Threshold Interphase Delay for Bipolar Pulses to Prevent Cancellation Phenomenon during Electrochemotherapy.

Electroporation-based procedures employing nanosecond bipolar pulses are commonly linked to an undesirable phenomenon known as the cancelation effect. The cancellation effect arises when the second pulse partially or completely neutralizes the effects of the first pulse, simultaneously diminishing cells' plasma membrane permeabilization and the overall efficiency of the procedure. Introducing a temporal gap between the positive and negative phases of the bipolar pulses during electroporation procedures may help to overcome the cancellation phenomenon; however, the exact thresholds are not yet known. Therefore, in this work, we have tested the influence of different interphase delay values (from 0 ms to 95 ms) using symmetric bipolar nanoseconds (300 and 500 ns) on cell permeabilization using 10 Hz, 100 Hz, and 1 kHz protocols. As a model mouse hepatoma, the MH-22a cell line was employed. Additionally, we conducted in vitro electrochemotherapy with cisplatin, employing reduced interphase delay values (0 ms and 0.1 ms) at 10 Hz. Cell plasma membrane permeabilization and viability dependence on a variety of bipolar pulsed electric field protocols were characterized. It was shown that it is possible to minimize bipolar cancellation, enabling treatment efficiency comparable to monophasic pulses with identical parameters. At the same time, it was highlighted that bipolar cancellation has a significant influence on permeabilization, while the effects on the outcome of electrochemotherapy are minimal.

The Effects of Bipolar Cancellation Phenomenon on Nano-Electrochemotherapy of Melanoma Tumors: In Vitro and In Vivo Pilot.

The phenomenon known as bipolar cancellation is observed when biphasic nanosecond electric field pulses are used, which results in reduced electroporation efficiency when compared to unipolar pulses of the same parameters. Basically, the negative phase of the bipolar pulse diminishes the effect of the positive phase. Our study aimed to investigate how bipolar cancellation affects Ca2+ electrochemotherapy and cellular response under varying electric field intensities and pulse durations (3-7 kV/cm, 100, 300, and 500 ns bipolar 1 MHz repetition frequency pulse bursts, n = 100). As a reference, standard microsecond range parametric protocols were used (100 µs × 8 pulses). We have shown that the cancellation effect is extremely strong when the pulses are closely spaced (1 MHz frequency), which results in a lack of cell membrane permeabilization and consequent failure of electrochemotherapy in vitro. To validate the observations, we have performed a pilot in vivo study where we compared the efficacy of monophasic (5 kV/cm × ↑500 ns × 100) and biphasic sequences (5 kV/cm × ↑500 ns + ↓500 ns × 100) delivered at 1 MHz frequency in the context of Ca2+ electrochemotherapy (B16-F10 cell line, C57BL/6 mice, n = 24). Mice treated with bipolar pulses did not exhibit prolonged survival when compared to the untreated control (tumor-bearing mice); therefore, the bipolar cancellation phenomenon was also occurrent in vivo, significantly impairing electrochemotherapy. At the same time, the efficacy of monophasic nanosecond pulses was comparable to 1.4 kV/cm × 100 µs × 8 pulses sequence, resulting in tumor reduction following the treatment and prolonged survival of the animals.

Synthesis, Characterization and Bioassay of Novel Substituted 1-(3-(1,3-Thiazol-2-yl)phenyl)-5-oxopyrrolidines.

Thiazole derivatives attract the attention of scientists both in the field of organic synthesis and bioactivity research due to their high biological activity. In the present study, thiazole ring was obtained by the interaction of 1-(4-(bromoacetyl)phenyl)-5-oxopyrrolidine-3-carboxylic acid with thiocarbamide or benzenecarbothioamide, as well as tioureido acid. A series of substituted 1-(3-(1,3-thiazol-2-yl)phenyl)-5-oxopyrrolidines with pyrrolidinone, thiazole, pyrrole, 1,2,4-triazole, oxadiazole and benzimidazole heterocyclic fragments were synthesized and their antibacterial properties were evaluated against Gram-positive strains of Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa, Escherichia coli and Salmonella enterica enteritidis. The vast majority of compounds exhibited between twofold and 16-fold increased antibacterial effect against the test-cultures when compared with Oxytetracycline.

Mitochondrial depolarization and ATP loss during high frequency nanosecond and microsecond electroporation.

It is predicted that ultra-short electric field pulses (nanosecond) can selectively permeabilize intracellular structures (e.g., mitochondria) without significant effects on the outer cell plasma membrane. Such a phenomenon would have high applicability in cancer treatment and could be employed to modulate cell death type or immunogenic response. Therefore, in this study, we compare the effects of 100 µs x 8 pulses (ESOPE - European Standard Operating Procedures on Electrochemotherapy) and bursts of 100 ns pulses for modulation of the mitochondria membrane potential. We characterize the efficacies of various protocols to trigger permeabilization, depolarize mitochondria (evaluated 1 h  after treatment), the extent of ATP depletion and generation of reactive oxygen species (ROS). Finally, we employ the most prominent protocols in the context of Ca2+ electrochemotherapy in vitro. We provide experimental proof that 7.5-12.5 kV/cm x 100 ns pulses can be used to modulate mitochondrial potential, however, the permeabilization of the outer membrane is still a prerequisite for depolarization. Similar to 100 µs x 8 pulses, the higher the permeabilization rate, the higher the mitochondrial depolarization. Nevertheless, 100 ns pulses result in lesser ROS generation when compared to ESOPE, even when the energy input is several-fold higher than for the microsecond procedure. At the same time, it shows that even the short 100 ns pulses can be successfully used for Ca2+ electrochemotherapy, ensuring excellent cytotoxic efficacy.

Effects of buffer composition and plasmid toxicity on electroporation-based non-viral gene delivery in mammalian cells using bursts of nanosecond and microsecond pulses.

Gene electrotransfer (GET) is non-viral gene delivery technique, also known as electroporation-mediated gene delivery or electrotransfection. GET is a method used to introduce foreign genetic material (such as DNA or RNA) into cells by applying external pulsed electric fields (PEFs) to create temporary pores in the cell membrane. This study was undertaken to examine the impact of buffer composition on the efficiency of GET in mammalian cells Also, we specifically compared the effectiveness of high-frequency nanosecond (ns) pulses with standard microsecond (µs) pulses. For the assessment of cell transfection efficiency and viability, flow cytometric analysis, luminescent assays, and measurements of metabolic activity were conducted. The efficiency of electrotransfection was evaluated using two different proteins encoding plasmids (pEGFP-N1 and Luciferase-pcDNA3). The investigation revealed that the composition of the electroporation buffer significantly influences the efficacy of GET in CHO-K1 cell line. The different susceptibility of cell lines to the electric field and the plasmid cytotoxicity were reported. It was also shown that electroporation with nanosecond duration PEF protocols ensured equivalent or even better transfection efficiency than standard µsPEF. Additionally, we successfully performed long-term transfection of the murine 4T1 cell line using high-frequency nanosecond PEFs and confirmed its' applicability in an in vivo model. The findings from the study can be applied to optimize electrotransfection conditions.

Application of Gold Nanoparticles for Improvement of Electroporation-Assisted Drug Delivery and Bleomycin Electrochemotherapy.

Background/Objectives: Electrochemotherapy (ECT) is a safe and efficient method of targeted drug delivery using pulsed electric fields (PEF), one that is based on the phenomenon of electroporation. However, the problems of electric field homogeneity within a tumor can cause a diminishing of the treatment efficacy, resulting only in partial response to the procedure. This work used gold nano-particles for electric field amplification, introducing the capability to improve available elec-trochemotherapy methods and solve problems associated with field non-homogeneity. Methods: We characterized the potential use of gold nanoparticles of 13 nm diameter (AuNPs: 13 nm) in combination with microsecond (0.6-1.5 kV/cm × 100 µs × 8 (1 Hz)) and nanosecond (6 kV/cm × 300-700 ns × 100 (1, 10, 100 kHz and 1 MHz)) electric field pulses. Finally, we tested the most prominent protocols (microsecond and nanosecond) in the context of bleomycin-based electrochemotherapy (4T1 mammary cancer cell line). Results: In the nano-pulse range, the synergistic effects (improved permeabilization and electrotransfer) were profound, with increased pulse burst frequency. Addi-tionally, AuNPs not only reduced the permeabilization thresholds but also affected pore resealing. It was shown that a saturated cytotoxic response with AuNPs can be triggered at significantly lower electric fields and that the AuNPs themselves are non-toxic for the cells either separately or in combination with bleomycin. Conclusions: The used electric fields are considered sub-threshold and/or not applicable for electrochemotherapy, however, when combined with AuNPs results in successful ECT, indicating the methodology's prospective applicability as an anticancer treatment method.

Susceptibility of various human cancer cell lines to nanosecond and microsecond range electrochemotherapy: Feasibility of multi-drug cocktails.

Electrochemotherapy (ECT) involves combining anticancer drugs with electroporation, which is induced by pulsed electric fields (PEFs), while the effects vary in effectiveness based on the specific parameters of the electrical pulses and susceptibility of the cells to a specific drug. In this work, we utilized conventional microsecond electroporation protocols (0.8 - 1.5 kV/cm × 100 µs × 8, 1 Hz) and the new modality of nanosecond pulses (4 and 8 kV/cm × 500 ns × 100, 1 kHz and 1 MHz), which are compressed into a high frequency burst. Sensitive and resistant lung, breast and ovarian human cancer cell lines were used in the study. In order to overcome drug-resistance, we have investigated the feasibility to use anticancer drug cocktails i.e., bleomycin and cisplatin combinations with metformin, vinorelbine and Dp44mT. The different susceptibility of various human cancer cells lines to electric pulses was determined, the efficacy of ECT was characterized and the type of cell death depending on the combinations of drugs was investigated. The results indicate that synergistic effects of PEFs with drug cocktails may be used to overcome drug-resistance in cancer, while the application of nsPEF provides more flexibility in parametric protocols and modulation of cancer cell death.

Calcium Electrochemotherapy for Tumor Eradication and the Potential of High-Frequency Nanosecond Protocols.

Calcium electroporation (CaEP) is an innovative approach to treating cancer, involving the internalization of supraphysiological amounts of calcium through electroporation, which leads to cell death. CaEP enables the replacement of chemotherapeutics (e.g., bleomycin). Here, we present a standard microsecond (µsCaEP) and novel high-frequency nanosecond protocols for calcium electroporation (nsCaEP) for the elimination of carcinoma tumors in C57BL/6J mice. We show the efficacy of CaEP in eliminating tumors and increasing their survival rates in vivo. The antitumor immune response after the treatment was observed by investigating immune cell populations in tumors, spleens, lymph nodes, and blood, as well as assessing antitumor antibodies. CaEP treatment resulted in an increased percentage of CD4+ and CD8+ central memory T cells and decreased splenic myeloid-derived suppressor cells (MDSC). Moreover, increased levels of antitumor IgG antibodies after CaEP treatment were detected. The experimental results demonstrated that the administration of CaEP led to tumor growth delay, increased survival rates, and stimulated immune response, indicating a potential synergistic relationship between CaEP and immunotherapy.

The prevalence of facial and dentolabial parameters among students of the Faculty of Dentistry of Lithuanian University of Health Sciences.

OBJECTIVE: Nowadays esthetic checklist is an indispensable assistance for the dental clinics. Processing the information recorded on it and integrating the esthetic evaluation with biologic and functional parameters will really allow dentist to formulate a correct diagnosis and to choose most suitable treatment plan for the patient. The aim of this study was to evaluate the prevalence of facial and dentolabial parameters among students of the Faculty of Dentistry of Lithuanian University of Health Sciences and to identify relationship of all facial and dentolabial parameters results. MATERIAL AND METHODS: The study is carried out at the Department of Dental and Maxillofacial Orthopedics of Lithuanian University of Health Sciences. The facial and dentolabial parameters of students are examined by visual examination using medical gloves and rulers. Participants were sitting on a chair at a distance of 150 cm in front of the researchers. RESULTS: The thick type of lips was the most popular for normal profile face, of thin lips - concave, of medium lips type - convex. The most popular of profile face types were normal and convex. The most popular tooth exposure among all smile line types was less than half teeth. Type when more than half of teeth was diagnosed in the most of high "Gummy smile". CONCLUSIONS: Was found relationship between: the profile of face and type of lips; the tooth exposure at rest and type of smile line; the commisural line vs horizon and interpupillary line vs horizon; the facial midline and occlusal plane vs commisural line/horizon; the type of smile line and type of lips; the labial corridor and 10 teeth number of smile width type.

The relationship between pathological wear of teeth and temporomandibular joint dysfunction.

OBJECTIVE: Temporomandibular joint dysfunction (TMJD) is quite frequent pathology in population. In scientific literature pathological tooth wear (PTW) is listed among the predisposing factors of it. MATERIAL AND METHODS: During the survey 189 primary patients of dental department in polyclinic of Panevezys city were examined. According to the tooth wear degree 2 groups have been formed: control group (grade 0-1) withouth TMJ dysfunction; and experimental group (grade 2-4) with presumable TMJD. RESULTS: In experimental group tooth wear of anterior and posterior region increases with age respectively (p<0.05). It was also found that centric occlusion (CO) and relative rest heights difference increases with age in the control group (p<0.05) as well as the experimental group (p=0.001). In patients with diagnosed TMJD the difference between heights in CO and relative rest was bigger than in patients without TMJD (p=0.039). In the experimental group TMJD was diagnosed the most of patients grade-4 in 90%. The most common symptom is joint sounds was observed in 72.2%. CONCLUSIONS: 35-50 and over 50 age groups of patients had a higher first molars degree of wear than the younger ones. In patients with diagnosed TMJD the difference between heights in CO and relative rest was bigger than in patients without TMJD. TMJD symptoms and of posterior teeth pathological wear interface hypothesis confirmed. In the experimental group TMJD was diagnosed the most common in 90% of patients grade-4. The most common symptom is "Nutcracker" of experimental group patients 77.2%.

In vitro color and roughness stability of different temporary restorative materials.

OBJECTIVE: The aim of this study was to evaluate the effects of staining solutions on the color stability and roughness of different provisional resin material. MATERIAL AND METHODS: Three different materials were tested (two polymethylmethacrylate and one bis-acryl composite resins) in cola and coffee and for 1 and 4 weeks. 240 specimens were used - half of them polished and other half not. Color measurements were made before and after immersions using CIE L*a*b*. Surface roughness was measured using profilometer. Data was analyzed by ANOVA and Tukey HSD multiple comparison tests. RESULTS: The highest ΔE values were observed in the coffee. Unpolished and polished bis-acryl resin showed the highest ΔE values (p<0.00) in both intervals. The i-TEMP had the lowest or one of the least color changes. The time factor had influence on the color stability of provisional materials (p<0.01). The highest Ra values of polished specimens were observed in the cola; i-TEMP exposed the best result (p<0.00). The Pearson Correlation test showed a strong correlation between ΔE and Ra in provisional restorative materials in coffee and weak-moderate correlation in cola. CONCLUSIONS: The higher color stability of polished and unpolished specimens was shown by polymethylmethacrylate than bis-acryl resins. All polished specimens shown better color stability properties than unpolished except for the hot polymerization provisional materials (i-CAB). Polished polymethylmethacrylate showed better results of average roughness in comparison with bis-acryl resin. Cold-polymerization polymethylmethacrylate had better results of color and roughness stability than hot-polymerization polymethylmethacrylate.