Inside and outside of virus-like particles HBc and HBc/4M2e: A comprehensive study of the structure.

Hepatitis B virus core antigen (HBc) with the insertion of four external domains of the influenza A M2 protein (HBc/4M2e) form virus-like particles whose structure was studied using a combination of molecular modeling and cryo-electron microscopy (cryo-EM). It was also shown that self-assembling of the particles occurs inside bacterial cells, but despite the big inner volume of the core shell particle, purified HBc/4M2e contain an insignificant amount of bacterial proteins. It was shown that a fragment of the M2e corresponding to 4M2e insertion is prone to formation of amyloid-like fibrils. However, as the part of the immunodominant loop, M2e insertion does not show a tendency to intermolecular interaction. A full-atomic HBc-4M2e model with the resolution of about 3 Å (3.13 Å for particles of Т = 4 symmetry, 3.7 Å for particles of Т = 3 symmetry) was obtained by molecular modeling methods based on cryo-EM data.

Old dog, new tricks: Influenza A virus NS1 and in vitro fibrillogenesis.

The influenza NS1 protein is involved in suppression of the host immune response. Recently, there is growing evidence that prion-like protein aggregation plays an important role in cellular signaling and immune responses. In this work, we obtained a recombinant, influenza A NS1 protein and showed that it is able to form amyloid-like fibrils in vitro. Using proteolysis and subsequent mass spectrometry, we showed that regions resistant to protease hydrolysis highly differ between the native NS1 form (NS1-N) and fibrillar form (NS1-F); this indicates that significant structural changes occur during fibril formation. We also found a protein fragment that is capable of inducing the process of fibrillogenesis at 37 °C. The discovery of the ability of NS1 to form amyloid-like fibrils may be relevant to uncovering relationships between influenza A infection and modulation of the immune response.

Cold and distant: structural features of the nucleoprotein complex of a cold-adapted influenza A virus strain.

Two influenza A nucleoprotein variants (wild-type: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was distance between nucleoprotein complex strands. The isolated complexes of two ribonucleoprotein variants were characterized by transmission electron microscopy and differential scanning fluorimetry (DSF). Presence of the E292G substitution was shown by DSF to affect nucleoprotein complex melting temperature. In the filament interface peptide model, it was shown that the peptide corresponding in primary structure to the wild-type NP (SGYDFEREGYS) is prone to temperature-dependent self-association, unlike the peptide corresponding to E292G substitution (SGYDFGREGYS). It was also shown that the SGYDFEREGYS peptide is capable of interacting with a monomeric nucleoprotein (wild type); this interaction's equilibrium dissociation constant is five orders of magnitude lower than for the SGYDFGREGYS peptide. Using small-angle neutron scattering (SANS), the supramolecular structures of isolated complexes of these proteins were studied at temperatures of 15, 32, and 37 °C. SANS data show that the structures of the studied complexes at elevated temperature differ from the rod-like particle model and react differently to temperature changes. The data suggest that the mechanism behind cold adaptation with E292G is associated with a weakening of the interaction between strands of the ribonucleoprotein complex and, as a result, the appearance of inter-chain interface flexibility necessary for complex function at low temperature.Communicated by Ramaswamy H. Sarma.

Ultrastructural Changes in the Air-Blood Barrier of Rats in Acute Intoxication with Furoplast Pyrolysis Products.

Rats were exposed to fluoroplast-4 pyrolysis products (sample weight 2.6 g, pyrolysis temperature 440-750°C, pyrolysis duration 4 min) containing perfluoroisobutylene over 15 min. Lung tissue samples for histological and electron microscopic examination were isolated in 3 and 30 min after intoxication and processed routinely. Histological examination revealed no structural changes in the lungs. In ultrathin sections of rat lungs, some changes in the structure of type I pneumocytes were detected in 3 min after the exposure: detachment of cytoplasmic processes and the appearance of transcytosis pores. These changes attested to impaired cell-cell interactions and their adhesion to the basement membrane, where structural disorganization and edema of the collagen matrix were observed. In 30 min following exposure, the signs of damage to type I pneumocytes became more pronounced. The increase in the equivalents of transcellular and paracellular permeability in the alveolar lining profile was observed. No changes in the pulmonary capillary endotheliocytes were detected, which suggest that type I pneumocytes are the primary target of the toxic effect of perfluoroisobutylene. The vulnerability of a particular cell population, in view of specific metabolism of these cells, can be the key to deciphering of the mechanisms of the toxic effect of pyrolysis products of fluorinated polymer materials.

[Pneumocephaly: an expert assessment in determining the severity of harm to health]. / Pnevmotsefaliya: ekspertnaya otsenka opredeleniya tyazhesti vreda zdorov'yu.

The purpose of the work is the analysis of etiology and pathogenesis of pneumocephaly in various injuries, diseases and pathological conditions for the reasonable determination of the severity of health damage during forensic medical examinations. The analysis of literature data on the causes and mechanisms of pneumocephaly is given. Attention is paid to the assessment of pneumocephaly in determining the health damage in patients with traumatic brain injury or if it is suspected. It is proposed to consider pneumocephaly as an indirect sign of a skull fracture. In case of pneumocephaly, a radiologically confirmed fracture is necessary to justify serious health damage. The medical documents of the victim should be examined to exclude concomitant diseases and pathological conditions that can lead to pneumocephaly.

COMPARISON OF INFLUENZA A VIRUS INHIBITION IN VITRO BY SIRNA COMPLEXES WITH CHITOSAN DERIVATIVES, POLYETHYLENEIMINE AND HYBRID POLYARGININE-INORGANIC MICROCAPSULES.

Anti-influenza drugs and vaccines have a limited effect due to the high mutation rate of virus genome. The direct impact on the conservative virus genome regions should significantly improve therapeutic effectiveness. The RNA interference mechanism (RNAi) is one of the modern approaches used to solve this problem. In this work, we have investigated the antiviral activity of small interfering RNA (siRNA) against the influenza A/PR/8/34 (H1N1), targeting conserved regions of NP and PA. Polycations were used for intracellular siRNA delivery: chitosan's derivatives (methylglycol and quaternized chitosan), polyethyleneimine, lipofectamine, and hybrid organic/non-organic microcapsules. A comparative study of these delivery systems with fluorescent labeled siRNA was conducted. The antiviral activity of three small interfering RNAs targeting the NP (NP-717, NP-1496) and PA (PA-1630) influenza A viruses genes was demonstrated, depending on the chosen carrier. The most effective intracellular delivery and antiviral activity were observed for hybrid microcapsules.

[Effects of the of renal warm ischemia time on the recovery of filtration function in the experiment].

AIM: To investigate experimentally ultrastructural and biochemical signs of acute injury to the renal parenchyma after warm renal ischemia of various duration and subsequent reperfusion. MATERIALS AND METHODS: The experiments were performed on 44 healthy conventional female rabbits of the "Chinchilla" breed weighted 2.6-2.7 kg, which were divided into four groups. In the first, control, group included pseudo-operated animals. In the remaining three groups, an experimental model of warm ischemia of renal tissue was created, followed by a 60-minute reperfusion. The renal warm ischemia time was 30, 60 and 90 minutes in the 2nd, 3rd and 4th groups, respectively. Electron microscopy was used to study ultrastructural disturbances of the renal parenchyma. Biochemical signs of acute kidney damage were detected by measuring the following blood serum and/or urine analytes: NGAL, cystatin C, KIM-1, L-FABP, interleukin-18. The glomerular filtration was evaluated by creatinine clearance, which was determined on days 1, 5, 7, 14, 21 and 35 of follow-up. RESULTS: A 30-minute renal warm ischemia followed by a 60-minute reperfusion induced swelling and edema of the brush membrane, vacuolation of the cytoplasm of the endothelial cells of the proximal tubules, and microvilli restructuring. The observed disorders were reversible, and the epithelial cells retained their viability. After 60 minutes of ischemia and 60 minutes of reperfusion, the observed changes in the ultrastructure of the epithelial cells were much more pronounced, some of the epithelial cells were in a state of apoptosis. 90 min of ischemia and 60 min of reperfusion resulted in electron-microscopic signs of the mass cellular death of the tubular epithelium. Concentration in serum and/or biochemical urine markers of acute renal damage increased sharply after ischemic-reperfusion injury. Restoration of indicators was observed only in cases when the renal warm ischemia time did not exceed 60 minutes. The decrease in creatinine clearance occurred in the first 24 hours after the intervention, lasting not less than two weeks after a 30-minute warm ischemia, at least 3 weeks after a 60-minute warm ischemia and continued more than a month after a 90-minute renal artery occlusion. CONCLUSION: Intraoperative warm ischemia and subsequent reperfusion are the actual reasons for the alteration of the ultrastructure of the renal tissue and the impairment of the filtration function. The severity of the disorders depends on the duration of the damaging factors. After a 30-60-minute ischemia, the structural and functional changes in the renal tissue are reversible. The mass death of nephrocytes-effectors is possible only after warm renal ischemia longer than 60 min.

[The application of forensic medical knowledge for the reconstruction of historical events].

The objective of the present study was to summarize the results of many year investigations on the application of forensic medical methods and experience for the reconstruction of historical events including identification of the ancient Russian saints' hallows and statesmen's remains, elucidation of the genuine causes of death of the members of the Russian Imperial House of Romanovs based on the recently discovered archival materials, restoration of the character of the injuries suffered by Aleksander II, M.I. Kutuzov, P. Demidov, G. Gapon., and G. Rasputin, the attribution A.S. Pushkin's memorial belongings based on the biological traces, and the like.

[FUNCTIONAL LINK BETWEEN TRANSPORT PARAMETERS OF MDCK1 CELLS MONOLAYER AND THEIR ACTIN CYTOSKELETON ORGANIZATION].

On MDCK1 cell monolayer, dynamics of the spatial organization of actin cytoskeleton and dynamics of trans-epithelial electrical resistance (TEER) have been studied upon exposure of arginine-vasopressin (AVP) and protein-kinase A (PKA) activator forskolin. It has been found that exposure to these physiologically active compounds causes fibrillary actin depolymerization (both in apical and in basal cytoplasm) and, simultaneously, significant decrease in the cell monolayer trans-epithelial electrical resistance. TEER decrease indicates the stimulation of ions and water flow across the cell monolayer. In order to clarify pathways of movement of ions and water across MDCK monolayer, we have carried out an immunofluoresence study of claudin 1 and 2 localization in the tight junctions of MDCK ATCC cells (low TEER) and MDCK1 cells (high TEER). We have demonstrated that in the tight junctions of MDCK ATCC cells both claudin 1 and claudin 2 are present. In MDCK1 cells tight junctions, claudin 1 is localized and pore-forming claudin 2 is completely lacking. Under forskolin exposure to MDCK1 cells, no alterations in studied claudins distribution has been found. These data indicate that forskolin-induced TEER decrease is linked with alterations in trans-cellular, not in para-cellular, permeability of monolayer.

[Morphological features of the myometrium in connective tissue dysplasia in women with uterine inertia].

OBJECTIVE: to reveal the morphological features of the lower uterine segment myometrium in connective tissue dysplasia (CTD) in women with uterine inertia. MATERIAL AND METHODS: Histological, immunohistochemical (with antibodies against collagen types I and III, matrix metalloproteinases 1 and 9 (MMR-1, MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), fibronectin; fibulin-5, connexin-43), electron microscopic, and electron immunocytochemical studies with morphometry of myometrial fragments from 15 parturient women with CTD and uterine inertia (a study group) and those from 10 women without CTD (a control group). RESULTS: The myometrium in CTD exhibited the decreased expression of connextin-43, fibulin-5, TIMP-1, collagens types I and III with collagen type III predominance and the unchanged levels of fibronectin and MMP-1 and MMP-9. Electron microscopy and immunocytochemistry showed fewer intercellular contacts and the dramatically lower expression of connexin-43 than in the control. CONCLUSION: A set of found myometrial changes in women with uterine inertia is a manifestation of CTD.

[Influence of protamine on expression of tight junction proteins in Caco-2].

Action of polycation protein protamine on the expression of tight junction proteins (claudins-1, -2, -3 and occludin) which contribute to paracellular transport function was investigated on cellular models of tight (MDCK I cell line) and leaky (Caco-2 cell line) epithelium. The expression of claudins-1,-3 and occludin was observed in both cell lines by methods of immunocytochemistry. Influence of protamine (100 microg/ml; 30 min; apical) on fluorescence intensity of claudins-1, -3 was different in MDCK I and Caco-2 cells. Addition ofprotamine to the incubation medium of Caco-2 cells resulted in significant increase of claudin-3 expression by 45 % (p <0.01) in comparison with control, whereas claudin-1 and occludin expression did not alter. On the contrary, in MDCK I cells protamine induced the significant decrease ofclaudin-1 and -3 expression by 25 % (p <0.001) and 15 % (p < 0.01) respectively, whereas occludin expression did not alter. It was confirmed by the methods of confocal laser scanning microscopy that protamine alter the expression of claudins-1, -3 directly in the tight junctions. Our results suggest that charged chyme components may alter paracellular permeability of epithelium.

[Modeling of kidney collecting ducts function in the cell culture conditions].

Continuous cell lines originating from the kidney collecting duct represent a powerful tool for the modeling of water and ions reabsorbtion processes. Present review considers the basic methodical approaches being utilized to study vasopressin-induced water transport mechanisms in the cell culture conditions--microscopical methods, electrophysiological measurements, various ways of evaluation of water flow across the cell monolayer, transfections of native and mutant proteins, GFP-technology. The results of the highest significance for the understanding of collecting ducts function which were obtained with usage of these methods are analyzed in the review.

[Ultrastructure of coelomic epithelium and coelomocytes of intact and wounded starfish Asterias rubens L].

Samples of coelomic epithelium and coelomocytes suspension of intact and wounded starfishes Asterias rubens L. were analyzed by electron microcopy. It has been demonstrated that coelomic epithelium is composed of three types of cells: flagellar (approx. 60%), secretory (approx. 3%) and myoepithelial (approx. 37%). Flagellar and secretory cells form the apical surface of coelomic epithelium. Secretory cells are represented by two subtypes: granular and mucous secretory cells. Myoepithelial cells are located in the basal zone of the epithelium. Adjacent flagellar cells are separated by intercellular gaps of various size in 4-5% of cases. These gaps are apparently the lacunae left by the flagellar cells after their departure to the coelomic cavity. The morphological pattern of transition of coelomic epithelium flagellar cells to the coelomocytes has been characterized. No significant structural alterations in organization of the coelomic epithelium were revealed after moderate wounding used in the present study. Small round-shaped young coelomycytes (approx. 3%) and bigger mature coelomocytes (approx. 97%) were found in coelomocytes suspension. A flagellum was revealed on the surface of one of the young coelomocytes. Surface of the mature coelomocytes forms the processes of various size and structure; their cytoplasm contains lysosomes and fagocytic vacuoles of different size. After wounding, activation of coelomocytes was noted finding expression in the sharp rise in the number and the length of their surface filopodia, and in the multicellular aggregates formation. By the sum of the ultrastructural data, histogenesis of coelomocytes from the flagellar cells of the coelomic epithelium is supposed to be a process of cellular transdifferentiation.

[Arginin-vasopressin-induced alterations in the structure of MDCK cells].

Alterations in the structural organization of MDCK cells under arginin-vasopressin (AVP) action were studied by electron and fluorescent microscopy. Electron-microscopical evidence was obtained that MDCK cells within monolayer form structurally distinct apical and basolateral surfaces separated by well-developed intercellular contact zones. It was proved that AVP specifically bound to the receptors on the basolateral surface of the cells, and was internalized from the surface in 10-15 min. AVP action resulted in fragmentation of Golgi apparatus and swelling of Golgi cisterns caused by initiation of osmotic water flow across the monolayer. Significant depolymerization of cells actin cytoskeleton was revealed under AVP or forskolin (an adenylate cyclase activator) exposure. Functional role and regulatory mechanisms of described structural alterations are discussed.

[Principles of radiographic and endoscopic diagnosis of colorectal cancer]. / Printsipi luchevoi i éndoskopicheskoi diagnostiki opukholei tolstoi kishki.

An evaluation of the potential of X-ray and endoscopy diagnosis and preoperative staging of colorectal cancer has shown that the best results are to be obtained by their complex multistage and purposeful use. Timely examination heeding the specific features of either component will permit detection and identification of colorectal tumors according to International TNM classification.

[Potentialities of computed tomography in the diagnosis of hiatal hernia]. / Vozmozhnosti komp'iuternoi tomografii v diagnostike gryzh pishchevodnogo otverstiia diafragmy.

An analysis of the results of 98 studies has demonstrated the potentialities of X-ray computed tomography in the diagnosis of hiatal hernia (HH), provided the X-ray computed tomography (XCT) semiotics of HH. It has found that XCT may directly visualize and objectively evaluate anatomic structures, such as diagraphmatic crus and esophageal foremen. It has emphasized that when XCT of abdominal and thoracic organs is performed, it is necessary to include the areas of diagragmatic curs and esophageal foramen into the list of anatomic structures binding for visual assessment and characterization, which in combination with other studies will assist in the early diagnosis of hiatal hernia and eventually expand the potentialities of XCT to a greater extent.

[Potential of abdominal computed tomography and ultrasonography for the complex diagnosis of early gastric cancer]. / Vozmozhnosti transabdominal'nogo ul'trazvukovogo issledovaniia i rentgenovskoi komp'iuternoi tomografii v kompleksnoi diagnostike rannego raka zheludka.

The potential of computed tomography and ultrasonography in making complex diagnosis of "small forms" of early gastric cancer was demonstrated by evaluation of the data on examination of 116 patients and the pivotal role of the extent of intramural invasion was established. The semiotics of computed tomography and ultrasonography for intramural invasion and the sequence of X-ray and endoscopic procedures were developed.

[Potentialities of transabdominal ultrasound study in the diagnosis of gastric peptic ulcer]. / Vozmozhnosti transabdominal'nogo ul'trazvukovogo issledovaniia v diagnostike iazvennoi bolezni zheludka.

The results of examination in 44 patients with gastric peptic ulcer were used to consider the potentialities of a transabdominal ultrasound study in the diagnosis and monitoring of gastric ulcerations. The ultrasound semiotics of gastric ulcers is described in the paper. A role of transabdominal ultrasound study in the algorithm of radiation and instrumental studies of gastric ulcer is defined. The inclusion of this study into the algorithm of diagnosis and monitoring of gastric peptic ulcer will make it possible not only to diagnose gastric ulcerations better, but to follow up their cicatrization. It is expedient to include a transabdominal ultrasound study as one of the diagnostic techniques for gastric peptic ulcer.

[Capacities of transabdominal ultrasound study in the diagnosis of polyps of the stomach and colon]. / Vozmozhnosti transabdominal'nogo ul'trazvukovogo issledovaniia v diagnostike polipov zheludka i tolstoi kishki.

The data obtained from studies of 84 patients with polyps of the stomach and colonic were used to consider the capacities of transabdominal and transrectal ultrasound studies and their diagnostic efficiency in the diagnosis of polyps of the stomach and colon. The ultrasound semiotics of polyps of the gastric and colon was described. A place of transabdominal ultrasound sonography (US) in the algorithm of radiation and endoscopic studies was established. This investigation led to the conclusion that transabdominal US, along with indirect diagnosis, permits solution of number of fundamental problems facing prior to polypectomy--these are primarily to define the nature of a detected polyp (epithelial and non-epithelial), based on an analysis of the laminar structure of the wall of an organ at the polyp base, a possible polyp malignancy and the degree of invasion into the depths of the gastric wall, as well as to detect a feeding vessel (in the CDC mode) and to predict the likelihood of possible complications. It is advisable to use transabdominal US in a complex of diagnostic techniques for polyps of the stomach and colon.