Optogenetic cytosol acidification of mammalian cells using an inward proton-pumping rhodopsin.

Ion gradients are a universal form of energy, information storage and conversion in living cells. Advances in optogenetics inspire the development of novel tools towards control of different cellular processes with light. Rhodopsins are perspective tools for optogenetic manipulation of ion gradients in cells and subcellular compartments, controlling pH of the cytosol and intracellular organelles. The key step of the development of new optogenetic tools is evaluation of their efficiency. Here, we used a high-throughput quantitative method for comparing efficiency of proton-pumping rhodopsins in Escherichia coli cells. This approach allowed us to show that an inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR) is a powerful tool for optogenetic control of pH of mammalian subcellular compartments. Further, we demonstrate that NsXeR can be used for fast optogenetic acidification of the cytosol of mammalian cells. This is the first evidence of optogenetic cytosol acidification by an inward proton pump at physiological pH values. Our approach offers unique opportunities to study cellular metabolism at normal and pathological conditions and might help to understand the role of pH dysregulation in cellular dysfunctions.

[Cardiac ruptures in acute myocardial infarction]. / Razryvy serdtsa pri ostrom infarkte miokarda.

Presented in the article are the generalized data of the Russian and foreign literature addressing the currently important problem of myocardial ruptures as one of the most dangerous complications of infarction, also analysing the results of clinical studies on interconnection of heart ruptures with systemic thrombolytic therapy and with a percutaneous coronary intervention. This is followed by describing the mechanisms that may lead to myocardial rupture during thrombolytic therapy and surgical endovascular treatment, underlying the necessity of pharmacological pre- and post-conditioning for prevention of reperfusion myocardial lesions. The article also touches upon the clinical and instrumental diagnosis of myocardial ruptures, as well as approaches to surgical treatment depending on the type of rupture and necessity of myocardial revascularization.

[Acute cerebral circulation impairment associated with percutaneous coronary intervention]. / Ostroe narushenie mozgovogo krovoobrashcheniia, assotsiirovannoe s chreskozhnym koronarnym vmeshatel'stvom.

The article deals with generalized literature data on the current importance of the problem concerning acute cerebral ischemia occurring during percutaneous coronary interventions. Pathophysiological mechanisms of the development of acute cerebral circulation impairment are described, with their classification. Also considered are various risk factors which may cause this formidable complication, followed by describing modern methods of its surgical treatment. It is noted that the main risk factors for neurological complications appearing after revascularization of coronary arteries include age, accompanying diseases (instability of arterial pressure, diabetes mellitus, atrial fibrillation, atherorosclerotic lesions of the aorta and brachiocephalic arteries, previous stroke), as well as systolic dysfunction, leukocytosis, and a low level of cognitive function prior to operation. The development of acute cerebral circulation impairment during and after percutaneous coronary intervention is an indication for using methods of endovascular treatment. It is most appropriate to use a combination of direct percutaneous thrombectomy and a stent retriever. It is underlined in the article that removal of the thrombus form the cerebral artery is not an independent surgical procedure but rather a methodology envisaging an operation to be performed from the simplest technique to the most complicated one. As the first procedure, it is necessary to perform thrombaspiration and then, if ineffective, a second stage consisting in thrombextraction with the help of a stent retriever. Besides, in order to increase efficacy of thrombaspiration there have been worked out several techniques combining the use of direct thrombextraction and a stent retriever. Described in detail are 4 most commonly used techniques.

[The Bleeding Safety of Ticagrelor in Patients With ST-Elevation Acute Coronary Syndrome Treated With Fibrinolytic Therapy].

Aim      To compare hemorrhagic safety of ticagrelor and clopidogrel in patients with ST-segment elevation acute coronary syndrome (STEACS) after thrombolytic therapy (TLT).Material and methods  This nonrandomized study included 183 patients followed up for 30 days. Hemorrhagic safety was compared in a group of patients with STEACS (n=71) after a thrombolytic treatment with alteplase and early ticagrelor treatment (180 mg followed by switching to 90 mg twice daily) and in a group of patients (n=112) with STEACS receiving TLT with alteplase and clopidogrel (loading dose, 600 mg followed by switching to 75 mg daily). Primary endpoint was hemorrhage associated with TLT; patients were followed up for 30 days.Results During the follow-up period, TLT-associated hemorrhages were observed in 11.3% of patients in the ticagrelor treatment group and in 10.7% of patients in the clopidogrel treatment group (p=0.9; odds ratio, 1.06 at 95 % confidence interval, from 0.41 to 2.73). Intracranial hemorrhages and fatal hemorrhages were absent in both groups.Conclusion      There were no significant differences in hemorrhagic safety between patients with STEACS after the TLT treatment with alteplase and early treatment with ticagrelor or clopidogrel.

[Expression of ganglioside GD2 on colorectal adenocarcinoma cells]. / Ékspressiia gangliozida GD2 na kletkakh kolorektal'noi adenokartsinomy.

Using flow cytometry GD2 ganglioside expression was evaluated both on colorectal adenocarcinoma cell lines and on tumor tissue samples from colorectal cancer patients. The marker was found on EpCAM-positive tumor cells in 6 of 12 patients' samples but not on the HT29 and CaCo-2 cell lines. GD2 expression was not an exceptional feature of cancer stem cells, since its expression level was similar on CD133-positive and CD133-negative tumor cells. Thus, the presence of GD2 ganglioside was revealed on colorectal adenocarcinoma cells for the first time. This finding makes it possible to use targeted therapy to treat this disease.

[Reaction of connective tissue of arterial and venous walls to implantation of synthetic and biological grafts]. / Reaktsiia soedinitel'noi tkani arterial'noi i venoznoi stenok na implantatsiiu sinteticheskogo i biologicheskogo protezov.

BACKGROUND: 30% of patients in the remote period appear to develop restenosis of the anastomoses formed during reconstructive-restorative operations on vessels. Two major causes of restenosis have been determined: intimal hyperplasia and smooth muscle cell proliferation. The reaction of connective-tissue elements of vascular walls in response to implantation of grafts remains unstudied. OBJECTIVE: The study was undertaken to examine the reaction of connective tissue of the arterial and venous walls to implantation of synthetic and biological grafts. MATERIAL AND METHODS: We analysed the results of an experimental study performed on a total of 60 rabbits divided into 4 groups each consisting of 15 animals: in the first 2 groups we examined tissue reaction to implantation of synthetic and biological grafts on the wall of an artery and in the remaining 2 groups - on the wall of a vein. The synthetic grafts were made of polytetrafluoroethylene, with the biological ones made of bovine internal thoracic arteries. The portions of the vessels along with the implanted grafts were subjected to histological examination on postoperative days 14, 21 and 30. The specimens were studied by means of light microscopy after haematoxylin-eosin staining. We also carried out a morphometric study consisting in determining the quantitative ratio of the cellular composition of connective tissue. RESULTS: After implantation on the arterial wall, the connective-tissue capsule under the synthetic prosthesis was 1.8 times thicker than under the biological one, with predominance of the cellular component over fibrous one, which deformed the zone of implantation. Under the biological prosthesis, the capsule on the arterial wall was presented by fibrous structures with no deformation of the zone of implantation. The reparative reaction of the venous wall to implantation of the synthetic prosthesis was represented by a thin fibrous connective-tissue capsule. The reaction of the venous wall to the biological transplant was at all terms of the experiment characterised by significant inflammatory alterations with granulomatosis and immunomorphological shifts with formation of lymphoid follicles on the border with the transplant. CONCLUSION: Implantation of the synthetic polytetrafluoroethylene prosthesis on the arterial wall induced a hyperplastic reaction of the elements of connective tissue, and a normoplasic one on the venous wall. Implantation of the biological prosthesis on the arterial wall did not induce proliferation of connective-tissue elements, and on the venous wall - granulomatous inflammation.

Analysis of Anti-Glycan IgG and IgM Antibodies in Colorectal Cancer.

We propose an approach that allows simultaneous determination of the levels of M and G isotypes of antibodies to the panel of glycans using microarrays. The level of IgG antibodies to 3'-O-su-Lea glycan detects patients with colorectal cancer with a sensitivity of 69.77% and specificity of 62.75%. The percentage of correctly classified colorectal cancer patients with the use of a combination of two markers IgM antibodies to glycans 3'-sialyl-TF and 3'-O-su-Lea is as high as 74.23%. The levels of IgM antibodies to 3'-O-su-Lea glycan differ significantly in patients with and without regional metastases. The levels of some of antiglycan IgM or IgG antibodies differed significantly in patients with tumors of different location and differentiation.

[Overexpression of microRNAs miR-9, -98, and -199 Correlates with the Downregulation of HK2 Expression in Colorectal Cancer].

Glycolysis activation is one of the main features of energy metabolism in cancer cells that is associated with the increase in glycolytic enzyme synthesis, primarily, hexokinases (HKs), in many types of tumors. Conversely, in colorectal cancer (CRC) the decrease in the expression of HK2 gene, which encodes one of the key rate-limiting enzyme of glycolysis, was revealed, thus, the study of the mechanisms of its inhibition in CRC is of particular interest. To search for potential microRNAs, inhibiting the expression of HK2 in CRC, we have performed the analysis of data from "The Cancer Genome Atlas" (TCGA) and five microRNA-mRNA target interaction databases (TargetScan, DIANA microT, mirSVR (miRanda), PicTar, and miRTarBase) using original CrossHub software. Seven microRNAs containing binding site on mRNA HK2, which expression is negatively correlated with HK2 expression, were selected for further analysis. The expression levels of these microRNAs and mRNA HK2 were estimated by quantitative PCR on a set of CRC samples. It has been shown, that the expression of three microRNAs (miR-9-5p, -98-5p, and -199-5p) was increased and correlated negatively with mRNA level of HK2 gene. Thus, downregulation of HK2 gene may be caused by its negative regulation through microRNAs miR-9-5p, -98-5p, and -199-5p.

[Pancreatoduodenectomy: learning curve within single multi-field center]. / Pankreatoduodenal'naia rezektsiia: krivaia obucheniia na primere odnogo mnogoprofil'nogo tsentra.

AIM: To analyze learning curve by using of immediate results of pancreatoduodenectomy at multi-field oncology institute. MATERIAL AND METHODS: For the period 2010-2016 at Abdominal Oncology Department of Herzen Moscow Oncology Research Institute 120 pancreatoduodenal resections were consistently performed. All patients were divided into two groups: the first 60 procedures (group A) and subsequent 60 operations (group B). Herewith, first 60 operations were performed within the first 4.5 years of study period, the next 60 operations - within remaining 2.5 years. RESULTS: Learning curves showed significantly variable intraoperative blood loss (1100 ml and 725 ml), surgery time (589 min and 513 min) and postoperative hospital-stay (15 days and 13 days) in group A followed by gradual improvement of these values in group B. Incidence of negative resection margin (R0) was also significantly improved in the last 60 operations (70 and 92%, respectively). CONCLUSION: Despite pancreatoduodenectomy is one of the most difficult surgical interventions in abdominal surgery learning curve will differ from one surgeon to another.

[Hydrogel microchip as a tool for studying exosomes in human serum].

Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.

[Results of using various methods of autovenous femoropopliteal bypass grafting below the knee-joint fissure]. / Rezul'taty primeneniya razlichnykh sposobov autovenoznogo bedrenno-podkolennogo shuntirovaniya nizhe shcheli kolennogo sustava.

The objective of the study was to improve therapeutic outcomes in patients presenting with lower limb critical ischaemia by means of optimizing the technique of femoropopliteal bypass grafting. The authors analysed the results of comprehensive examination and treatment of a total of 90 patients presenting with critical lower limb ischaemia on the background of atherosclerotic lesions of arteries of the femoropopliteal and crural segments. Depending on the technology of performing femoropopliteal bypass grafting, the patients were subdivided into three groups each consisting of 30 subjects. The groups were comparable by gender, age, concomitant diseases, degree of damage to arteries of the femoro-popliteal-crural segment, and the state of the distal vascular channel. Group One patients underwent conventional autovenous bypass grafting with a reversed great saphenous vein. Group Two patients endured surgery according to the "in situ" technique. Group Three patients were subjected to an original technology of bypass grafting using a free autovenous graft with destroyed valves without reversion of the vein. It was demonstrated that the use of a free autovenous transplant with destroyed valves increased the volumetric blood flow through the shunt in the remote postoperative period 2.4-fold as compared with the reversed vein and 1.7-fold as compared with the "in situ" autovein. The proposed technique of femoropopliteal bypass grafting made it possible in the immediate postoperative period to decrease the rate of early postoperative complications by 23.4% as compared with the option of using the reversed vein and by 13.3% as compared with the "in situ" technique, as well as to lower the incidence rate of late shunt thromboses by 40 and 13.3%, respectively, and the number of limb amputations by 30 and 6.7%, respectively. The use of a free autovenous graft with destroyed valves is pathogenetically justified and makes it possible to optimize the results of treatment of patients.

[Free autovenous graft with destructed valves in treatment of severe ischemia of lower extremities]. / Lechenie kriticheskoi ishemii nizhnikh konechnostei s ispol'zovaniem svobodnogo autovenoznogo transplantata s razrushennymi klapanami.

AIM: to improve treatment of patients with severe atherosclerotic lesion of lower extremities arteries followed by critical ischemia by optimization of femoropopliteal bypass surgery. MATERIAL AND METHODS: Treament and survey of 60 patients with severe atherosclerotic lesion of femoropopliteotibial segment and critical lower limb ischemia were analyzed. Patients were divided into 2 groups depending on technique of femoropopliteal bypass. Conventional in situ autovenous technique was used in group 1. In the second group we used original method of free autovenous graft with destructed valves. RESULTS: Technique of free autovenous graft with destructed valves decreases incidence of early postoperative complications by 13.3%, remote thrombosis of graft by 13.3%. Also it increases physical and mental components of health by 7.9% and 3.1% respectively. CONCLUSION: Use of free autovenous graft with destructed valves is reasonable and improves results of treatment.

[Present condition and perspective of development of self-care decontamination products].

Present condition and perspective of development of self-care decontamination products. This work provides data describing the current state and perspectives of development of self-care decontamination products for neutralization and removal of poisonous and highly toxic biologic and radioactive agents from open human body surfaces and close- fitting cloth. The history of self-care decontamination products creation in Russia and foreign countries is represented. The detailed performance characteristics of the developed individual anti-gas kits IPP-3, IPP-51, IPP-8, IPP-10, IPP-11, which were accepted to the Army and the Navy supply in the 1920-1930s, during the Great Patriotic war, in post-war period and now, are given. The individual anti-gas kits RSDL, M291, M295 and MKI for government-issue equipment of North Atlantic nations' armies are described in detail. It was showed that the general limitation both of the native and foreign anti-gas kit models is the poor efficacy against exposure of vesicants from the group of blister warfare agents. The most perspective research guidelines in creation of new self-care decontamination products. are validated.

EFFICACY OF FREE AUTOVENOUS TRANSPLANT IN FEMORAL-POPLITEAL BYPASS BELOW THE JOINT SPACE OF THE KNEE.

The article presents an analysis of complex examination and treatment of 60 patients with critical ischemia of the lower extremities due to atherosclerotic lesions of femoral-poplitealtibia segment. Typical traditional autovenous bypass of reverse big saphena was performed for the first (control) group of patients. The operation, which used an original technology by free autovenous transplant with collapsed valves without vein reverse, was completed for the second (experimental) group of patients. An application of original treatment technology allowed an increase of blood flow volume in the extremity in 1,5 times, an arterial blood flow - in 1,6 times, rate of microcirculation - in 1,3 times. An early postoperative complications were decreased on 23,4%, the late shunt thrombosis - on 40%. Long-term shunt passability was improved in 2,6 times and physical component of health - on 10,6%, psychological - on 4,3%. The shunt functioned in 36,7% of the first group and in 83,3% patients of the second group after two years since operation. The extremity was saved in 60% of the first group and 90% patients of the second group.

[Downregulation of OGDHL expression is associated with promoter hypermethylation in colorectal cancer].

Cell metabolic reprogramming is one of the cancer hallmarks. Glycolysis activation, along with suppression of oxidative phosphorylation and, to a lower extent, the TCA cycle, occurs in the majority of malignant tumors. A bioinformatics search for the glucose metabolism genes that are differentially expressed in colorectal cancer (CC) was performed using the data of The Cancer Genome Atlas (TCGA) Project. OGDHL for an oxoglutarate dehydrogenase complex subunit, which is involved in the TCA cycle and is indirectly responsible for the induction of apoptosis, was identified as one of the most promising candidates. A quantitative PCR analysis showed, on average, an eightfold downregulation of OGDHL in 50% (15/30) of CC samples. Based on the TCGA data, promoter hypermethylation was assumed to be a major mechanism of OGDHL inactivation. Bisulfite sequencing identified the OGDHL promoter region (+327 ... +767 relative to the transcription start site) that is often methylated in CC samples with downregulated ODGHL expression (80%, 8/10) and is possibly crucial for gene inactivation. Thus, frequent and significant OGDHL downregulation due to hypermethylation of a specific promoter region was demonstrated for CC. The OGDHL promoter methylation pattern was assumed to provide a marker for differential diagnosis of CIMP+ (CpG island methylator phenotype) tumors, which display dense hypermethylation of the promoter region in many genes.

[The use of the new loads of expendable medical supplies by the medical service of the Armed Forces].

The new loads of expendable medical supplies adopted by the Armed Forces of the Russian Federation and included into regulating documents are the most important elements of the authorized equipment system. Nine loads of expendable medical supplies, combined into two classification groups, are provided for the medical service. The use of these loads improves the effectiveness of medical supply for all stages of medical evacuation, medical continuity during medical and evacuation procedures and allows to deliver medical aid to patients on the basis of modern and innovative medical technologies.

CD133+ human colorectal adenocarcinoma cells are resistant to staining with fluorescent dyes used for analysis of ABC transporter activities.

Flow cytometry measurement of the expression of surface marker CD133 simultaneously with the analysis of fluorescent dye exclusion was performed in order to develop new methods for detection of cancer stem cell populations in tumor tissue samples from patients with colorectal adenocarcinoma. No correlation was found between the count of CD133(+) cancer cells and the volume of the "population" formed from cells actively pumping off the fluorescent dye. On the other hand, the fluorescence distribution plot showed predominant location of CD133(+) cancer cells among cells stained with neither DyeCycle Violet DNA-binding dye, nor rhodamine 123 mitochondrial dye. These cells did not show the properties of the classical "side population", because they did not shift to the area of stained cell after treatment with ionic channel blocker verapamil.

Total mesorectal excision with water-jet dissection in patients with rectal cancer: surgical and morphological aspects.

AIM: This publication will describe our own experience of using the ERBEJET2(®) water-jet dissector during surgical interventions for rectal cancer. METHOD: We utilized the water-jet dissection technique to obtain tissue specimens in 10 patients with rectal cancer. All patients thus underwent nerve-sparing low anterior resection of the rectum along with para-aortic lymphadenectomy. No intraoperative complications were registered. The postoperative period went uncomplicated in all patients. No dysuria was observed. Obtained tissue specimens were examined morphologically. Macroscopic examination included assessments of the preservation of the rectal fascia propria and the amount of cellular tissue along the anterior, posterior, and lateral surfaces of the rectum. We performed microscopy of the circumferential resection margin to characterize the surgical clearance and the intensity and depth of damage to the mesorectal tissue. On morphological examination, the quality of mesorectal excision was found to be good (Grade 3) in all 10 patients. RESULTS: As the results of our study demonstrate, the depth of lateral tissue damage is minimal with the water-jet dissector. CONCLUSION: Water-jet dissectors have their own place in the long list of armamentarium used in surgical interventions performed for rectal cancer and contribute to improving oncological and functional outcomes of surgical treatment in this patient population.

[Prophylaxis of hepatic insufficiency after major hepatic resections].

UNLABELLED: The article deals with a problem of prophylaxis of hepatic insufficiency in oncological patients after liver resections. MATERIALS AND METHODS: We analyzed data of effectiveness and safety of the use of Remaxol in oncological patients with hepatic metastasis of colorectal cancer--dynamics of indicators of cytolysis and cholestasis, hepatic protein synthesis, exchange of pigments, pro- and antioxidant system and the level of endogen intoxication in postoperative period. RESULTS: Use of Remaxol allows decreasing the duration of postoperative rehabilitation and intensive care unit staying.