Generation and analysis of mouse embryonic stem cells with knockout of the Mcph1 (microcephalin) gene.

Chromatin is not randomly distributed within the nucleus, but organized in a three-dimensional structure that plays a critical role in genome functions. Сohesin and condensins are conserved multi-subunit protein complexes that participate in mammalian genome organization by extruding chromatin loops. The fine temporal regulation of these complexes is facilitated by a number of other proteins, one of which is microcephalin (Mcph1). Mcph1 prevents condensin II from associating with chromatin through interphase. Loss of Mcph1 induces chromosome hypercondensation; it is not clear to what extent this reorganization affects gene expression. In this study, we generated several mouse embryonic stem cell (mESC) lines with knockout of the Mcph1 gene and analyzed their gene expression profile. Gene Ontology analyses of differentially expressed genes (DEGs) after Mcph1 knockout revealed gene categories related to general metabolism and olfactory receptor function but not to cell cycle control previously described for Mcph1. We did not find a correlation between the DEGs and their frequency of lamina association. Thus, this evidence questions the hypothesis that Mcph1 knockout-mediated chromatin reorganization governs gene expression in mESCs. Among the negative effects of Mcph1 knockout, we observed numerous chromosomal aberrations, including micronucleus formation and chromosome fusion. This confirms the role of Mcph1 in maintaining genome integrity described previously. In our opinion, dysfunction of Mcph1 may be a kind of "Rosetta stone" for deciphering the function of condensin II in the interphase nucleus. Thus, the cell lines with knocked-out Mcph1 can be used to further study the influence of chromatin structural proteins on gene expression.

Antiplatelet and Antithrombotic Properties of Compound L-36, a 6H-1,3,4-Thiadiazine Derivative.

Compound L-36, a new derivative of 6H-1,3,4-thiadiazine, was studied in in vitro and in vivo experiments. This compound exhibits high antiplatelet and antithrombogenic activity. In in vitro experiments, compound L-36 by its antiplatelet activity (by IC50) was superior to acetylsalicylic acid by 9.4 times. In in vivo experiments, compound L-36 by its ED50 value was close to the comparison drug. On the model of pulmonary artery thrombosis, compound L-36 ensured better survival of experimental animals than acetylsalicylic acid. Morphological studies showed that compound L-36 effectively attenuated the thrombosis processes in the pulmonary tissue induced by intravenous injection of a thrombogenic mixture (epinephrine and collagen).

Evaluation of the Possibility of Axial Psoriatic Arthritis Patients Meeting Classification Criteria for Axial Spondyloarthritis and Ankylosing Spondylitis.

The objective of the study was to analyze whether axial psoriatic arthritis (axPsA) patients meet classification criteria for axial spondyloarthritis (axSpA) and ankylosing spondylitis (AS). A total of 104 patients (66 men and 38 women) with PsA according to CASPAR criteria were examined, all patients had back pain. Patients were evaluated for presence of inflammatory back pain (IBP) by ASAS criteria. Back pain not meeting the ASAS criteria was taken to be chronic back pain (chrBP). Patients underwent hands, feet and pelvis, cervical spine and lumbar spine X-rays. Erosions, osteolysis, and juxta-articular new bone formation were evaluated. Definite radiographic sacroiliitis (d-rSI) was defined as bilateral grade ≥ 2 or unilateral grade ≥ 3. Nineteen patients without d-rSI underwent sacroiliac joints MRI. Ninety-three patients underwent HLA B27 examination. The number of patients who met the criteria for axSpA (ASAS) and the modified New York (mNY) criteria for AS was determined. IBP was identified in 67 (64.4%) patients; chrBP, in 37 (35.6%) patients; 31 (29.8%) patient were of older age (over 40) at the onset of IBP/chrBP; 57 (58.8%) patients had d-rSI; 6 (31.6%) patients had MRI-SI; syndesmophytes were detected in 57 (58.8%) cases. Among 40 patients without d-rSI, 19 (47.5%) had syndesmophytes. In 38/97 (39.2%) patients d-rSI was detected along with syndesmophytes, while 19/97 (19.6%) patients had isolated d-rSI without spondylitis, and 19/97 (19.6%) patients had isolated syndesmophytes without d-rSI. HLA B27 was present in 28 (30.1%) cases. 51 (55.4%) patients met criteria for axSpA. Forty-one (44.6%) patients did not meet criteria for axSpA; however, 27 (65.9%) of them had syndesmophytes. Forty-eight (48.5%) PsA patients met mNY criteria for AS. Among these patients, a set of specific features was revealed: 18 (37.5%) had no IBP, 18 (37.5%) were of older age (over 40) at the onset of IBP/chrBP, 34 (70.8%) had dactylitis, 38 (79.2%) had erosive polyarthritis, 23 (48.8%) had juxta-articular new bone formation, 14 (30.2%) had osteolysis, 23 (48.9%) had "chunky" non-marginal syndesmophytes, and 40 (82.6%) had nail psoriasis; 28 (66.6%) patients were HLA-B27 negative. Forty-five percent of axPsA patients do not meet criteria for axSpA. Characteristic features have been identified to differentiate axPsA from AS.

[Fatal case of rhinocerebral mucormycosis on the background of type II diabetes mellitus]. / Letal'nyi sluchai rinotserebral'nogo mukormikoza na fone sakharnogo diabeta 2-go tipa.

Mucormycosis is a disease caused by fungi of the Mucorales family, widespread in the environment, with pronounced angiotropism and the ability to angioinvasion, leading to thrombosis with surrounding necrosis. The main triggers for the development of mucormycosis are: immunodeficiency states, use of glucocorticosteroid drugs, decompensation of diabetes mellitus, concomitant diseases, age > 65 years. We present a clinical case of rhinocerebral mucormycosis in a 79-year-old patient against the background of uncontrolled type 2 diabetes mellitus with ketoacidosis, a condition after previous glucocorticosteroid therapy for COVID-19 (according to the severity of the disease). After suffering a new coronavirus infection caused by the SARS-CoV-2 virus, she was admitted to the hospital with complaints characteristic of mucormycosis. On the 5th day of hospital stay, the patient's condition worsened significantly, despite the correction of the therapy, and on the 12th day the patient died. According to the results of the autopsy, it was established that the rhinocerebral mucormycosis was complicated by thrombosis of the anterior and posterior left cerebral arteries with subsequent infarctions in the frontal lobe and parieto-occipital region of the brain left hemisphere, cerebral edema, which was the immediate cause of death.

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.

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.

[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.

[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.