[The errors in intravital pathologicoanatomic diagnostic: a publications review].

The review presents analysis of scientific publications considering medical errors in intravital pathologicoanatomic diagnostic. The examples of classification, rate of diagnostic errors and possible ways of decreasing the number of diagnostic errors in pathological anatomy are considered.

Characterization of CoCas9 nuclease from Capnocytophaga ochracea.

Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from Capnocytophaga ochracea, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5'-NRRWC-3' PAM and efficiently cleaves DNA in vitro. Functional characterization of CoCas9 opens ways for genetic engineering of C. ochracea using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.

[Type II CRISPR-Cas System Nucleases: A Pipeline for Prediction and In Vitro Characterization].

The use of CRISPR-Cas bacterial adaptive immunity system components for targeted DNA changes has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editors are based on the Cas9 effectors of the type II CRISPR-Cas systems. In complex with guide RNAs, Cas9 proteins are able to directionally introduce double-stranded breaks into DNA regions that are complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an important task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search for and subsequent characterization of new Cas9 nucleases developed in our laboratory. Detailed protocols describing the bioinformatical search, cloning, and isolation of recombinant Cas9 proteins, testing for the presence of nuclease activity in vitro, and determining the PAM sequence, which is required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered.

[Post-traumatic stress disorder in the focus of international research: from soldier heart to ICD-11]. / Posttravmaticheskoe stressovoe rasstroistvo v tsentre mezhdunarodnykh issledovanii: ot «soldatskogo serdtsa¼ k MKB-11.

The article presents the evolution of the post-traumatic stress disorder (PTSD) definition from various war syndromes to PTSD definition as a sovereign disorder and complex PTSD appearance in ICD-11 as distinct diagnosis. It is focused on the epidemiology, gender issues, clinical features and differential diagnostic PTSD aspects. It is considered elaboration of the new international clinical guidelines for the anxiety disorders including PTSD, the new pharmaceutical and psychotherapeutic treatment algorithms for PTSD based on the evidence based research data is presented. Additionally as an illustration case vignette is described.

[Time perspective perception specific and maladaptation in elderly under uncertainty conditions during pandemic emergency.]

The purpose of the study - to determine the specifics of experiencing psychological stress, the time perspective and symptoms of adaptation disorders in older people in a pandemic emergency compared to the active adult population. At the first stage, 587 people were examined using the PSM-25 scale, then 100 respondents with maladaptive stress levels (sum ≥155 points) were selected. The main group consisted of 50 people aged 60 to 74 years (average age 65±2,7 years), 50 people aged 18 to 44 years (average age 32±3,8 years) were included in comparison group, who were examined with using the SCL-90-R questionnaire for assessing psychopathological symptoms and the ZTPI questionnaire for assessing the perception of time perspective. The presence of adaptation disorders in both groups was revealed. In the elderly group, the activation of the perception of the positive past was established, which can be an adaptive resource, and the greater severity of the fatalistic perception of the present, which causes passive overcoming with stress and narrows the adaptive capabilities of the elderly. The results of the study make it possible to identify potential «targets¼ of psychotherapeutic work with the population in conditions of pandemic stress, taking into account the age factor.

Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album.

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that - at a subsequent stage of transfection into yeast cells - it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein's secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 µg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

[Pandemic COVID-19 and mental disorders international experience and therapeutic approaches]. / Psikhicheskie narusheniya, svyazannye s pandemiei COVID-19 (mezhdunarodnyi opyt i podkhody k terapii).

The article considers the COVID-19 pandemic impact on the population mental health, the main psychogenic and somatogenic mental disorders associated with COVID-19 are distinguished. The comorbid PTSD clinical features are described. The mental health risk factors for health workers are presented. The psychotherapeutic and psychopharmaceutical approaches are proposed.

[The impact of the ones sexuality and autonomy awareness on the insomnia severity in patients with anxiety neurotic disorders]. / Vliyanie vospriyatiya sobstvennoi seksual'nosti i avtonomnosti na vyrazhennost' insomnii u patsientov s trevozhnymi rasstroistvami nevroticheskogo urovnya.

OBJECTIVE: To evaluate an impact of perception of ones sexuality and autonomy on the level of insomnia in patients with neurotic/anxiety disorders. MATERIAL AND METHODS: A sample included 123 (93 women and 30 men) patients with neurotic/anxiety disorders (F40, F41, F43, F45) in the age from 25 to 50 years old, anxiety was the main syndrome in the clinical structure of the disorder. Test battery included Pittsburgh Sleep Quality index for the subjective sleep quality evaluation, Insomnia Severity index for stratification of patients into groups with different insomnia level. Anxiety was evaluated with the Hamilton Anxiety Rating Scale. Personality traits, sexuality and sovereignty of psychological space were assessed with the G. Ammon Ego-structure test and the Sovereignty of the individual psychological space questionnaire. Multiple regression analysis was used to study the interrelationship between insomnia severity and psychological characteristics. Insomnia severity index was chosen as the dependent variable. RESULTS AND CONCLUSION: An analysis of the regression model has shown the increase in insomnia index with the simultaneous augmentation of the following scales «Physical body sovereignty¼, «Sovereignty of objects¼, «Destructive aggression¼, «Deficient aggression¼, «Constructive narcissism¼, and decrease of the scales «Territory sovereignty¼, «Deficient narcissism¼, «Constructive sexuality¼. The study results imply the importance of the use of the level of sexual functioning and autonomy as the marker of identity maturity for psychotherapeutic targets in the treatment of neurotic/anxiety disorders. The use of anxiety and insomnia as the main complaints can be explained by its societal approval and need further active evaluation by the specialist for the successful psychotherapeutic treatment.

[Pandemic and mental adjustment disorders. Therapy options]. / Pandemiya i adaptatsionnye trevozhnye rasstroistva: vozmozhnosti terapii.

The article considers the main triggers of adjustment and anxiety disorders in the time of pandemic that brings significant changes in all life dimensions with the high uncertainty level. The article highlights a role of coronavirus infodemic in the development of anxiety disorders. The main targets for cognitive, emotional and behavioral interventions are suggested. Recommendations for anxiety adjustment disorders psychopharmacotherapy with the use of tranquillizers as treatment of choice are given. Prevention measures for medical doctors working in conditions of pandemic are proposed.

[Comorbid posttraumatic stress disorder diagnostic improvement in oncology elderly patients after surgical treatment in relation to rehabilitation tasks.]

The cancer incidence increase in elderly patients determines the actuality of this study, because oncological illnesses are often accompanied by mental disturbances. This article considers clinical psychological risk factors of the posttraumatic stress disorder (PTSD) onset in prostate and rectal cancer elderly patients after surgical treatment. The study results showed that lack of social support, deficit of the successful stress coping experience in the past, maladaptive internal disease model, and destructive personality profile proved to be statistically significant risk factors and consequently psychotherapeutic interventions targets in PTSD treatment and prevention. Consideration of the elderly oncology patient clinical, psychological and social characteristics allows to elaborate effective personalized strategies of the complex medical-psychological care in geriatric medicine.

Assessing exterior egg quality indicators using machine vision.

1. The objective of this study was to develop a machine vision method for analysing exterior parameters of chicken eggs to automate the stage of primary sorting. 2. The developed algorithm based on predetermined thresholds calculated egg quality indicators, including geometric dimensions, shape index and the mottling grade. The algorithm was implemented with an experimental setup that combined the image-based and the candling methods. A total of 400 egg samples were analysed. 3. Comparison of results of the algorithm with those obtained using the traditional manual method showed that mean value of radii values difference was 0.095 ± 0.058 mm for the sharp and 0.080 ± 0.047 mm for the blunt end of the egg, with standard deviations of 0.58 mm and 0.49 mm, respectively. 4. The correlation coefficient between the shape index values determined by the two methods was 0.93; the standard deviation of absolute differences between corresponding values was 1.05%. 5. The results of mottling grade estimation were compared using F-measure and confusion matrix. 6. The results allow the possibility to perform the assessment of egg exterior quality factors in an automatic mode, independent of the expertise of a grader.

[A comparative study of the efficacy of personality-oriented (reconstructive) and cognitive-behavioral psychotherapy in neurotic anxiety disorders with insomnia]. / Sravnitel'noe issledovanie éffektivnosti lichnostno-orientirovannoi (rekonstruktivnoi) i kognitivno-povedencheskoi psikhoterapii pri trevozhnykh rasstroistvakh nevroticheskogo urovnia s insomniei.

AIM: To study personality-oriented (reconstructive) and cognitive-behavioral psychotherapy in patients with neurotic anxiety disorders with insomnia. MATERIAL AND METHODS: Ninety-eight patients with disorders coded in ICD-10 as F 40.0; F40.1; F41.0; F41.1; F48.0 were studied. Clinical, psychometric, experimental-psychological methods and polysomnography were used. RESULTS AND CONCLUSION: Phenomenological insomnia types characteristic of neurotic anxiety disorders and their relationship with types of intrapersonal conflicts are described. A quantitative analysis of the dynamics of symptomatic complains, anxiety level, and insomnia is presented. The efficacy of the both psychotherapeutic treatments is shown. However the personality oriented (reconstructive) psychotherapy has demonstrated better and more stable results. This can be explained first of all by its pathogenesis-oriented nature and involvement of the cognitive emotional and behavioral spheres as well as the resolution of the intrapersonal conflicts, which are the cornerstone of neurotic disorders.

[The use of spitomine (buspirone) in the combined therapy of panic disorder].

OBJECTIVE: To explore the efficacy of spitomine (buspirone) in the combined therapy of panic disorder, we studied the dynamics of clinical parameters and personality traits associated with development and maintenance of panic disorder during the treatment. MATERIAL AND METHODS: Thirty patients of a treatment setting for out-patients with panic disorder were enrolled in the study. Patients received combined treatment with spitomine (buspirone) in the dose of 30 mg/daily (10 mg three times a day) during 6 weeks and individual short-term direct cognitive-behavioral therapy. Anxiety and panic symptoms were measured with HARS, CGI, MADRS and STAI-A. RESULTS: There was a significant reduction in panic and anxiety symptoms (by 50% on the HARS) that reached on average 9.73 scores to the end of the study. This finding indicates the absence of anxiety and, therefore, therapeutic efficacy of the treatment. Scores on state anxiety of STAI-A were significantly lower compared to baseline though the level of anxiety remained relatively high. The partial decrease in trait anxiety, though its level was high as well, was noted. CONCLUSION: The combined therapy of panic disorder with spitomine (buspirone) and cognitive-behavioral therapy is an optimal treatment, though not without the limitations of short-term methods. Good tolerability of the drug allowed all patients to complete the study.

New-generation Skulachev ions exhibiting nephroprotective and neuroprotective properties.

A mitochondria-targeted chimeric compound consisting of a rhodamine derivative linked to a plastoquinone molecule (10-(6'-plastoquinonyl)decylrhodamine, SkQR1) was studied under conditions of acute brain or kidney damage. A protective effect of this compound was demonstrated in a model of focal brain ischemia, rat kidney ischemia/reperfusion, myoglobinuria (rhabdomyolysis, or crush syndrome), and pyelonephritis. We found that a single intraperitoneal injection of SkQR1 diminishes the size of the ischemic zone in the brain and improves performance of a test characterizing neurological deficit in ischemic animals. Control substance not containing plastoquinone appeared to be not neuroprotective. The data show that SkQR1 is a nephroprotectant and neuroprotectant, which can be due to the antioxidative action of this Skulachev cation.

Inhibition of GSK-3ß decreases the ischemia-induced death of renal cells.

Pharmacological preconditioning with insulin and lithium ions prevented the death of renal cells under conditions of ischemia/reperfusion. Preincubation of cells with insulin or lithium ions decreased production of reactive oxygen species after ischemia/reoxygenation. These agents also prevented the development of mitochondrial dysfunction in renal cells induced by ischemia/reoxygenation. It was hypothesized that the protective effects of these agents are related to inhibition of glycogen synthase kinase-3(. This enzyme is inactivated upon phosphorylation of serine residue in position 9. We found that in vivo administration of lithium ions to animals before renal ischemia prevents the development of kidney failure.

Interrelations of mitochondrial fragmentation and cell death under ischemia/reoxygenation and UV-irradiation: protective effects of SkQ1, lithium ions and insulin.

Mitochondria-targeted antioxidant 10-(6-plastoquinonyl)decyltriphenyl-phosphonium (SkQ1) as well as insulin and the inhibitor of glycogen-synthase kinase, Li(+) are shown to (i) protect renal tubular cells from an apoptotic death and (ii) diminish mitochondrial fission (the thread-grain transition) induced by ischemia/reoxygenation. However, SkQ1 and LiCl protected the mitochondrial reticulum of skin fibroblasts from ultraviolet-induced fission but were ineffective in preventing a further cell death. This means that mitochondrial fission is not essential for apoptotic cascade progression.

Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture.

The goals of the study were: (1) to explore the communication between human mesenchymal stem cells (MSC) and rat cardiac myocytes resulting in differentiation of the stem cells and, (2) to evaluate the role of mitochondria in it. Light and fluorescence microscopy as well as scanning electron microscopy revealed that after co-cultivation, cells formed intercellular contacts and transient exchange with cytosolic elements could be observed. The transport of cytosolic entity had no specific direction. Noticeably, mitochondria also could be transferred to the recipient cells in a unidirectional fashion (towards cardiomyocytes only). Transmission electron microscopy revealed significant variability in both the diameter of intercellular contacting tubes and their shape. Inside of these nanotubes mitochondria-resembling structures were identified. Moreover, after co-cultivation with cardiomyocytes, expression of human-specific myosin was revealed in MSC. Thus, we speculate that: (1) transport of intracellular elements to MSC possibly can determine the direction of their differentiation and, (2) mitochondria may be involved in the mechanism of the stem cell differentiation. It looks plausible that mitochondrial transfer to recipient cardiomyocytes may be involved in the mechanism of failed myocardium repair after stem cells transplantation.

The mitochondrion as janus bifrons.

The signaling function of mitochondria is considered with a special emphasis on their role in the regulation of redox status of the cell, possibly determining a number of pathologies including cancer and aging. The review summarizes the transport role of mitochondria in energy supply to all cellular compartments (mitochondria as an electric cable in the cell), the role of mitochondria in plastic metabolism of the cell including synthesis of heme, steroids, iron-sulfur clusters, and reactive oxygen and nitrogen species. Mitochondria also play an important role in the Ca(2+)-signaling and the regulation of apoptotic cell death. Knowledge of mechanisms responsible for apoptotic cell death is important for the strategy for prevention of unwanted degradation of postmitotic cells such as cardiomyocytes and neurons.

The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney.

Reoxygenation following ischemia causes tissue oxidative stress. We studied the role of oxidative stress caused by kidney ischemia/reperfusion (I/R) on the mitochondria of renal tissue slices. I/R caused the mitochondria to be swollen, fragmented, and have lower membrane potential. The mitochondria generated more reactive oxygen species (ROS) and nitric oxide (NO) in situ as measured by fluorescence of ROS- and NO-sensitive probes. Infusion of lithium ion, an inhibitor of glycogen kinase synthase-3, caused phosphorylation of its Ser-9 and restored the membrane potential and decreased ROS production of the mitochondrial fraction. Ischemic kidney and hypoxic rat preconditioning improved mitochondrial membrane potential and lowered ROS production caused by subsequent I/R similar to lithium ion infusion. Preconditioning normalized NO production in mitochondria as well. The drop in the mitochondrial membrane potential was prevented by NO synthase inhibition, demonstrating a strong contribution of NO to changes in mitochondrial energy metabolism during the I/R transition. Mitochondria in the I/R-stressed kidney contained less cytochrome c and more pro-apoptotic Bax, consistent with apoptotic degradation.

Transplantation or alternative surgical treatment of patients with ischemic dilative cardiomyopathy and aneurysmatic dilation of the left ventricular cavity.

BACKGROUND: Patients with terminal ischemic heart disease (IHD), severely depressed pump function with large LV dyskinesis with or without fibrosis do not benefit from revascularization alone; in time they are listed for transplantation. The long waiting list and lack of organ donors have imposed implementation of Direct Circular Repair (DCR) with total revascularization as an alternative. METHODS: DCR was performed on 17 patients with terminal IHD, after total revascularisation. The resected dyskinetic tissue was pathohistologicaly examined. Transesophageal ultrasound was performed pre- and early post-operatively and hemodynamic parameters measured invasively. RESULTS: Pathohistology showed that even in macroscopically viable myocardium where only dyskinesia without fibrosis persists, there are irreversible lesions on the ultrastructural level. Along with revascularization, with the application of DCR the LV spherical geometry was reconstructed with hemodynamic improvement. CONCLUSION: Total revascularisation with DCR offers an alternative to transplantation in patients with wide anterior wall dyskinesia with or without fibrosis due to terminal IHD, the most frequent group listed for transplantation.