial tumor nodules. In this paper, we present the first case of deep-seated tumor electrochemotherapy based on numerical treatment planning.
The aim of our study was to treat a melanoma metastasis in the thigh of a patient. Treatment planning for electrode positioning and electrical pulse parameters was performed for two different electrode configurations: one with four and another with five long needle electrodes. During the procedure, the four electrode treatment plan was adopted and the patient was treated accordingly by electrochemotherapy with bleomycin. The response to treatment was clinically and radiographically evaluated. Due to a partial response of the treated tumor, the metastasis was surgically removed after 2 months and pathological analysis was performed.
A partial response of the tumor to electrochemotherapy was obtained. Histologically, the metastasis showed partial necrosis due to electrochemotherapy, estimated to represent 40-50% of the tumor. Based on the data obtained, we re-evaluated the electrical treatment parameters in order to correlate the treatment plan with the clinical response. Electrode positions in the numerical model were updated according to the actual positions during treatment. We compared the maximum value of the measured electric current with the current predicted by the model and good agreement was obtained. Finally, tumor coverage with an electric field above the reversible threshold was recalculated and determined to be approximately 94%. Therefore, according to the calculations, a small volume of tumor cells remained viable after electrochemotherapy, and these were sufficient for tumor regrowth.
In this, the first reported clinical case, deep-seated melanoma metastasis in the thigh of the patient was treated by electrochemotherapy, according to a treatment plan obtained by numerical modeling and optimization. Although only a partial response was obtained, the presented work demonstrates that treatment of deep-seated tumor nodules by electrochemotherapy is feasible and sets the ground for numerical treatment planning-based electrochemotherapy.
EudraCT:2008-008290-54.
Electroporation is a method in which electric field of sufficient amplitude and duration applied to cells causes transient permeabilization of the cell membrane. Due to possibility, that ...microenvironment in applications of electroporation in vivo is acid, we studied effects of acid electroporation and recovery media. We observed no difference in the permeabilization threshold of cells which were electroporated and allowed to recover in growth (pH 7.8) or acid (pH 6.5) media. In contrast, statistically significant difference was observed in survival of cells that were exposed to pulse amplitudes greater than permeabilization threshold. At equal voltages applied survival of cells was greater if acid electroporation and recovery media were used compared to survival after electroporation and recovery in growth media. Our results imply that there is difference in membrane repair effectiveness depending on extracellular pH.
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