[Genome Editing in Species of the Tribe Triticeae with the CRISPR/Cas System].

The tribe Triticeae includes important agricultural crops, such as bread wheat, durum wheat, barley, rye, and triticale. Research in the field of reverse genetics and genetic engineering of Triticeae received a new impetus as the CRISPR/Cas genome editing system came into broad use. The review describes and analyzes the data on recent advances in genomic editing of cultivated plants of the tribe Triticeae and tools used in the field. The tools most commonly used for genome editing in Triticeae include the codon-optimized Cas9 gene under the control of the maize ubiquitin gene promoter and guide RNAs under the control of Pol III promoters U6 and U3 in one or more binary vectors. Phosphinothricin and hygromycin resistance genes are used as selectable genes. Agrobacterium-mediated transformation and biolistics are performed to obtain genome-edited plants, and immature embryos are used as explants. Approaches developed to overcome the problem of low regenerative capacity of Triticeae include in planta transformation of shoot apical meristems, transformation of microspores and pollen grains, and the use of haploinductors. Bread wheat and barley were subject to genomic editing in the majority of studies published to date, and durum wheat and triticale were recently used in CRISPR/Cas knockout studies of target genes. Further progress in the development of genome editing of cultivated plants of the tribe Triticeae should be aimed at expanding the range of species and varieties involved and overcoming the problems of low regenerative capacity. This will allow genetic modification of elite varieties, which will be in demand in agricultural production.

A Model of Short Bowel Syndrome in Rodents in a Long-Term Experiment.

Simulation of short bowel syndrome (SBS) in experimental animals is of great interest for studies in translational medicine. The processes of intestinal adaptation are most often studied in rats aged 8-10 weeks with up to 70% resection the small bowel. In this case, the animals are euthanized in 2 weeks after the surgery; further development of the pathological process remains practically unexplored. In our experiment, a number of blood biochemical parameters and morphological signs of intestinal adaptation were assessed within 34 weeks after surgery. It was shown that after resection of 70% of the small bowel in rats (180±15 g), clinical symptoms are observed only during the first 2 weeks, however, the mortality in the group was observed throughout the entire period of the study in the absence of clear clinical signs. Morphological compensatory changes in the walls of the small intestine continued up to 34 weeks after resection, and their morphometry can be used to describe adaptation in the dynamics of the study.

Alpha-B-Crystallin Effect on Mature Amyloid Fibrils: Different Degradation Mechanisms and Changes in Cytotoxicity.

Given the ability of molecular chaperones and chaperone-like proteins to inhibit the formation of pathological amyloid fibrils, the chaperone-based therapy of amyloidosis has recently been proposed. However, since these diseases are often diagnosed at the stages when a large amount of amyloids is already accumulated in the patient's body, in this work we pay attention to the undeservedly poorly studied problem of chaperone and chaperone-like proteins' effect on mature amyloid fibrils. We showed that a heat shock protein alpha-B-crystallin, which is capable of inhibiting fibrillogenesis and is found in large quantities as a part of amyloid plaques, can induce degradation of mature amyloids by two different mechanisms. Under physiological conditions, alpha-B-crystallin induces fluffing and unweaving of amyloid fibrils, which leads to a partial decrease in their structural ordering without lowering their stability and can increase their cytotoxicity. We found a higher correlation between the rate and effectiveness of amyloids degradation with the size of fibrils clusters rather than with amino acid sequence of amyloidogenic protein. Some external effects (such as an increase in medium acidity) can lead to a change in the mechanism of fibrils degradation induced by alpha-B-crystallin: amyloid fibers are fragmented without changing their secondary structure and properties. According to recent data, fibrils cutting can lead to the generation of seeds for new bona fide amyloid fibrils and accelerate the accumulation of amyloids, as well as enhance the ability of fibrils to disrupt membranes and to reduce cell viability. Our results emphasize the need to test the chaperone effect not only on fibrillogenesis, but also on the mature amyloid fibrils, including stress conditions, in order to avoid undesirable disease progression during chaperone-based therapy.

[Oxidative stress in focal symptomatic and cryptogenic epilepsy in young patients]. / Okislitel'nyi stress pri fokal'noi simptomaticheskoi i kriptogennoi epilepsii v molodom vozraste.

OBJECTIVE: To study oxidative stress in young patients with focal symptomatic and cryptogenic epilepsy and after the new-onset epileptic seizures. MATERIAL AND METHODS: Patients, aged from 19 to 44 years, were distributed into three groups, 30 patients in each: patients after a few (first) epileptic seizures, patients in the clinical remission that has lasted at least one year, and patients with treatment-resistant epileptic seizures. The control group included 20 healthy people. Parameters of the pro-oxidant status (TBA-reactive products) and the antioxidant defense (total superoxide-scavenging activity, catalase, total antioxidant activity, and reduced thiols (SH groups)) were measured in the blood plasma. RESULTS: No significant differences in the concentrations of TBA-reactive products were identified in patients with epilepsy compared with healthy controls while concentrations of reduced SH groups, total superoxide-scavenging activity, catalase activity and the total antioxidant activity were significantly decreased in patients. In addition, some of the parameters displayed significant differences between different groups of patients. CONCLUSION: In patients with epilepsy, the changes in the free-radical processes are seen already after the first seizures and persist in the treatment-resistant epilepsy and in clinical remission. Since the parameters of the activity of the antioxidant defense are significantly different in different groups of patients, one can assume that different elements of the oxidative stress are present after the first epileptic seizures and in different courses of the disease.

[Design of Guide RNA for CRISPR/Cas Plant Genome Editing].

CRISPR/Cas technology of genome editing is a powerful tool for making targeted changes in the DNA of various organisms, including plants. The choice of the precise nucleotide sequence (protospacer) in the gene to be edited is important in the design of guide RNA, which can be carried out by specialized software. We review and compare all the known on-line and off-line resources for guide RNA design, with special attention paid to tools capable of searching for off-target edits sites in plant genomes. The use of Cas12a may be preferable to Cas9. Techniques allowing C→T and G→A base editing without DNA cleavage are discussed along with the basic requirements for the design of effective and highly specific guide RNAs. Ways for improving guide RNA design software are presented. We also discuss the lesser risks of off-target editing in plant genomes as opposed to animal genomes. Examples of edited plant genomes including those that do not lead to the creation of transgenic plants are reviewed.

[Endothelial damage and circadian blood pressure profile in rheumatoid arthritis].

AIM: To study the influence of the state of endothelium on the daily profile of arterial pressure (AP) in patients with rheumatoid arthritis (RA). MATERIALS AND METHODS: In 70 RA pts carried out C-reactive protein (CRP), vascular endothelial adhesion molecule type 1 (sVCAM-1), antigen von Willebrand Factor (AG WF), interleukin-8 (Il-8), rheumatoid factor (RF), IgG, endotheline-1 (ET-1), number of desquamated endotheliocytes cells (DE), VS, activity of renin by immunoenzyme analysis. The dysfunction of endothelium was evaluated by calculation of DE. The functional methods included the daily monitoring of arterial pressure (AP). RESULTS: Arterial hypertension (AH) occurred in 40 (57.1%) pts. RA pts are revealed the signs of endothelial dysfunction, about which significant differences among the indices of activation of endothelium in comparison with control group testify. ET-1, sVCAM-1, vWF AG, Il-8, CRP content was higher in RA pts. Reliably above there was a number of DE. Reliable differences according to these indices depending of RA activity were discovered. With conducting of correlation analysis it is revealed, that markers of the activation of endothelium: sVCAM-1, vWF AG positively correlated with increasing RF IgG and indices of the immune inflammation: CRP, and DE number. In patients suffering from RA, showed signs of endothelial dysfunction. The positive correlation between endothelial damage and daily profile of AP show the relationship of these processes. CONCLUSION: Positive correlations between the damage of endothelium and disturbance of AP daily profile testify about the interrelation of these processes.

Yeast red pigment modifies cloned human α-synuclein pathogenesis in Parkinson disease models in Saccharomyces cerevisiae and Drosophila melanogaster.

Recently, we identified the yeast red pigment (RP), a polymer of 1-(5'-Phosphoribosyl)-5-aminoimidazole, as a novel potential anti-amyloid agent for the therapy of neurodegenerative diseases. The purpose of this study was to further validate RP for treatment of Parkinson's disease (PD) and to clarify molecular mechanisms involved in the reduction of amyloid cytotoxicity. We investigated RP effects in vivo using Saccharomyces cerevisiae and Drosophila melanogaster PD models. Western blot analysis revealed reduction in the levels of insoluble α-synuclein in both models, while soluble α-synuclein decreased only in Drosophila. In both models RP significantly reduced α-synuclein cytotoxicity, as was revealed by immunohistochemistry in Drosophila (p < 0.001, n = 27 flies per genotype/assay) and by flow cytometry in yeast (p < 0.05). Data obtained from the yeast PD model suggests that RP antitoxic effects are associated with a drop in ROS accumulation, and slower cellular transition from the early to late apoptotic stage. Using Drosophila brain tissue sections, we have demonstrated that RP helps to compensate for an α-synuclein-mediated reduction in the number of dopaminergic neurons and leads to better performance in animal climbing tests (p < 0.001, n = 120-150 flies per genotype/assay). Taken together, these results demonstrate the potential of RP for the treatment of PD, at least in model systems.

CHARACTERISTICS OF AMYLOID DEPOSITS DETECTED IN THE INTERNAL ORGANS OF mdx MICE.

The dystrophin-deficient mdx mouse is the most commonly used experimental model of Duchenne muscular dystrophy (DMD). Although the amyloid has been shown in the muscle biopsies of patients with different types of muscular dystrophies, there are no data on the amyloid accumulations in the biopsy of DMD patients or mdx mouse. Therefore, the aim of the present study was to testify the hypothesis of probable accumulation of amyloid in the visceral organs of mdx mouse. Specimens of myocardium, kidneys, and liver of male and female mdx mice aged from 2 months to 1.5 years (n = 9) were used in the study. The histochemical staining with Congo red demonstrated amyloid accumulations in the studied organs of the mdx mice. Morphology and localization of the found accumulations were described in details and analyzed. The mass-spectrometric study determined the vitronectin and apolipoprotein A-II as the most probable components of the amyloid accumulations in the mdx mouse.

[Endogenous erythropoietin, acute kidney injury, and prognosis in patients with acute coronary syndrome].

AIM: To investigate the prognostic value of serum endogenous erythropoietin (EPO) in patients with acute coronary syndrome (ACS), including that in the development of acute kidney injury (AKI). SUBJECTS AND METHODS: Eighty-four patients (46 men, 38 women; mean age 63 ± 11 years) with ACS were examined. Twenty-one (25%) patents were diagnosed with ECG ST-segment elevation acute myocardial infarction (STSEAMI), 12 (14%) had ECG non-STSEAMI, and 51 (61%) had unstable angina. Thrombolytic therapy was performed in 10 (48%) patients with STSEAMI. The patients whom had not undergone coronarography were included in the investigation to exclude the nephrotoxic effect of X-ray contrast agents. RESULTS: AKI was observed in 7 of the patients with acute myocardial infarction and in only 1 of those with unstable angina. Four (5%) patients died during hospitalization. The EPO level of > 10.5 IU/ml predicted the development of AKI in the ACS patients with a sensitivity of 71% and a specificity of 67%. That of > 13.7 IU/ml was associated with hospital death in the ACS patients with a sensitivity of 100% and a specificity of 75% (AUC = 0.93%). CONCLUSION: High serum EPO levels in an ACS patent during his hospital stay may serve as a biomarker for a high risk for AKI and high death rates.

[Problems in the diagnosis of acute kidney injury in patients with ST-segment elevation myocardial infarction].

AIM: To estimate the frequency and severity of acute kidney injury (AKI) in patients with ST-segment elevation acute myocardial infarction (STSEAMI), to specify whether the changes in diuresis and serum creatinine levels are equally sensitive diagnostic criteria for AKI, and to define their prognostic value. SUBJECTS AND METHODS: Three hundred and nineteen patients (249 (78%) men and 70 (22%) women; age 58 +/- 10 years) with STSEAMI who received thrombolytic therapy (TLT) were examined. The diagnosis of STSEAMI, indications for and contraindications to TLT, evaluation of its efficiency were made in accordance with the All-Russian Scientific Society of Cardiology guidelines (2007). AKI was diagnosed and classified using the KDIGO guidelines (2012). RESULTS: AKI was diagnosed in terms of diuresis, calculated creatinine levels, and creatinine level changes in 107 (34%), 73 (23%), and 68 (22%) patients, respectively. Among the patients with AKI diagnosed in view of diuresis, in-hospital death rates were higher than in those without AKI (chi2 = 25.46; p < 0.001); the similar pattern was seen in patients with AKI diagnosed in terms of calculated creatinine levels (chi2 = 3.99; p = 0.045). Logistic regression analysis indicated that regardless of gender, age, and time interval between onset of clinical manifestation and hospital admission, the in-hospital death rates were associated with the presence of AKI in view of diuresis (relative risk 14; 95% confidence interval, 4.03 to 52.08; p < 0.001). CONCLUSION: The STSEAMI patents receiving TLT exhibited a high rate of AKI. The major problem in the early detection of AKI is associated with difficulties in the differential diagnosis of AKI and chronic kidney disease. AKI diagnosed in view of diuresis is of greater prognostic value for in-hospital mortality than that diagnosed in terms of creatinine levels. The diagnosis of renal dysfunction in view of basal creatinine levels is prognostically important despite the fact that this cannot differentiate AKI from chronic kidney disease in the early stage.

[Pharmacological treatment of hemorheological disorders in children with acute respiratory viral infection].

This article presents the results of monitoring in a group of children with severe forms of acute respiratory viral infection and toxic encephalopathy. Hemorheological disorder in the form of hyperviscosity syndrome has been detected in all patients with severe forms of acute respiratory viral infection. It was suggested the inclusion of cytoflavin in the complex therapy for correcting the hemorheological status. The administration of cytoflavin led to reduction of erythrocyte aggregation and general improvements in rheological indices of children with severe forms of acute respiratory viral infection and toxic encephalopathy.

[Acute respiratory viral infection in children: clinical pattern, hemorheology disorders, methods of therapy].

Clinical and laboratory indices of intoxication and hemorheology disorders that arise during acute viral infection in children have been studied. It is established for the first time that reamberine is capable of reducing the plasma and blood viscosity and can be used in the program of infusion therapy of high blood viscosity syndrome in children with acute respiratory viral infections. One of the positive mechanisms of reamberin clinical performance is its positive impact on microcirculation and hemorheology. Thus, reamberin can be considered as a means of pathogenetic therapy in acute period of infectious toxicosis during acute respiratory viral infections in children.

[The effects of RH 421 on the activity of amphotericin B in cell and model membranes].

The combined action of polyene macrolide antibiotic amphotericin B and styryl dye RH 421 in model lipid membranes and in yeast Saccharomyces cerevisiae has been studied. RH 421 insertion inti the ergosterol-containing lipid bilayers led to the increase of the steady-state number of open amphotericin channels by 8.5 +/- 3.2 times. Paper disk agar diffusion method has demonstrated that RH 421 enhanced the antimicrobial effect of amphotericin B leading to an increase in the zone of growth inhibition of S. cerevisiae strain by 1.5 times. Our data suggest that RH 421 is a potential synergist of amphotericin B and can be used in new formulations with improved pharmacological properties.

[Acute kidney injury in patients with myocardial infarction and efficacy of thrombolytic therapy].

Aim of the study was to assess rate and severity of acute kidney injury (AKJ) (RIFLE and AKIN criteria) in patients with ST-elevation myocardial infarction (STEMI), to determine relationship between AKJ, mortality and effectiveness of thrombolytic therapy (TLT). We examined 146 patients (117 men, 29 women, mean age 56.7+/-10.8 ) with STEMI subjected to TLT with streptokinase. AKJ was diagnosed and classified according to RIFLE and AKIN criteria by creatinine (RIFLECr, AKINCr) and diuresis (RIFLEou, AKINou). TLT was effective in 104(71%) patients. AKJ was found in 74 (51%) according to RIFLECr and in 86 (59%) - AKINCr, in 51 (35%) - RIFLEou and AKINou criteria. Eight patients 8 (5%) died. Results of logistic regression analysis showed that AKJ according to RIFLEou or AKINou irrespective of sex, age, and time after appearance of symptoms to hospitalization, was associated with mortality (relative risk [RR] 12.9, 95% confidence interval [95%CI] 1.45-115,58, p=0.002). Thus more than 50% of STEMI patients have AKJ according to RIFLECr AKINCr criteria. Frequency of AKJ according to RIFLEou and AKINou was by 40% and one third less than that according to AKINCr and RIFLECr, respectively. Presence of AKJ was associated with TLT inefficacy and elevation of mortality.

[Hemorheological disorders in Epstein-Barr virus mononucleosis in children and methods of their correction].

The results of observations in a group of 80 young patients aged 1-14 years with diagnosis of primary Epstein-Barr virus (EBV) infection are presented. Hemorheological disorders arising from the EBV mononucleosis were identified. The results of using reamberin in the infusion therapy for the correction of hemorheological disorders have been analyzed. It is established that reamberine infusion reduces the plasma viscosity and blood viscosity, rapidly improves the condition of children, and restores hemodynamic indices. The use of reamberine helped to reduce the duration of hospital treatment for patients with severe EBV mononucleosis.

[Combined physiotherapy of patients with dorsopathies and concomitant osteoarthrosis].

The combined treatment of patients presenting with dorsopathies and concomitant osteoarthrosis using magnetic stimulation, ultrasonic and peloidotherapy was shown to promote the recovery of the locomotor function of the vertebral column and the affected limb joints, alleviate pain sensation, and exert anti-inflammatory effect. Moreover, it contributed to the improvement of the overall orthopedic situation in the region. The inclusion of vibrotraction in the combined treatment of patients presenting with dorsopathies and concomitant osteoarthrosis resulted in a marked decrease of the severity of static and dynamic disturbances of the spine, increase in the activity of spinal muscles, extension of the range of locomotor movements, and improvement of working ability of the patients.

[The effect of red pigment of Saccharomyces cerevisiae on insulin fibril formation in vitro].

The effect of the yeast red pigment, the result of polymerization of AIR, and of its low molecular weight derivative (presumably devoid of phosphoribosyl moiety) on the formation of amyloid fibrils in vitro was studied. Both the red pigment and its derivative, the result of acid hydrolysis of the original pigment, were shown to diminish the intensity of amyloid bound Thioflavine T fluorescence. Correlation between the decrease of the intensity of Thioflavine T fluorescence and the concentration of both forms of the red pigment was demonstrated. Both forms were also able to compete with Thioflavine T for amyloid fibrils. Electron microscopy permitted to visualize a drop of fibril size in the case of red pigments presence during their formation.

[Efficacy of reamberin in treating heavy acute intestinal infection in children].

The current health conditions dictate the need to reduce the time of patient treatment in hospital and require rational use of drugs. Reduction of the duration of intoxication syndrome in severe forms of intestinal infections in children depends on infusion therapy and choice of solutions for intravenous administration. Reamberin is generation IV infusion preparation for intensive care, representing a balanced isotonic detoxicant infusion solution based on succinic acid. Using reamberin ensures a significant decrease in the time of stay in hospital for children with severe forms of intestinal infections, which is achieved by reducing the duration of endogenous intoxication.

[Correction of antioxidant system disorders in children with acute intestinal infection].

Administration of reamberin leads to rapid reduction of the intoxication symptoms, improves general condition, and reduces fever stage duration. The dynamics of inflammatory symptoms is characterized by decreasing duration of hemocolitis in comparison that in patients receiving glucose-salt solutions. Reamberin accelerates normalization of the chemiluminescence indices, reduces the intensity of oxidative processes (to within 3-5 days) in patients with high level of free-radical oxidation, and leads to decreasing endogenous intoxication on early terms of the disease. Removal of the dysbalance between oxidation and antioxidant systems, especially in cases of serious shigellosis and in patients with high level of lipid peroxidation, favors a decrease in the damaging action of free radicals and eliminates metabolic disturbances in children within early reconvalesce period.