Effects of stearic acid on the embryo cryopreservation in mouse.

BACKGROUND: Intracellular lipids are sensitive to freezing. Lipidome modification is an important tool for studying the role of intracellular lipids in cryotolerance of mammalian oocytes and preimplantation embryos. OBJECTIVE: To study the effects of in vitro exposure of murine embryos to saturated stearic acid (SA) on the lipid content, embryo development and cryotolerance. MATERIALS AND METHODS: In vivo derived mouse embryos were cultured with 100 uM SA for 48 h up to the morula/blastocyst stage. Some of the SA-treated embryos were chosen for the evaluation of their development competence and the change in the lipidome, and other embryos were either slowly frozen or rapidly vitrified. RESULTS: Nile red staining combined with confocal laser scanning microscopy revealed a decrease in the total amount of lipids in the SA-treated embryos. Raman measurements showed that the lipid unsaturation was lower in embryos after in vitro SA culture. The addition of SA did not affect the embryo development before cryopreservation, but negatively affected the results of slow freezing cryopreservation and vitrification. CONCLUSION: In vitro SA exposure lowered the total amount of intracellular lipids and unsaturation in mouse embryos. The changes were accompanied with a significantly lower efficacy of embryo cryopreservation. https://doi.org/10.54680/fr24110110512.

Irreversible lipid phase transition detected in a porcine oocyte at chilling.

Under physiological conditions, the membranes and lipid droplets of germ cells are in a conformationally disordered phase. Typically, during cooling, lipids undergo the transition to ordered phases and, upon heating, melt into a disordered phase. In this communication, we report the lipid phase transition in lipid droplets observed in porcine oocytes. Upon cooling, a sharp lipid phase transition from conformationally disordered to ordered state was detected within the temperature range between 20 and 15 °C. Subsequent heating to 45 °C does not return lipids to their original phase state. To the best of our knowledge, this is the first observation of an irreversible phase transition in lipid droplets of biological cells with native lipid composition.

Cryopreservation increases accumulation of exogenous stearic acid in mouse embryos.

Cryopreservation of preimplantation embryos is a widely used technique, but this procedure might impact the subsequent embryo development. The effect of slow freezing and vitrification on the lipid metabolism in preimplantation mammalian embryos is not well studied. In this work, we applied Raman spectroscopy of isotopically labeled molecules to address the effects of cryopreservation on fatty acid accumulation in mouse embryos. Embryos after slow freezing or vitrification were cultured for 20 h in a medium supplemented with bovine serum albumin saturated with deuterated stearic acid (dSA). After this period the concentration of dSA estimated from Raman spectra of frozen-thawed and vitrified-warmed embryos at the morula stage was almost twice higher compared to non-cryopreserved morulas. At the same time, frozen-thawed and vitrified-warmed 4-cell embryos did not demonstrate any difference in the level of stearic acid uptake from non-cryopreserved embryos of the same stage. After an additional 24 h culture, cryopreserved and non-cryopreserved embryos demonstrated similar dSA uptake.

Clinical Assessment of Perfusion Techniques During Surgical Repair of Coarctation of Aorta With Aortic Arch Hypoplasia in Neonates: A Pilot Prospective Randomized Study.

We aimed to compare the safety and efficacy of 3 perfusion methods primarily used in aortic arch reconstruction in infants, namely, deep hypothermic circulatory arrest, selective antegrade cerebral perfusion, and double arterial cannulation. Forty-five infants with aortic arch obstruction and biventricular anatomy were enrolled in this pilot prospective study (ClinicalTrials.gov registration number: NCT02835703). Patients were randomly assigned into 3 groups according to the perfusion strategy (deep hypothermic circulatory arrest, n = 15; selective antegrade cerebral perfusion, n = 15; double arterial cannulation, n = 15). The primary composite endpoint was the incidence of adverse events in the early postoperative period (acute kidney injury [KDIGO criteria], new brain magnetic resonance imaging (MRI) findings, and in-hospital mortality). The secondary endpoints were intensive care unit length of stay, vasoactive-inotropic score index, and cardiopulmonary bypass duration. All patients underwent aortic arch reconstruction under cardiopulmonary bypass and were monitored with near-infrared spectroscopy during surgery. No significant differences in the baseline characteristics and cardiopulmonary bypass duration were observed among the groups. The incidence of unfavorable events was lower in the double arterial cannulation group (P = 0.041). Acute kidney injury was observed in 8, 6, and 5 patients from the deep hypothermic circulatory arrest, selective antegrade cerebral perfusion, and double arterial cannulation groups, respectively (P = 0.64). Twelve patients from the deep hypothermic circulatory arrest group had new brain MRI findings (P = 0.019). There were 5 in-hospital deaths with no significant difference among the groups (P = 0.70). The "head" and "lumbar" values on near-infrared spectroscopy during aortic arch reconstruction were significantly higher in the selective antegrade cerebral perfusion and double arterial cannulation groups than in the deep hypothermic circulatory arrest group. Patients in the double arterial cannulation group had a significantly lower vasoactive-inotropic score index 24 hours postoperatively than the deep hypothermic circulatory arrest group (P = 0.03). Vasoactive-inotropic score index >12 was found to be a risk factor for acute kidney injury and early mortality. Continuous regional perfusion during aortic arch reconstruction decreases the risk of new brain MRI findings in infants and the need for postoperative inotropic support. Although values of near-infrared spectroscopy during the procedure were significantly higher with continuous perfusion strategies, these methods do not reduce the acute kidney injury incidence compared to that with deep hypothermic circulatory arrest. Double arterial cannulation significantly reduces the need for inotropic support.

[Thoracoscopic clipping of patent ductus arteriosus: position of surgery in the era of transcatheter procedures]. / Torakoskopicheskoe klipirovanie otkrytogo arterial'nogo protoka: mesto khirurgii v épokhu transkateternykh tekhnologii.

AIM: To describe single-center evolution of the procedure and to evaluate the results of thoracoscopic clipping of patent ductus arteriosus (PDA) with diameter over 3,0 mm in term infants weighting over 4,0 kg. MATERIAL AND METHODS: Thoracoscopic clipping of PDA has been performed in 140 patients for the period from March 2012 to March 2018 in Meshalkin National Medical Research Center. Mean age was 4.0 years (range 3 months - 13 years), mean body mass index - 15.4±2.2 kg/m2. INCLUSION CRITERIA: PDA size 3.5-10 mm, Qp/Qs >1,3/1,0, weight 4.0-40 kg. Mean PDA size was 4.6±0.9 mm (range 3.5-8.0 mm), mean pulmonary artery pressure - 34.3±5.8 mm Hg, mean systemic/pulmonary flow Qp/Qs - 1.6±0.3. All patients underwent successful PDA closure through four-port technique under endotracheal general anesthesia and no need for pleural drainage. RESULTS: Mean procedure time was 24.5±15.5 min. In 29 (20,7%) cases we used titanium clips, in 11 (79.3%) - polymer locking ligating clips. There was 1 conversion to mini-thoracotomy. There were no deaths, bleeding or any other life-threatening complications. 94 (67.1%) patients were weaned from ventilator within operating theatre, in other 46 (32.9%) patients mean ventilation time in ICU was 1.3±1.0 hours. In-hospital postoperative complications: pneumothorax - 2 (1.4%) cases, recurrent laryngeal nerve dysfunction - 1 (0.7%), false croup - 1 (0.7%). There were 2 residual leakages in 2 (1.4%) patients in 10 and 6 months after titanium clip deployment. Both of them underwent transcatheter closure using the coil. Considering these cases all following patients underwent PDA closure by polymer locking ligating clips with no cases of residual leakage. CONCLUSION: Thoracoscopic PDA closure by polymer locking ligating clip is safe and effective technique for surgical management of PDA with diameter over 3.0 mm in term infants weighting over 4.0 kg.

[New biophotonics methods for improving efficiency and safety of laser modification of the fibrous tunic of the eye]. / Novye metody biofotoniki dlia povysheniia éffektivnosti i bezopasnosti lazernykh tekhnologii modifikatsii fibroznoi obolochki glaza.

The article describes a newly developed and tested diffractive optical element (DOE) that converts non-uniform radiation of the laser output into a homogeneous ring. The Gerchberg-Saxton algorithm is shown to be well suited for achieving annular intensity distribution. Testing this ring transducer on threshold-plasticity cornea demonstrated the reversibility of axisymmetric changes in the cornea. Atomic-Force microscopy of the area of maximum stresses in the corneal periphery showed no significant changes in the structure of the cornea when irradiated in the selected mode. Measurement of Young's modulus of the corneal surface areas after their irradiation also revealed no changes in the elastic properties, while examination of the corneal structure demonstrated the absence of significant structural changes in irradiated samples compared with intact ones.

Diagnosis of traumatic brain injury using miRNA signatures in nanomagnetically isolated brain-derived extracellular vesicles.

The accurate diagnosis and clinical management of traumatic brain injury (TBI) is currently limited by the lack of accessible molecular biomarkers that reflect the pathophysiology of this heterogeneous disease. To address this challenge, we developed a microchip diagnostic that can characterize TBI more comprehensively using the RNA found in brain-derived extracellular vesicles (EVs). Our approach measures a panel of EV miRNAs, processed with machine learning algorithms to capture the state of the injured and recovering brain. Our diagnostic combines surface marker-specific nanomagnetic isolation of brain-derived EVs, biomarker discovery using RNA sequencing, and machine learning processing of the EV miRNA cargo to minimally invasively measure the state of TBI. We achieved an accuracy of 99% identifying the signature of injured vs. sham control mice using an independent blinded test set (N = 77), where the injured group consists of heterogeneous populations (injury intensity, elapsed time since injury) to model the variability present in clinical samples. Moreover, we successfully predicted the intensity of the injury, the elapsed time since injury, and the presence of a prior injury using independent blinded test sets (N = 82). We demonstrated the translatability in a blinded test set by identifying TBI patients from healthy controls (AUC = 0.9, N = 60). This approach, which can detect signatures of injury that persist across a variety of injury types and individual responses to injury, more accurately reflects the heterogeneity of human TBI injury and recovery than conventional diagnostics, opening new opportunities to improve treatment of traumatic brain injuries.

[Diverticula and Congenital Aneurysms of the Left Ventricle: Anatomical Features, Pathophysiology, Clinical Presentation and Treatment Strategy].

Congenital aneurysms and diverticula of the left ventricle represent a rare group of anomalies in the spectrum of congenital heart disease. Although natural histories of these anomalies are considerably different and characterized by different rates of lifethreatening events, similarity of their clinical presentation and diagnostic criteria do not allow to differentiate this anomalies at routine examination. Data on etiology, methods of diagnosis and treatment published by various authors is controversial. In this review we present relevant aspects of etiology, pathophysiology and treatment strategy of patients with left ventricular diverticula and congenital aneurysms.

Endoventriculoplasty of the Left Ventricle for Congenital Diverticulum.

Congenital left ventricular diverticulum is an extremely rare heart defect. Here, we report a case of successful endoventriculoplasty using a Dacron patch in an infant who was followed up for one year.

[Genetic Differentiation among Natural Populations of the Lizard Complex Darevskia raddei as Inferred from Genome Microsatellite Marking].

The article presents the genetic parameters of the populations of lizards of the Darevskia raddei complex (D. raddei nairensis and D. raddei raddei) and the populations of D. valentini calculated on the basis of the analysis of variability of 50 allelic variants of the three nuclear genome microsatellite-containing loci of 83 individuals. It was demonstrated that the F(st) genetic distances between the populations of D. raddei nairensis and D. raddei raddei were not statistically significantly different from the F(st) genetic distances between the populations of different species, D. raddei and D. valentini. At the same time, these distances were statistically significantly higher than the F(st) distances between the populations belonging to one species within the genus Darevskia. These data suggest deep divergence between the populations of D. raddei raddei and D. raddei nairensis of the D. raddei complex and there arises the question on considering them as separate species.

[FIRST EXPERIENCE OF APPLICATION OF TECHNOLOGY OF A TWO-STREAM LOW-FREQUENCY ULTRASOUND TECHNOLOGY IN ABDOMINAL SURGERY].

Biophysical peculiarities of action on tissues of a two-strem low-frequency ultrasound (TSLFU) technology, elaborated by "Arobella Medical LLC" (USA) firm, were studied. Capacity of ultrasound to separate a pathologically-changed and healthy tissues, to divide the structures in accordance to their bioacoustical parameters constitutes the technology peculiarities. The presence of such a biophysical effect permits to achieve high resectability (R0) in patients with oncological diseases. Antibacterial effect and stimulation of intraorgan microcirculation with ultrasound irradiation were noted. Biophysical peculiarities of TSLFU were successfully applied in surgical treatment of 48 patients, suffering inflammatory and oncological diseases of the abdominal cavity organs.

[Thermodynamic characterization of di-, tri-, and tetranucleotide loci in parthenogenetic lizards Darevskia unisexualis].

Allelic polymorphism of three microsatellite loci from the genome of parthenogenetic lizard Darevskia unisexualis was characterized using analysis of free energy (Gibbs energy) of the DNA/DNA duplex formation within the stepwise mutational model. It was demonstrated that the number of microsatellite cluster monomericic units would change to decrease the mean free energy of the locus. In addition, based on the analysis of nucleotide composition, the GC content of each locus was evaluated, and belonging of the loci examined to certain isochore families was suggested.

Attenuated total reflection Fourier transform infrared and polarization spectroscopy of in vivo human skin ablated, layer by layer, by erbium:YAG laser.

The results of an experimental study of the possibilities of monitoring erbium yttrium aluminum garnet laser-mediated ablation of human epidermis with the use of Fourier transform infrared (FTIR) spectroscopy and spectral polarization techniques are presented. The attenuated total reflection (ATR) method was used for FTIR spectroscopic measurements. Spectral polarization monitoring of the ablation was carried out by analyzing the spectra of the degree of residual linear polarization of a probe light diffusely reflected from the laser-treated region of skin. It was found that the analysis of FTIR spectra allows monitoring of the water and protein contents in the subsurface layers of the treated skin, while the degree of residual polarization measured at the wavelengths of maximal absorption of hemoglobin is sensitive to changes in the epidermis thickness and the blood content in the dermal layer (the degree of erythema).

A prospective randomised study of laser reshaping of cartilage in vivo.

Laser reshaping using low laser energy levels was performed on the cartilage of ten porcine ears. The ears were examined up to 4 months after laser reshaping and the stability of the reshaping was assessed by photography and casts obtained from alginate impressions of the ears. The cartilage was also studied histologically in three animals at 3 weeks, 8 weeks, and 4 months using haematoxylin and eosin (H&E), periodic-acid-Schiff (PAS) and alcian blue stains as well as Mib-1 antibody stain to detect cells in cycle. These ears were compared with the other control or non-irradiated ear. All the treated ears initially demonstrated a controlled alteration in their shape. Four were exposed to a pulse energy of 2 J/cm2 at 20 Hz with a spot diameter of 1.1 mm for 7 s and retained their shape for up to 14 days. However, the three exposed to the same levels of energy for 9 s showed tissue necrosis whereas the three which were treated for less than 4 s returned to their original shape after 24 h. Histological examination revealed that there was a dramatic eccentric proliferation of perichondrium away from the centre of irradiation with a noticeable absence of any inflammation. In all laser-irradiated areas there was a loss of staining with PAS and a dramatic increase in staining with alcian blue within the cartilage matrix. Viable chondrocytes were absent from the irradiated cartilage. Staining with a Mib-1 antibody that detects cells in cycle showed positive staining in a small fraction of proliferated perichondrial cells where new cartilage had been produced.

Temperature dependence of cloned mammalian and salmonid cardiac Na(+)/Ca(2+) exchanger isoforms.

The cardiac Na(+)/Ca(2+) exchanger (NCX), an important regulator of cytosolic Ca(2+) concentration in contraction and relaxation, has been shown in trout heart sarcolemmal vesicles to have high activity at 7 degrees C relative to its mammalian isoform. This unique property is likely due to differences in protein structure. In this study, outward NCX currents (I(NCX)) of the wild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressed in oocytes were measured to explore the potential contributions of regulatory vs. transport mechanisms to this observation. cRNA was transcribed in vitro from both wild-type cDNA and was injected into Xenopus oocytes. I(NCX) of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30 degrees C using the giant excised patch technique. The I(NCX) for both isoforms exhibited Na(+)-dependent inactivation and Ca(2+)-dependent positive regulation. The I(NCX) of NCX1.1 exhibited typical mammalian temperature sensitivities with Q(10) values of 2.4 and 2.6 for peak and steady-state currents, respectively. However, the I(NCX) of NCX-TR1.0 was relatively temperature insensitive with Q(10) values of 1.2 and 1.1 for peak and steady-state currents, respectively. I(NCX) current decay was fit with a single exponential, and the resultant rate constant of inactivation (lambda) was determined as a function of temperature. As expected, lambda decreased monotonically with temperature for both isoforms. Although lambda was significantly greater in NCX1.1 compared with NCX-TR1.0 at all temperatures, the effect of temperature on lambda was not different between the two isoforms. These data suggest that the disparities in I(NCX) temperature dependence between these two exchanger isoforms are unlikely due to differences in their inactivation kinetics. In addition, similar differences in temperature dependence were observed in both isoforms after alpha-chymotrypsin treatment that renders the exchanger in a deregulated state. These data suggest that the differences in I(NCX) temperature dependence between the two isoforms are not due to potential disparities in either the I(NCX) regulatory mechanisms or structural differences in the cytoplasmic loop but are likely predicated on differences within the transmembrane segments.

Inhibition of Na+/Ca2+ exchange by KB-R7943: transport mode selectivity and antiarrhythmic consequences.

The Na+/Ca2+ exchanger plays a prominent role in regulating intracellular Ca2+ levels in cardiac myocytes and can serve as both a Ca2+ influx and efflux pathway. A novel inhibitor, KB-R7943, has been reported to selectively inhibit the reverse mode (i.e., Ca2+ entry) of Na+/Ca2+ exchange transport, although many aspects of its inhibitory properties remain controversial. We evaluated the inhibitory effects of KB-R7943 on Na+/Ca2+ exchange currents using the giant excised patch-clamp technique. Membrane patches were obtained from Xenopus laevis oocytes expressing the cloned cardiac Na+/Ca2+ exchanger NCX1.1, and outward, inward, and combined inward-outward currents were studied. KB-R7943 preferentially inhibited outward (i.e., reverse) Na+/Ca2+ exchange currents. The inhibitory mechanism consists of direct effects on the transport machinery of the exchanger, with additional influences on ionic regulatory properties. Competitive interactions between KB-R7943 and the transported ions were not observed. The antiarrhythmic effects of KB-R7943 were then evaluated in an ischemia-reperfusion model of cardiac injury in Langendorff-perfused whole rabbit hearts using electrocardiography and measurements of left ventricular pressure. When 3 microM KB-R7943 was applied for 10 min before a 30-min global ischemic period, ventricular arrhythmias (tachycardia and fibrillation) associated with both ischemia and reperfusion were almost completely suppressed. The observed electrophysiological profile of KB-R7943 and its protective effects on ischemia-reperfusion-induced ventricular arrhythmias support the notion of a prominent role of Ca2+ entry via reverse Na+/Ca2+ exchange in this process.

Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na(+)-Ca(2+) exchanger.

Ion transport and regulation of Na(+)-Ca(2+) exchange were examined for two alternatively spliced isoforms of the canine cardiac Na(+)-Ca(2+) exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na(+)-Ca(2+) exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na(+)(i)- (i.e., I(1)) and Ca(2+)(i)- (i.e., I(2)) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current-voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I(1) and I(2) regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with alpha-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I(1) inactive state of the exchanger than does the A exon of NCX1.4. With respect to I(2) regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca(2+)(i), NCX1.3 showed a prominent decrease at higher concentrations (>1 microM). This does not appear to be due solely to competition between Ca(2+)(i) and Na(+)(i) at the transport site, as the Ca(2+)(i) affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca(2+)(i) had only modest effects on Na(+)(i)-dependent inactivation of NCX1.3, whereas I(1) inactivation of NCX1.4 could be completely eliminated by Ca(2+)(i). Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na(+)-Ca(2+) exchange to fulfill tissue-specific requirements of Ca(2+) homeostasis.

Functional role of ionic regulation of Na+/Ca2+ exchange assessed in transgenic mouse hearts.

Na+/Ca2+ exchange is the primary mechanism mediating Ca2+ efflux from cardiac myocytes during diastole and, thus, can prominently influence contractile force. In addition to transporting Na+ and Ca2+, the exchanger is also regulated by these ions. Although structure-function studies have identified protein regions of the exchanger subserving these regulatory processes, their physiological importance is unknown. In this study, we examined the electrophysiological and mechanical consequences of cardiospecific overexpression of the canine cardiac exchanger NCX1.1 and a deletion mutant of NCX1.1 (Delta680-685), devoid of intracellular Na+ (Na+i)- and Ca2+ (Ca2+i)- dependent regulatory properties, in transgenic mice. Using the giant excised patch-clamp technique, normal ionic regulation was observed in membrane patches from cardiomyocytes isolated from control and transgenic mice overexpressing NCX1.1. In contrast, ionic regulation was nearly abolished in mice overexpressing Delta680-685, indicating that the native regulatory processes could be overwhelmed by expression of the transgene. To address the physiological consequences of ionic regulation of the Na+/Ca2+ exchanger, we examined postrest force development in papillary muscles from NCX1.1 and Delta680-685 transgenic mice. Postrest potentiation was found to be substantially greater in Delta680-685 than in NCX1.1 transgenic mice, supporting the notion that ionic regulation of Na+/Ca2+ exchange plays a significant functional role in cardiac contractile properties.