KI Membranų biofizikos laboratorija (10.07)

Essential oils (EOs) from plants exhibit various biological activities. The major ingredients of EOs are terpenes composed of isoprene subunits. In our recent studies, we have determined several terpenes as new inhibitors of intercellular communication through gap junctions (GJ). Moreover, we showed that the potency of the monoterpene α-pinene depended on connexin 43 (Cx43) phosphorylation. In the current work, using molecular modeling, we selected terpenes composed of different number of isoprene subunits and having different number of cycles. Using patch-clamp techniques, we examined their potency to inhibit Cx43 GJ conductance. We found that the selected terpenes inhibited the Cx43 GJ conductance with IC50ies ranging from 2.6 to 38µM; the sesquiterpenes but not monoterpenes exhibited extremely long delay until the onset of GJ conductance recovery after their washout; the potency of sesquiterpenes to inhibit the membranous Na+ and L-type Ca2+ currents in human atrial myocytes was more than 10-fold lower. These properties of sesquiterpenes raised the idea of using them for verifying the controversial ephaptic excitation that we carried-out by microelectrode recording and optical mapping in Langendorff-perfused rabbit's heart. High doses of farnesene that were supposed to completely block GJ intercellular communication did not block the spread of electrical excitation in the ventricles and only produced mild changes in action potential parameters comparable to farnesene impact on membranous Na+ and Ca2+ currents. Our findings suggest that ephaptic transmission may play a major role in cardiac excitation.

The use of cardioplegia not only achieves cardiac arrest but also minimizes ischemic/reperfusion (I/R) injury, potentially improving short- or long-term outcomes. The aim of this study was to evaluate the impact of different cardioplegic solutions - del Nido, Custodiol HTK and St. Thomas on genes expression and cytokines response in an immature rat heart model of I/R using the Langendorff preparation. Expression of genes which are involved in cell cycle, proliferation, apoptosis resistance and response to hypoxia were determined in cardiac tissue, as well as levels pro/anti-inflammatory cytokines were measured.

Stents are medical devices used to treat the narrowing of the blood vessel, most commonly caused by atherosclerosis. Currently used bare-metal, drug-eluting stents are limited in size and architectural complexity, and there are a few risks associated with these medical devices. In some cases stents can cause thrombosis or even death. Furthermore, said risk increases while stenting relatively small vessels. This paper shows that vascular stents for relatively small vessels and/or for patient-specific stenting applications can be printed using two-photon polymerization (2PP). 2PP is an additive manufacturing technique with sub-μm resolution and unlimited 3D geometry potential. These capabilities were used to produce stents for small blood vessels, which are 5 mm tall and 0.7 or 0.9 mm in diameter, with 3D struts as thin as 50 μm. Several novel approaches were introduced to accommodate the printing of such a structure like voxel elongation and printing in stereolithography-like vat-sample holder configuration. Furthermore, the produced stents were tested mechanically proving their mechanical resilience to most common types of mechanical deformations. Experimental results were also compared to mathematical modeling, showing excellent agreement, hinting at the possibility of designing and testing complex micro-stent geometries before fabrication in silico. Finally, biocompatibility experiments were performed, in which rats survived the 7-day incubation period and showed no significant biocompatibility issues. Overall, the presented work gives an outlook on all aspects related to the 3D printing of stents - design, manufacturing, mechanical, and biological testing. We show that 2PP aligns very well with all these aspects and is a promising technique for the mass manufacturing of vascular stents for small vessels or specifically for the patient.

In recent years, approximately 20 000 sheep have been used every year in biomedical research across Europe. Especially orthopaedic studies frequently rely on sheep because of the weight and size of the animal, as well as the regeneration time of healing like in humans. Here, surgical interventions are commonly used, and researchers are obliged to minimize any kind of suffering according to the 3R principle (reduce, refine, replace) and animal’s welfare legislation. There is responsibility to ensure appropriate anaesthesia and/or analgesia for all ruminants undergoing painful procedures. The aim of this study is to evaluate how the heart rate and noninvasive arterial pressure of sheep change during traumatological operations. Twenty female Lithuania black-headed sheep at the age of 13–14 months and 30–35 kg of body weight were obtained and handled at the Biological Research Centre of the Lithuanian University of Health Sciences. Permission for the study was obtained from national regulatory bodies. After arrival, the animals were given 2 weeks of habituation to the staff members and regular handfeeding. Prior to surgery, the sheep were not fasted. The sheep underwent general anaesthesia. The sheep were pre-treated with xylazine (0.05 mL/kg, i.m.) and, after 5 min, with fentanyl (1 mL i.m.) before general anaesthesia was induced using propofol CRI (20 mg/kg/h, i.v.). Following orotracheal intubation, anaesthesia was maintained with propofol (20 mg/kg/h), fentanyl (3 mL/h), 21% of oxygen, and 1 L/min of ambient air by inhalation. Oxygen saturation, temperature, heart rate (HR), respiratory rate (RR) and non-invasive blood pressure were monitored continuously. For local anaesthesia, 2% lidocaine (max. 4 mg/kg,) was administered along the incision sites. As soon as the animals breathed spontaneously, they were ex-tubated and the rumen tube was removed. Regarding pain management, ketoprofen (2 mg/kg, i.v.) was administered following 3 days post-surgery. In this study, it was observed that only after premedication the heart rate was lower than after starting the procedure after 15 min, 95.8 ± 27.63 and 104.4 ± 31.1 beats per minute, respectively. This can be influenced by the intubation of the animal, because approximately after 10–15 min from laying the animal on the table, the animal was intubated. The heart rate remained within normal range for young sheep throughout the operation. The average duration of surgery was 52 ± 22.5 min. Vital parameters were monitored every 15 min during surgery. During data processing, the dynamics of mean non-invasive arterial pressure were observed, MAP was highest at the beginning, 127 mmHg ± 27.3 mmHg, and slowly decreased during the entire surgery. This is possibly the influence of anaesthetics, as all drugs used for anaesthesia dilate blood vessels and thus reduce arterial pressure. Blood pressure remained higher than normal range during the entire procedure. The study found that the blood pressure of the sheep remained high even though signs of pain such as the pinch test, reflexes and pupillary movement were negative, ensuring adequate analgesia. An anaesthesia protocol, ensuring analgesia and adequate anaesthesia were used, but closer monitoring is needed in future studies.

Baseline echocardiographic measurements are essential for accurate and reliable diagnostics of cardiovascular diseases. To avoid misinterpretation of echocardiographic images and measurements, establishing standardized reference values is necessary. Several studies have highlighted the suitability of sheep as models for cardiovascular research due to their morphological similarities to humans, including comparable adult heart size, cardiomyocyte nuclei count, venous drainage patterns, and physiological responses to cardiovascular disease induction [1]. Consequently, sheep are widely used and considered reliable in biomedical imaging studies [2, 3]. Additionally, the increasing popularity of sheep as companion animals, appreciated for their gentle nature and ease of care, has elevated the need for precise cardiovascular diagnostics in veterinary medicine [4]. The aim of this study was to establish cardiac ultrasound methodology of two Lithuanian sheep breads to asses baseline cardiac ultrasound measurements. Physically mature Lithuanian blackhead sheep (LBS) (N = 12) aged 300 to 310 days weighing 33.1 ± 1.47 kg and 20 Romanov sheep (RS) (N = 20) aged 180 to 200 days weighing 22.6 ± 1.80 kg were analyzed in this study. Two-dimensional, M-mode and pulsed wave Doppler echocardiographic studies were accomplished while measuring normal cardiac dimensions, time indices and blood volumes. A 4–5 MHz phased-array transducer was used to acquire the images. The transducer was pressed to the skin approximately 2–3 cm dorsal to the fourth and fifth right intercostal spaces. All the echocardiographic measurements were significantly correlated with body weight and breed. This study demonstrated that it is possible to perform a cardiac ultrasound examination on standing sheep with ease, without disrupting physiology or causing stress to the animal. The sheep remain calm while standing, able to ruminate and breathe without interruption. No anesthesia was used in this study, meaning the physiological parameters of the heart were not affected. This study results showed that it is possible to measure the left ventricle ejection fraction, which was significantly higher in LBS. The left ventricular posterior wall index (LVPi) and the intraventricular septum index were measured, and LVPi was significantly higher in Romanov sheep. Using ultrasound examination on standing sheep makes it possible to calculate early and late diastolic pressure ratio, which was higher in Romanov sheep. The human cardiac ultrasound protocol was adapted for sheep ultrasound measures, and aortic flow acceleration time as well as velocity time integral were observed in both sheep breeds. Depending on the protocol, the left ventricular diastolic and systolic volume as well as the stroke volume were measured. The observed measurements were higher in LBS. This study concluded that echocardiography could be used as a tool in diagnosing and further researching cardiac diseases and disorders of Lithuanian blackhead and Romanov sheep.

The meat industry is observing an increase in the population of purebred animals from the finest meat breeds globally, due to the introduction of innovative solutions in animal husbandry, feeding practices, breeding techniques, and overall management [1]. Ultrasound can be added in breeding programs to select animals with the potential to pass on superior meat qualities [2]. Key indicators of good quality include fat thickness, the area of the longissimus dorsi muscle, hide thickness, the percentage of intramuscular fat, and the marbling score [3]. The aim of this study was to determine the relationship between daily body weight and indicators of quality meat using ultrasound imaging. During the study, a total of 603 cattle scans were performed. Daily weight gain was calculated by deriving the average of age and weight. Ultrasound scan was performed using MyLabOneVET ultrasound equipment (Esaote, Germany). During the scan, the sensor was pressed deep into the muscle for a few seconds until the image on the screen was smooth and consistent. Different structures are visible during the examination: subcutaneous fat, transverse section of the long back muscle, and fat layer near the rib cage. After the examination, the created images are saved. After the examination, the stored images are evaluated and adipose tissue measurements are performed in the scanner, then the muscle area and the fat layer near the ribs are evaluated in this way. The data were calculated using installed formulas, according to which it is possible to accurately calculate the yield percentage while the animal is still alive. The measurement data were coded and compiled using an Excel (MsOffice, USA) calculator. To calculate the relationship between the outcome variables, the Pearson correlation test was performed, and the correlation coefficient R was calculated, which does not depend on the measurement units of the variables. This study was conducted in six different farms in Lithuania. Before each ultrasound examination, the cattle were weighed, and their heads were fixed accurate scanning. After analyzing the collected data, it was found that the daily weight of the animals varied from 0.51 kg/d up to 2.7 kg/d, on average 1.41 kg/d. A weak positive correlation (r = 0.372) was found in correlation tests between daily body weight and long back muscle area, P < 0.01. The study also analyzed the relationship between yield grade and daily weight gain. It was found that there was no significant correlation between these two parameters. A similar trend was observed when analyzing the relationship between daily body weight and intermuscular fat percentage (IMF): the correlation was very weak, but positive. Analysis of the data on fat thickness and daily weight demonstrated that there was no correlation between these parameters. This shows that the daily weight does not have a significant effect on the thickness of subcutaneous fat. However, a weak positive correlation (r = 0.383) was found between daily body weight and the thickness of the longus back muscle, P < 0.01. The results of this study do not prove the benefits of ultrasound scanning. The study found only a weak relationship between the measured parameters. The study has several methodological limitations, so further research is needed to accurately assess the benefits of ultrasound scanning.

Introduction. Cardioplegic solutions are employed during heart surgery to halt myocardial contraction. Their main purpose is to minimize myocardial oxygen demand by inducing electromechanical quiescence and cooling of the heart, thereby reducing ischemic effects. This process helps prevent degenerative changes, decreases the metabolic demands of the cardiac muscle, and protects cardiac myocytes from apoptosis and cell death [1]. Ischemic injury in the heart can be characterized by various molecular alterations within cells. BCL-2 family proteins play a crucial role in regulating mitochondrial apoptosis, either facilitating or preventing cell death [2]. In addition, FOS proteins have been associated with cell transformation, differentiation, proliferation [3]. The BNIP2 gene interacts with various proteins to protect cells from externally induced cell death [4]. Meanwhile, HIF1α serves as a key regulator of cellular and systemic homeostasis in response to hypoxia [5]. Aim. To assess the effects of three different cardioplegic solutions on immature rat heart tissues, we analyzed gene expression associated with the cell cycle, proliferation, apoptosis resistance, and hypoxia response. Methods. The animals were sacrificed, and the heart was removed through a median sternotomy, then transferred to a cold perfusion buffer. Within 5 min, the heart was mounted on a Langendorff system and perfused retrogradely. Cardioplegia was administered using St. Thomas (CP1), Custodiol HTK (CP2), or del Nido (CP3) solutions, with re-plegia intervals of 30 min (CP1), 90 min (CP3), or a single dose (CP2). As a positive control, fully perfused heart tissue was used (K-PRF). Ventricular biopsies were homogenized in liquid nitrogen to extract total RNA, which was then reverse-transcribed and analyzed for gene expression using TaqMan assays. Results. CP3-treated tissue had an average 50% lower BAX/BCL2 ratio than CP1 and CP2 tissue, however, these changes were not statistically significant. Following CP treatment, HIF1α gene was significantly overexpressed CP3 group compared to, CP1, and CP2 groups (P<0.05). In all cases, FOS expression was significantly decreased compared to the K-PRF after CP1 and CP2 treatment. After 2 and 4 h of incubation with CP3 solution, FOS gene expression remained substantially higher compared to CP1 (P<0.05) and CP2 (P<0.05). Similar results were observed in BNIP2 gene expression. Based on gene expression, CP3 treated cardiac tissue was comparable to K-PRF heart tissue in all timestamps. Conclusions. The CP3 solution exhibited the most effective cell-protective properties, comparable to heart perfusion. However, further studies are required to assess the electrophysiological and metabolic changes in heart tissue.

Background Invasive pulmonary aspergillosis (IPA) is a life-threatening infection in immunocompromised patients. Daily liposomal amphotericin B (LAMB) is effective against IPA; however, its efficacy, pharmacokinetics/pharmacodynamics (PK/PD), and safety with less frequent dosing regimens are not well understood. Utilizing the concentration-dependent pharmacodynamics of amphotericin B, we hypothesized that the high peak plasma concentrations and extended tissue concentrations of LAMB may allow dosing every two, three, or four days, delivering the same total drug amount as daily dosing. If successful, this dosing strategy could enable less frequent dosing in clinical practice, improving patient quality of life, reducing hospitalization costs, and allowing for outpatient management. Methods Six New Zealand white rabbits per dosage group received LAMB 24h after endotracheal inoculation of 1x108 Aspergillus fumigatus and continued for up to 12 days. Treatment groups: LAMB at 3 mg/kg Q24h (LAMB3), 6 mg/kg Q48h (LAMB6), 9 mg/kg Q72h (LAMB9), 12 mg/kg Q96h (LAMB12), or untreated controls (UC). PK parameters and multiple efficacy variables were evaluated to compare treatment groups and UC. Plasma PK parameters were determined by non-compartmental equations. Results PK analysis demonstrated a dose-proportional increase in Cmax and AUCt. There was a significant reduction of residual fungal burden (logCFU/g) (p≤0.01), lung weights (p≤0.01) and pulmonary infarct scores (p≤0.001) as measures of organism-mediated pulmonary injury, and prolonged survival (p≤0.01) in all treatment groups vs UC. There were no significant differences among treatment regimens. These data correlated directly with a significant decline of GMI antigenemia and serum BDG levels during therapy in comparison to progressive GMI and BDG levels of UC (p<0.01). Blood chemistry demonstrated comparable safety parameters in all treatment groups. All data are presented in Table and Figure. Conclusions This study demonstrated that LAMB exhibits dose-proportional and concentration-dependent PK/PD properties against IPA and establishes the preclinical foundation for patients to receive less frequent dosing, improved quality of life, reduced in-patient costs, and new options for outpatient care.

The rapid emergence of multidrug-resistant bacterial species poses a critical threat by reducing the efficacy of antibiotics and complicating infection treatment. Bacteriocins, such as klebicin KvarM, have emerged as promising alternatives to traditional antibiotics due to their targeted antimicrobial activity. In this study, we evaluated the therapeutic potential of Eudragit-coated klebicin KvarM in a mouse model of Klebsiella pneumoniae intestinal colonization, assessing both its antimicrobial effectiveness and impact on commensal gut microbiota.

The use of non-invasive technologies is among the most accurate methods for assessing meat quality parameters. Beef tenderness and palatability are strongly influenced by the amount of intramuscular fat, commonly referred to as marbling. Marbling is widely used in breeding programs to select animals with superior meat quality. In this study, we analyzed 236 Angus bulls and 22 Angus heifers from various farms in Lithuania, all aged 450 ± 112 days. Ultrasound examinations were performed using a linear transducer with a silicone standoff to evaluate the loin eye area. Key meat quality indicators, such as fat thickness, loin area and thickness, and intramuscular fat, were found to increase with the weight of bulls, ranging from 365 ± 12.70 kg to 825.57 ± 11.75 kg (p < 0.001), and heifers, ranging from 273 ± 20.71 kg to 767 ± 41.01 kg (p < 0.001). The highest intramuscular fat values were observed at 7.50 ± 0.75% (p < 0.05) to 6.9 ± 1.36% (p < 0.1) in bulls weighing 431.33–825.57 kg and 7.98 ± 0.28% (p < 0.001) in heifers weighing 603 kg. These findings demonstrate the feasibility of selecting Angus cattle for improved meat quality traits across different weight categories.