Assessing cell lines with inducible depletion of cohesin and condensins components through analysis of metaphase chromosome morphology.

One of the most productive strategies for finding the functions of proteins is to study the consequences of loss of protein function. For this purpose, cells or organisms with a knockout of the gene encoding the protein of interest are obtained. However, many proteins perform important functions and cells or organisms could suddenly lose fitness when the function of a protein is lost. For such proteins, the most productive strategy is to use inducible protein degradation systems. A system of auxin-dependent protein degradation is often implemented. To use this system, it is sufficient to introduce a transgene encoding a plant-derived auxin-dependent ubiquitin ligase into mammalian cells and insert a sequence encoding a degron domain into the gene of interest. A crucial aspect of development of cell lines engineered for inducible protein depletion is the selection of cell clones with efficient auxin-dependent degradation of the protein of interest. To select clones induced by depletion of the architectural chromatin proteins RAD21 (a component of the cohesin complex) and SMC2 (a component of the condensin complex), we propose to use the morphology of metaphase chromosomes as a convenient functional test. In this work, we obtained a series of clones of human HAP1 cells carrying the necessary genetic constructs for inducible depletion of RAD21 and SMC2. The degradation efficiency of the protein of interest was assessed by flow cytometry, Western blotting and metaphase chromosome morphology test. Based on our tests, we showed that the clones we established with the SMC2 degron effectively and completely lose protein function when induced by auxin. However, none of the HAP1 clones we created with the RAD21 degron showed complete loss of RAD21 function upon induction of degradation by auxin. In addition, some clones showed evidence of loss of RAD21 function even in the absence of induction. The chromosome morphology test turned out to be a convenient and informative method for clone selection. The results of this test are in good agreement with flow cytometry analysis and Western blotting data.

Design and testing of a high perveance sheet beam electron gun.

This paper presents the design, prototype engineering, and initial testing of a relatively low-voltage, high aspect ratio electron gun for versatile pulsed power applications. The sheet beam, diode electron gun is designed for (23-25) kV voltage and 6 µA/V1.5, as is required for x-band klystron, as a part of a compact Klylac delivering electron beam energy in the MeV range. The gun prototype was engineered, built, and tested using a Scandinova M1-0.5 modulator. Beam loss on the anode aperture is evaluated from transient waveforms and equivalent circuit modeling.

Growth Induction of Solid Ehrlich Ascitic Carcinoma in Mice after Proton Irradiation of Tumor Cells Ex Vivo.

This study presents data on the growth rate and frequency of induction of the solid form of Ehrlich's ascites carcinoma (EAC) in mice in the short and long term after inoculation of ascitic cells irradiated ex vivo with a proton beam in the dose range of 30-150 Gy. It was shown that the growth rate of solid tumors after inoculation of irradiated cells ex vivo coincided with the growth of tumors in the control group. The frequency of tumor induction in mice after inoculation of EAC cells irradiated at a dose of 30 Gy was 80%, 60 Gy-60%, 90 Gy-25%, and 120 Gy-10%; at irradiation at a dose of 150 Gy, no tumors appeared during the entire observation period. Thus, we determined the dose of proton radiation required to eliminate tumor cells and/or signaling factors that can lead to the induction of tumor growth of EAC in mice.

[Treatment of ventral hernias in patients with morbid obesity]. / Taktika lecheniya ventral'nykh gryzh u patsientov s morbidnym ozhireniem.

The review is devoted to the treatment of ventral hernias in patients with morbid obesity. This issue is important due to significant number of such patients and no unambiguous clinical recommendations. The advantages of simultaneous surgery (with bariatric intervention) are obvious, i.e. lower risk of postoperative hernia incarceration and no need for re-hospitalization with another intervention. High risk of bariatric population makes it necessary to minimize surgery time and surgical trauma. A staged approach with reducing body weight surgically or conservatively before hernia repair is often chosen. Hernia repair should be performed using laparoscopic or robotic techniques with obligatory use of mesh implants. Panniculectomy or abdominoplasty as the main surgery is a valid option. Currently, it is necessary to develop clear criteria for selecting patients with morbid obesity for staged and simultaneous treatment of ventral hernias.

Universal Maximum Strength of Solid Metals and Alloys.

Interstitial electron density ρ_{o} is offered as a direct metric for maximum strength in metals, arising from universal properties derived from an electron gas. ρ_{o} sets the exchange-correlation parameter r_{s} in density-functional theory. It holds also for maximum shear strength τ_{max} in polycrystals [M. Chandross and N. Argibay, Phys. Rev. Lett. 124, 125501 (2020)PRLTAO0031-900710.1103/PhysRevLett.124.125501]. Elastic moduli and τ_{max} for polycrystalline (amorphous) metals are linear with ρ_{o} and melting T_{m} (glass-transition T_{g}) temperature. ρ_{o} or r_{s}, even with rule-of-mixture estimate, predicts relative strength for rapid, reliable selection of high-strength alloys with ductility, as confirmed for elements to steels to complex solid solutions, and validated experimentally.

[Models and sculptural casts in forensic medicine and medical criminology]. / Mulyazhi i skul'pturnye slepki v sudebnoi meditsine i meditsinskoi kriminalistike.

The study objective is to present facts from the history of casting art and biographical information about the casting artists S.P. Fiveysky and M.A. Kurbatov, the founders of the casting craft in Russia. The attributes of the technology for making death masks, their features, advantages, and disadvantages as objects that capture appearance are described. The role of models and death masks in forensic practice, forensic science, and pedagogical activity is addressed.

[COVID-19-associated mucormycosis in ophthalmological practice in the Volgograd region]. / COVID-19-assotsiirovannyi mukormikoz v oftal'mologicheskoi praktike Volgogradskogo regiona.

The problem of fungal infections in the era of COVID-19 has acquired special significance. This infection, directly or indirectly, through the use of glucocorticoids and antibiotics in its treatment, as well as poorer self-management of chronic diseases, has led to a wide spread of risk factors for fungal diseases among people who have had a novel coronavirus infection. The article presents two cases of COVID-19-associated mycosis, more related to mucormycosis, which were diagnosed by ophthalmologists in the Volgograd region. In the first case, the severe course of rhino-orbito-cerebral form of mucormycosis required a number of surgical interventions and prolonged treatment in the intensive care unit. In the second case, the patient asked for help without signs of aggravation of the general condition, but with irreversible local manifestations. In both cases, the eyeball was removed. Morphological examination revealed aseptal ribbon hyphae of different diameters, branching mainly at right angles, more typical for fungi of the Mucorales family. Due to the severe consequences of the disease, clinicians, including ophthalmologists, need to be especially alertness in patients with the described symptoms and risk factors in the post-COVID period.

Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration.

Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), ß-casein (Csn2), whey acidic protein (WAP), or ß-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a "precise" integration approach, placing the human granulocyte-macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2-11 µg/ml hGMCSF). This was probably caused by the absence of the 3'-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30-40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2-0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals' health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.

Development of a two-channel system for monitoring the mirror elements of the Millimetron space observatory.

The paper presents the results of the development of an onboard two-stage telescope monitoring (control) system of the Millimetron space observatory. A 3D laser scanner is used for the preliminary inspection of the reflector elements, and an image analyzer based on the Foucault knife method is used for the final one. The results of the calculation and modeling of the monitoring system for tuning the telescope elements into space are presented. The signals formed in the system are calculated, including using data on the actual shape of the manufactured mirror elements. Estimates of the expected measurement accuracy are given, and the test results of the developed system are presented.

Generation of two iPSC lines from healthy donor with a heterozygous mutation in the VPS13B gene.

The human induced pluripotent stem cell (iPSC) lines, iCS-MAF1-1 and iCS-MAF1-11, were generated from fibroblasts. The donor has a heterozygous mutation in the VPS13B gene, which manifests in her child as Cohen syndrome. It is a Golgi pathology, characterized by postnatal microcephaly and delayed growth and mental development. However, the process underlying pathological changes leading to the onset of the disease is still unknown. The use of iPSC will allow describing the early stages of neurogenesis, which is undoubtedly relevant for identifying key stages of development, at which phenotypic manifestations of mutations in the VPS13B gene are found.

Radioprotective Effect of Nanocerium by Irradiation of Mice with Carbon Ions in Medium and Lethal Doses.

The data of the study of the radioprotective properties of nanocerium (nCeO2) after total irradiation of mice with carbon ions in medium and lethal doses according to the micronucleus test and the criterion of 30-day survival are presented. A significant protective effect of nCeO2 upon irradiation at medium doses was observed at per os administration for 5 days before irradiation (that is, at long-term prophylactic use). Mouse survival data showed no protective effect of per os administration of nCeO2 in contrast to the micronucleus test results. After injections of both nCeO2 and saline solution 24 h before or immediately after irradiation, the radioprotective effect was detected using both methods. The data obtained revealed the dependence of the observed effects on the mode and time of nCeO2 administration, the influence of the solvent, the level of doses and the quality of radiation, as well as demonstrated the possibility of using nanocerium preparations to protect organisms from radiation with high LET values and the importance of further studies of the radioprotective properties of new nanomaterials.

[Abdominal pseudohernia: diagnosis and treatment]. / Abdominal'nye psevdogryzhi: diagnostika i lechenie.

OBJECTIVE: To present an effective treatment strategy for abdominal pseudohernia considering own clinical experience and literature data. MATERIAL AND METHODS: The authors report surgical treatment of 2 patients and therapeutic management of 1 patient with abdominal pseudohernia. Moreover, current state of this problem is analyzed according to literature data in Pubmed, Google and e-library databases. RESULTS. P: Seudohernia is followed by asymmetrical protrusion of abdominal wall, prolapsed site is not involved in breathing; patients cannot tense muscles in the affected area. T8-L2 skin sensitivity impairment or hyperesthesia may be observed. Denervation is confirmed by electroneuromyography. Ultrasound, CT and MRI reveal no defect in abdominal wall. However, its thinning within denervation area is visualized. Treatment is determined by the cause and reversibility of denervation. If mechanical damage (intersection) of T8-L2 nerves following trauma or any manipulation is absent, the main cause is herpes zoster neuropathy. Surgical treatment is not indicated. Antiherpetic therapy and physical therapy result functional restoration of abdominal wall muscles within 2-4 months. In case of mechanical cause of pseudohernia, it is advisable to wait at least a year after denervation for real assessment of situation. Partial or complete re-innervation is possible throughout this period. Surgical treatment is indicated if re-innervation is absent. An effective technique presented in this manuscript is reconstruction of the prolapsed area with several imbrication sutures in several planes up to symmetry with contralateral area with subsequent reinforcement with a mesh implant if necessary. There were no relapses in 2 our patients within 5 and 7 years, respectively. CONCLUSION: In our opinion, this report is the first manuscript devoted to systematic analysis of diagnosis and treatment of abdominal pseudohernia in available literature. Surgical treatment is not indicated for postherpetic pseudohernia. It is carried out for pseudohernia persisting for more than 1 year if its cause is trauma or any manipulation.

A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection.

Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes' expression are located in the promoter and 3'-untranslated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1-10 bp), but we detected several larger deletions as well (100-300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40-80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression.

[Features of pathological anatomy of COVID-19 according to autopsy results in the Volgograd Region]. / Osobennosti patologicheskoi anatomii COVID-19 po rezul'tatam autopsii v Volgogradskoi oblasti.

In connection with the ongoing pandemic of the novel coronavirus infection, the study of its morphopathology and the analysis of autopsy data are relevant. At the same time, attention should be paid to thromboses that play a significant role in the development of fatal outcomes in COVID-19, even taking into account the prescription of anticoagulant therapy to most patients. OBJECTIVE: To make an assessment of morphological changes and a statistical analysis of the structure of mortality in COVID-19 on the basis of autopsy results in the Volgograd Region in 2020. MATERIAL AND METHODS: The study was based on data from «The system for information on the work of the Volgograd Regional Autopsy Bureau¼ with a search for cases according to U07.1 code (the COVID-19 virus was identified) in January 1, 2020, to December 31, 2020, as well as on the autopsy materials of the Volgograd Regional Autopsy Bureau, and microscopic examination with photo fixation. Statistical processing was performed using the R programming language. RESULTS: During the above period, 1119 deaths were identified with a confirmed diagnosis of COVID-19. Anatomopathological examination of the autopsy material showed that 77.54% of cases had blood clots mainly in the vessels of the pulmonary microvasculature, often only in the single veins during the applied anticoagulant therapy.Analysis of variance indicated that the obtained result statistically significantly differed from the random distribution, and the probability of the presence of blood clots of specified localization was 3.17 times higher (CI 2.3-4.4; p<0.05) than their absence, as evidenced by logistic regression. In addition, perivascular and intra-alveolar diapedesis hemorrhages were noted in most fatal cases. CONCLUSION: Thus, this investigation has revealed that the high frequency of thrombosis detected in the presence of perivascular and intra-alveolar diapedesis hemorrhages in COVID-19 confirms the tendency of patients with a severe course of the disease to manifest hemostatic disorders, significant blood vascular endothelial injury, and obvious vascular impermeability.

[The social demographic aspects of vaccination of population of Russia in the context of COVID-19 pandemic].

The pandemic of the new corona-virus infection COVID-19 will be over only when population immunity will be developed. This condition can be achieved by both vaccination and surviving disease in natural way. The WHO recommends the first mode for achieving population immunity, since immune response to vaccine is similar to that one developed while surviving natural infection, but with minimal risks to health and life. The article presents the results of sociological survey organized with purpose of studying the attitude of Russians to vaccination against COVID-19 infection. The results of the survey demonstrated that at present, 22% of respondents are ready to be vaccinated i.e. they responded positively to the corresponding question. The following main factors determining population attitude to vaccination were identified. First, there is statistically significant correlation between vaccination readiness and education level: respondents with higher level of education are more likely to be vaccinated against COVID-19. Secondly, in general, the population is quite positive about the response of the National health care to the corona-virus pandemic. However, the percentage of population that is positive about vaccination can be higher if it would be possible to apply foreign-made vaccines in Russia. Thirdly, still there is large percentage of population believing that vaccination with medications that have not passed long-term clinical trials can result in health negative consequences. Fourthly, the majority of respondents experienced various manifestations of the pandemic on themselves: either they have been ill themselves, or their relatives and friends were ill or died and almost all of them somehow limited their common mobility.The survey results did not established statistically significant differences in distribution of responses among males and females. We assume that in order to develop positive attitude of population to vaccination, and specifically to Russian-made vaccines, more extensive information campaign is needed to be implemented. Also, significant increase of percentage of vaccinated population can be achieved if clearly defined requirements for availability of COVID-19 vaccination would be adopted on the international level. Actually, this is key condition for restoring international mobility and removing restrictions in national economies.

[Severe spine lesion following alkaptonuria. Case report]. / Tyazheloe porazhenie pozvonochnika pri alkaptonurii. Opisanie klinicheskogo sluchaya.

Alkaptonuria is a rare autosomal recessive disease. In these patients, melanin-like compounds as the final products of impaired metabolism of homogentisic acid are deposited mainly in connective tissue, including cartilage tissue of intervertebral discs. Similar to other degenerative spine diseases, lumbar segment is often damaged. The authors report a 67-year-old patient with alkaptonuria. Compression of cauda equina by damaged cartilage masses of intervertebral discs and spine ligaments with deposits of ochronotic pigment is described. Previously diagnosed alkaptonuria in this patient was confirmed by surgical findings (black pigmentation) and histological data.

[Urinary miRNA expression in cynomolgus macaques (Macaca fascicularis) fed high salt rations].

High food intake of sodium chloride is associated with damage not only the cardiovascular system, but also the kidneys. The mechanisms of the potential negative effects of high-salt diets on the kidneys have not been established. The aim of the study was to trace the changes in relative expression of miRNA-21, 203 and 133 in urine of cynomolgus macaques (Macaca fascicularis) fed high-salt diet with and without isolated soy proteins. Material and methods. The object of the study was 18 male cynomolgus macaques (Macaca fascicularis) aged 6-8 years with a body weight of 5.1-9.7 kg. The animals were divided in 3 groups (6 individuals each). The animals of the first (control) group received a standard diet (2 g NaCl/kg feed). The animals of the second group were fed high-salt diet (8 g NaCl/kg feed), of the third - high-salt diet combined with SUPRO 760 isolated soy protein (200 g/kg feed; instead of milk and egg proteins, corn gluten). Access to water was free. The follow-up period in this study was 4 months. In animals blood pressure (BP) and relative level of microRNA (miRNA) expression in urine were measured. Results and discussion. Keeping monkeys on the studied diets for 4 months did not lead to significant changes in systolic or diastolic BP compared with the initial level. In the control group, there were no distinct changes in the expression of miRNA-21 in urine during observation. In the other two groups, there was a significant increase (approximately equally) of this parameter in comparison with the initial values. Both high-salt diets resulted in a significant increase in the relative level of expression of miRNA-133 and miRNA-203 in urine compared to basal values. However, the increase in these parameters in the group of animals fed a high-salt diet in combination with soy isolate was significantly less than in monkeys fed only a high-salt diet. Conclusion. Possible, potentially negative effects of high-salt diets on kidney may be mediated by epigenomic mechanisms and partially modulated by the inclusion of isolated soy proteins in the diet.

[The preconditions of occurrence and social demographic consequences of pandemics].

The article considers the historical features of development and theoretical approaches to defining the concept of "pandemic" in social sciences. The approaches of demographic science to the study of the consequences of pandemics are outlined. The prerequisites of occurrence of major pandemics at various historical stages of humankind development are highlighted. The particular attention is paid to pandemics in the XXI century. The demographic and social economic consequences of pandemics for the countries all over the world, including the countries on post-Soviet territory, are considered. The characteristics of the COVID-19 pandemic are highlighted from the standpoint of social demographic consequences, including the impact on fertility and mortality. The key directions of pandemics control in some countries of the world are designated.

[Cancer of terminal part of common bile duct in a patient with heterotaxy syndrome]. / Rak terminal'nogo otdela obshchego zhelchnogo protoka u patsienta s sindromom geterotaksii.

Case report is devoted to successful pancreatectomy for cancer of terminal part of common bile duct in a patient with heterotaxy syndrome. The main difficulties during dissection of pancreaticoduodenal complex arose due to anatomical disorientation and the lack of standard topographic and anatomical landmarks. Preoperative computed tomography with assessment of visceral vessel anatomy is essential in all patients with biliopancreaticoduodenal tumors. If heterotaxy syndrome is suspected, additional examination is required to detect other potential abnormalities and prepare for unusual situation.