Papers by Saulius Satkauskas
Applied Sciences, 2021
There is growing concern regarding the nutritional value of processed food products. Although the... more There is growing concern regarding the nutritional value of processed food products. Although thermal pasteurization, used in food processing, is a safe method and is widely applied in the food industry, food products lack quality and nutritional value because of the high temperatures used during pasteurization. In this study, the effect of pulsed electric field (PEF) processing on whey protein content and bacterial viability in raw milk was evaluated by changing the PEF strength and number of pulses. For comparison, traditional pasteurization techniques, such as low-temperature long-time (LTLT), ultra-high temperature (UHT), and microfiltration (MF), were also tested for total whey protein content, bacterial activity, and coliforms. We found that, after treatment with PEF, a significant decrease in total bacterial viability of 2.43 log and coliforms of 0.9 log was achieved, although undenatured whey protein content was not affected at 4.98 mg/mL. While traditional pasteurization te...
Molecules
One of current applications of electroporation is electrochemotherapy and electroablation for loc... more One of current applications of electroporation is electrochemotherapy and electroablation for local cancer treatment. Both of these electroporation modalities share some similarities with radiation therapy, one of which could be the bystander effect. In this study, we aimed to investigate the role of the bystander effect following these electroporation-based treatments. During direct CHO-K1 cell treatment, cells were electroporated using one 100 µs duration square wave electric pulse at 1400 V/cm (for bleomycin electrotransfer) or 2800 V/cm (for irreversible electroporation). To evaluate the bystander effect, the medium was taken from directly treated cells after 24 h incubation and applied on unaffected cells. Six days after the treatment, cell viability and colony sizes were evaluated using the cell colony formation assay. The results showed that the bystander effect after bleomycin electrotransfer had a strong negative impact on cell viability and cell colony size, which decrease...
Biomedicines
Intracellular calcium ion delivery via sonoporation has been validated to be a substitute for cla... more Intracellular calcium ion delivery via sonoporation has been validated to be a substitute for classical chemotherapy. However, the mechanism behind calcium sonoporation remains unclear to this day. To elucidate the role of calcium in the process of sonoporation, we aimed to investigate the influence of different calcium concentration on cell membrane permeabilization and cell viability after sonoporation. In this study, we present experimental evidence that extracellular calcium plays a major role in cell membrane molecular transport after applying ultrasound pulses. Ultrasound-microbubble cavitation in the presence of different calcium concentration affects fundamental cell bio-physio-chemical conditions: cell membrane integrity, metabolic activity, and colony formation. Corresponding vital characteristics were evaluated using three independent viability tests: propidium iodide assay (20 min–3 h), MTT assay (48 h), and cell clonogenic assay (6 d). The results indicate instant cell ...
Biomedical Signal Processing and Control
International Journal of Molecular Sciences
Deregulated microRNA (miRNA) expression profiles and their contribution to carcinogenesis have be... more Deregulated microRNA (miRNA) expression profiles and their contribution to carcinogenesis have been observed in virtually all types of human cancer. However, their role in the pathogenesis of rare mesenchymal gastrointestinal stromal tumors (GISTs) is not well defined, yet. In this study, we aimed to investigate the role of two miRNAs strongly downregulated in GIST—miR-375-3p and miR-200b-3p—in the pathogenesis of GIST. To achieve this, miRNA mimics were transfected into GIST-T1 cells and changes in the potential target gene mRNA and protein expression, as well as alterations in cell viability, migration, apoptotic cell counts and direct miRNA–target interaction, were evaluated. Results revealed that overexpression of miR-375-3p downregulated the expression of KIT mRNA and protein by direct binding to KIT 3′UTR, reduced GIST cell viability and migration rates. MiR-200b-3p lowered expression of ETV1 protein, directly targeted and lowered expression of EGFR mRNA and protein, and negat...
Applied Sciences
Electroporation is an effective method for delivering plasmid DNA molecules into cells. The effic... more Electroporation is an effective method for delivering plasmid DNA molecules into cells. The efficiency of gene electrotransfer depends on several factors. To achieve high transfection efficiency while maintaining cell viability is a tedious task in electroporation. Here, we present a combined study in which the dynamics of both evaluation types of transfection efficiency and the cell viability were evaluated in dependence of plasmid concentration as well as at the different number of high voltage (HV) electric pulses. The results of this study reveal a quantitative sigmoidal (R2 > 0.95) dependence of the transfection efficiency and cell viability on the distance between the cell membrane and the nearest plasmid. We propose this distance value as a new, more accurate output parameter that could be used in further optimization studies as a predictor and a measure of electrotransfection efficiency.
Scientific Reports
The concurrent assessment of principal sonoporation factors has been accomplished in a single sys... more The concurrent assessment of principal sonoporation factors has been accomplished in a single systemic study. Microbubble sonodestruction dynamics and cavitation spectral characteristics, ultrasound scattering and attenuation, were examined in relation to the intracellular delivery of anticancer drug, bleomycin. Experiments were conducted on Chinese hamster ovary cells coadministered with Sonovue microbubbles. Detailed analysis of the scattering and attenuation temporal functions culminated in quantification of metrics, inertial cavitation dose and attenuation rate, suitable for cavitation control. The exponents, representing microbubble sonodestruction kinetics were exploited to derive dosimetric, microbubble sonodestruction rate. High intracorrelation between empirically-attained metrics defines the relations which indicate deep physical interdependencies within inherent phenomena. Subsequently each quantified metric was validated to be well-applicable to prognosticate the efficac...
Pharmaceutics
Electroporation—a transient electric-field-induced increase in cell membrane permeability—can be ... more Electroporation—a transient electric-field-induced increase in cell membrane permeability—can be used to facilitate the delivery of anticancer drugs for antitumour electrochemotherapy. In recent years, Ca2+ electroporation has emerged as an alternative modality to electrochemotherapy. The antitumor effect of calcium electroporation is achieved as a result of the introduction of supraphysiological calcium doses. However, calcium is also known to play a key role in membrane resealing, potentially altering the pore dynamics and molecular delivery during electroporation. To elucidate the role of calcium for the electrotransfer of small charged molecule into cell we have performed experiments using nano- and micro-second electric pulses. The results demonstrate that extracellular calcium ions inhibit the electrotransfer of small charged molecules. Experiments revealed that this effect is related to an increased rate of membrane resealing. We also employed mathematical modelling methods i...
International Journal of Molecular Sciences
Gastric cancer (GC) is one of the most common and lethal gastrointestinal malignancies worldwide.... more Gastric cancer (GC) is one of the most common and lethal gastrointestinal malignancies worldwide. Many studies have shown that development of GC and other malignancies is mainly driven by alterations of cellular signaling pathways. MicroRNAs (miRNAs) are small noncoding molecules that function as tumor-suppressors or oncogenes, playing an essential role in a variety of fundamental biological processes. In order to understand the functional relevance of miRNA dysregulation, studies analyzing their target genes are of major importance. Here, we chose to analyze two miRNAs, miR-20b and miR-451a, shown to be deregulated in many different malignancies, including GC. Deregulated expression of miR-20b and miR-451a was determined in GC cell lines and the INS-GAS mouse model. Using Western Blot and luciferase reporter assay we determined that miR-20b directly regulates expression of PTEN and TXNIP, and miR-451a: CAV1 and TSC1. Loss-of-function experiments revealed that down-regulation of miR...
Cancers
In this work, we have investigated the feasibility of sub-microsecond range irreversible electrop... more In this work, we have investigated the feasibility of sub-microsecond range irreversible electroporation (IRE) with and without calcium electroporation in vivo. As a model, BALB/C mice were used and bioluminescent SP2/0 myeloma tumor models were developed. Tumors were treated with two separate pulsed electric field (PEF) pulsing protocols PEF1: 12 kV/cm × 200 ns × 500 (0.006 J/pulse) and PEF2: 12 kV/cm × 500 ns × 500 (0.015 J/pulse), which were delivered with and without Ca2+ (168 mM) using parallel plate electrodes at a repetition frequency of 100 Hz. Both PEF1 and PEF2 treatments reduced tumor growth and prolonged the life span of the mice, however, the PEF2 protocol was more efficient. The delay in tumor renewal was the biggest when a combination of IRE with calcium electroporation was used, however, we did not obtain significant differences in the final mouse survival compared to PEF2 alone. Anti-tumor immune responses were also investigated after treatment with PEF2 and PEF2+Ca...
International Journal of Molecular Sciences
Electrochemotherapy is an efficient method for the local treatment of cutaneous and subcutaneous ... more Electrochemotherapy is an efficient method for the local treatment of cutaneous and subcutaneous metastases, but its efficacy as a systemic treatment remains low. The application of gene electrotransfer (GET) to transfer DNA coding for immune system modulating molecules could allow for a systemic effect, but its applications are limited because of possible side effects, e.g., immune system overactivation and autoimmune response. In this paper, we present the simultaneous electrotransfer of bleomycin and plasmid DNA as a method to increase the systemic effect of bleomycin-based electrochemotherapy. With appropriately selected concentrations of bleomycin and plasmid DNA, it is possible to achieve efficient cell transfection while killing cells via the cytotoxic effect of bleomycin at later time points. We also show the dynamics of both cell electrotransfection and cell death after the simultaneous electrotransfer of bleomycin and plasmid DNA. Therefore, this method could have applicat...
Scientific reports, Jan 5, 2018
A challenge for gene therapy is absence of safe and efficient local delivery of therapeutic genet... more A challenge for gene therapy is absence of safe and efficient local delivery of therapeutic genetic material. An efficient and reproducible physical method of electrospray for localized and targeted gene delivery is presented. Electrospray works on the principle of coulombs repulsion, under influence of electric field the liquid carrying genetic material is dispersed into micro droplets and is accelerated towards the targeted tissue, acting as a counter electrode. The accelerated droplets penetrate the targeted cells thus facilitating the transfer of genetic material into the cell. The work described here presents the principle of electrospray for gene delivery, the basic instrument design, and the various optimized parameters to enhance gene transfer in vitro. We estimate a transfection efficiency of up to 60% was achieved. We describe an efficient gene transfer method and a potential electrospray-mediated gene transfer mechanism.
Trends in biochemical sciences, Jun 1, 2018
Long-term transcriptional gene silencing has been hampered by delivery issues. A potential soluti... more Long-term transcriptional gene silencing has been hampered by delivery issues. A potential solution is the application of RNA viruses that generate small RNAs without any DNA intermediate. Long-term therapy for various diseases is expected after a single administration.
Scientific reports, Jan 19, 2018
Current electrotransfection protocols are well-established for decades and, as a rule, employ lon... more Current electrotransfection protocols are well-established for decades and, as a rule, employ long micro-millisecond range electric field pulses to facilitate DNA transfer while application of nanosecond range pulses is limited. The purpose of this paper is to show that the transfection using ultrashort pulses is possible by regulating the pulse repetition frequency. We have used 200 ns pulses (10-18 kV/cm) in bursts of ten with varied repetition frequency (1 Hz-1 MHz). The Chinese Hamster Ovary (CHO) cells were used as a cell model. Experiments were performed using green fluorescent protein (GFP) and luciferase (LUC) coding plasmids. Transfection expression levels were evaluated using flow cytometry or luminometer. It was shown that with the increase of frequency from 100 kHz to 1 MHz, the transfection expression levels increased up to 17% with minimal decrease in cell viability. The LUC coding plasmid was transferred more efficiently using high frequency bursts compared to single ...
Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference, 2018
Electrospray is a process based on creation and acceleration of small sized droplets based on ele... more Electrospray is a process based on creation and acceleration of small sized droplets based on electrostatic repulsion. Spraying plasmid containing liquids this process may be used to transfer genes into cells. Within this paper we report on a method for accessing and evaluating the spray modalities using high speed imaging system with a post processing of image data to obtain estimated volume and velocity of emerging droplets first. Second we investigate on the impact of different media on the spray modalities. Third we evaluate the impact of the spray on cell viability and on transfection efficiency of an eGFP plasmid as reporter gene obtained in an in vitro setup on alveolar epithelial like cells (A549).
Trends in Biotechnology
Recent discoveries have shown that self-replicating RNA viruses can produce small RNAs (sRNAs) in... more Recent discoveries have shown that self-replicating RNA viruses can produce small RNAs (sRNAs) in host cells. Given their potential to be modified to generate short-term transgene expression without integrating viral sequences into the host genome, these viruses could be used as safe delivery vehicles for sRNAs to induce long-term transcriptional gene silencing (TGS). This might open new avenues for therapeutic approaches, where a single administration would safely induce long-term therapeutic effects for various diseases. Here, we introduce and discuss the possible use of cytoplasmic alphaviruses, flaviviruses, Sendai virus (SeV), and nucleoplasmic Influenza A (IAV) and Borna disease (BoDV) viruses to induce long-term TGS.
Archives of Biochemistry and Biophysics
Electroporation is a widely established method for molecular delivery across electric field pertu... more Electroporation is a widely established method for molecular delivery across electric field perturbed plasma membrane. It can be used as a non-viral DNA transfection method, or as a way to achieve small molecule delivery to or extraction from cells. We examined the possibility of combining the DNA delivery to the cells with small molecule transport across electroporated plasma membrane. The results show that the presence of DNA in electroporation medium increases the extraction of fluorescent dye calcein from calcein-AM loaded cells as well as the delivery of small-molecule drug bleomycin to the cells. We propose that these results may have implications in enhanced drug delivery using electroporation both in vivo and in clinics.
CBU International Conference Proceedings
Gene electrotransfer, which designates the combination of gene transfer and electroporation, is a... more Gene electrotransfer, which designates the combination of gene transfer and electroporation, is a physical method for transfecting genes into cells and tissues. Many reports for the utilization of this techniques in animals confirmed that gene electrotransfer is a safe and efficient method. One of the major advantages of electrogene therapy is that it does not result in systemic toxicity. Gene electrotransfer (GET) of plasmids encoding cytokines has been shown to generate a potent anti-tumor effect. Delivery of plasmids encoding cytokines induces not only a local immune response but a systemic one as well. Cytokines can be used to stimulate host inflammatory responses and immunity to cancers. This review aims to summarize preclinically tested cytokine genes with the help of electroporation for cancer treatment.
Uploads
Papers by Saulius Satkauskas