The isolated proteolytic domain of Escherichia coli ATP-dependent protease Lon exhibits the peptidase activity

AbstractSelective protein degradation is an energy-dependent process performed by high-molecular-weight proteases. The activity of proteolytic components of these enzymes is coupled to the ATPase activity of their regulatory subunits or domains. Here, we obtained the proteolytic domain of Escherichia coli protease Lon by cloning the corresponding fragment of the lon gene in pGEX-KG, expression of the hybrid protein, and isolation of the proteolytic domain after hydrolysis of the hybrid protein with thrombin. The isolated proteolytic domain exhibited almost no activity toward protein substrates (casein) but hydrolyzed peptide substrates (melittin), thereby confirming the importance of the ATPase component for protein hydrolysis. Protease Lon and its proteolytic domain differed in the efficiency and specificity of melittin hydrolysis

Брыжеечные лимфатические узлы при моделировании рака прямой кишки и в условиях химиотерапии

The purpose of the study was to find out the typical morpho-functional changes in mesenteric lymph nodes of Wistar rats after modeling of rectal cancer and intraperitoneally injected cytostatic therapy with 5-fluorouracil and leucovorin. Malignant epithelial tumor (dimorphous cancer) invaded the wall of the rectum was detected 11 months after instillation of chemical carcinogens. Under conditions of tumor growth, significant changes in structural organization, activation of proliferative process and blast transformation in areas responsible for cellular and humoral immune response were found in mesenteric lymph nodes. Similar changes occurred against the background of intranodular redistribution of lymph to the cortical intermediate sinuses influencing on changes in cytoarchitectonics in areas responsible for detoxification processes and antitumor resistance. Under conditions of intraperitoneal injection of chemical agents, activity of proliferation and blast transformation in germinal centers and paracortical area was maintained, that, however, was followed by signs of suppression of immune responses in brain strands. Activation of indirect lymph flow and redistribution of lymph flow into cortical, intermediate and brain sinuses took place. Nonuniformity of structural transformations in 1 and 2 mesenteric lymph nodes related to different regions of lymph collection was noted. Increase in transport function of the mesenteric lymph nodes was observed.Цель исследования – выявление характерных морфофункциональных преобразований в брыжеечных лимфатических узлах при моделировании рака прямой кишки экспериментальным животным (крысы Вистар) и цитостатической терапии внутрибрю- шинным путем (5-фторурацил и лейковарин). Спустя 11 мес после инстилляции химического канцерогена в стенке прямой кишки выявлена злокачественная эпителиальная опухоль – диморфный рак. В условиях роста злокачественной опухоли прямой кишки в брыжеечных лимфатических узлах выявлены признаки значительных изменений структурно-клеточной организации, активации процессов пролиферации и бласттрансформации в зонах, ответственных за клеточное и гуморальное звенья иммунитета. Подоб- ные преобразования имели место на фоне внутриузлового перераспределения лимфы в корковые промежуточные синусы, что и повлияло на изменения цитоархитеконики в зонах органа, ответственных за процессы детоксикации и противоопухолевую рези- стентность. В условиях внутрибрюшинного способа введения химиопрепаратов наблюдалось сохранение активности процессов пролиферации и бласттрансфомации в герминативных центрах и паракортикальной зоне, что, однако, сопровождалось признаками подавления иммунологических процессов в мозговых тяжах. Имела место активация непрямого пути тока лимфы, перераспреде- ление ее в корковые промежуточные и мозговые синусы. Отмечается неравномерность структурно-клеточных преобразований в 1-м и 2-м брыжеечных лимфатических узлах, связанная с разными регионами сбора лимфы. Транспортная функция брыжеечных лимфатических узлов увеличилась

Аcinetobacter baumannii bv Tryptophandestruens bv nov. isolated from clinical samples

The aim of the study was to determine the taxonomic status of a group consisting of atypical strains of Acinetobacter baumannii, outline relevant characteristics and methods necessary for their identification. There were examined 10 strains of A. baumannii (6 of them primary comprised) bearing similar profile of atypical features isolated from clinical samples (urine, sputum) in 2017–2019 at the Military Medical Academy. Сlinical strains of typical A. baumannii (n = 36), Acinetobacter nosocomialis (n = 14), Acinetobacter pittii (n = 9) and 1 strain of Acinetobacter calcoaceticus isolated from the external environment were used in comparative studies. Atypical strains had the characteristics of A. calcoaceticus — A. baumannii (ACB) complex bacteria and were identified as A. baumannii. The utilization of substrates as the only carbon source was studied on a dense synthetic medium added with 0.2 % substrate during incubation for 72 hours at 37°C. Carbohydrate oxidation coupled to acid formation was detected on the Hugh–Leifson medium by using a micromethod. Aromatic amino acid biotransformation was carried out in liquid and dense nutrient media assessed in chromogenic reaction. The rpoB gene was used for strain genetic characterization. Amplification of two 940 and 1210 base pair (bp)-long fragments from the rpoB gene was performed by the routine polymerase chain reaction using primers with previously described sequences. Amplification products were sequenced by Sanger using Big Dye Terminator v3.1 (Applied Biosystems, USA) and capillary electrophoresis on an automatic sequencer ABI PRISM 3130 (Applied Biosystems, USA), followed by using methods for determining the similarity levels of sequenced fragments with the rpoB gene sequences of the reference strain A. baumannii ATCC 17978 (GenBank accession no. CP053098.1). It was found that all strains belonging to atypical A. baumannii spp. had a specific set of features that distinguish them from typical strains of A. baumannii as well as other types of the ACB complex: detected biotransformation of L-tryptophan (via anthranilate pathway) and anthranilic acid under unambiguous lack of such signs in other bacteria; lack of utilized sodium hippurate and L-arabinose being unambiguously evident in other bacteria; lack of utilized L-tryptophan, putrescine, L-ornithine being utilized in the majority of strains of belonging to other bacterial species. Genetic analysis showed that the control strains of typical A. baumannii displayed 99.20–99.21% similarity within the sequenced fragments of the rpoB gene with those from the rpoB gene of the reference strain. All 10 strains of atypical A. baumannii had similar features (99.20–99.21%). At the same time, parameters of control strains from other bacterial species significantly differed: A. nosocomialis (95.10–95.97%), A. pittii (94.63–94.92%), A. calcoaceticus (93.00%). Hence, the strains of atypical and typical A. baumannii are genetically homogeneous and belong to the same species. The data presented allow us to consider this group of atypical A. baumannii strains as a new biovar. We propose the name for this new biovar — tryptophandestruens (tryptophan-destroying) stemming from the Latin word destruens — destroying. Identification of A. baumannii bv. tryptophandestruens bacteria can be carried out in laboratory of any level by using tests for L-tryptophan biotransformation as well as sodium hippurate utilization

EXPERIMENTAL MODELING OF APOPTOSIS UNDER CONDITIONS OF STABLE STRONTIUM EXPOSURE

Apoptosis is defined as a highly regulated form of programmed cell death with typical morphological and biochemical features. A variety of factors, including heavy metals, may influence the intensity of programmed cell death. The aim of the work was to simulate apoptosis in an in vitrosystem under the conditions of stable strontium exposure. The children’s population consuming drinking water with high strontium (Sr2+) content (n = 49) was observed. The level of lymphocyte apoptosis was determined with flow cytometry technique, by means of labeled annexin V-FITC conjugate (AnnV-FITC) and propidium iodide (PI) staining. AnnV-FITC+PI- cells were regarded as early apoptotic forms, whereas late apoptotic and/or necrotic cells were AnnV-FITC+PI+. The isolated leukocytes were incubated with Sr2+ at a concentration of 7.0 mg/l, the maximal permitted concentration (MPC) for water of aqueous objects, for 4 hours at 37 ºC. Expression of CD95 and p53 apoptosis markers was performed by flow cytometry using labeled monoclonal antibodies.In vitroexposure to strontium was associated with significantly decreased expression of apoptosisregulating factors, i.e., membrane marker CD95 and intracellular transcription protein p53, 1.56- and 1.68-fold, respectively. Meanwhile, we revealed a significantly (4.68-fold) decreased amounts of AnnV-FITC+PI--cells, as well as a statistically significant (1.35-fold) increase of the AnnV-FITC+PI+-cells. Moreover, the amounts of AnnV-FITC+ PI--lymphocytes in all samples were below the physiological ranges and control values. The number of samples with higher contents of AnnV-FITC+PI+-lymphocyte exceeding the established standards and control values, was 30.8%. Thus, it has been experimentally proven that strontium, at a concentration corresponding to MPC for water objects may significantly inhibit cell death along apoptotic pathways, with switching to necrotic cell death mechanisms, according to phosphatidylserine contents, as detected by annexin V binding test. The data have revealed an ability of strontium to have a significant effect upon the parameters of regulation and maintenance of cellular homeostasis, by influencing the apoptosis intensity, due to shifting a balance towards necrosis and reducing expression of apoptosis-regulating factors. The results of this study may be used in order to identify some marker indexes of immune disorders potentially induced by external influence of strontium upon human health under specific environmental factors

Directional Sensitivity of the NEWSdm Experiment to Cosmic Ray Boosted Dark Matter

We present a study of a directional search for Dark Matter boosted forward when scattered by cosmic-ray nuclei, using a module of the NEWSdm experiment. The boosted Dark Matter flux at the edge of the Earth's atmosphere is expected to be pointing to the Galactic Center, with a flux 15 to 20 times larger than in the transverse direction. The module of the NEWSdm experiment consists of a 10 kg stack of Nano Imaging Trackers, i.e.~newly developed nuclear emulsions with AgBr crystal sizes down to a few tens of nanometers. The module is installed on an equatorial telescope. The relatively long recoil tracks induced by boosted Dark Matter, combined with the nanometric granularity of the emulsion, result in an extremely low background. This makes an installation at the INFN Gran Sasso laboratory, both on the surface and underground, viable. A comparison between the two locations is made. The angular distribution of nuclear recoils induced by boosted Dark Matter in the emulsion films at the surface laboratory is expected to show an excess with a factor of 3.5 in the direction of the Galactic Center. This excess allows for a Dark Matter search with directional sensitivity. The surface laboratory configuration prevents the deterioration of the signal in the rock overburden and it emerges as the most powerful approach for a directional observation of boosted Dark Matter with high sensitivity. We show that, with this approach, a 10 kg module of the NEWSdm experiment exposed for one year at the Gran Sasso surface laboratory can probe Dark Matter masses between 1 keV/c$^2$ and 1 GeV/c$^2$ and cross-section values down to $10^{-30}$~cm$^2$ with a directional sensitive search.Comment: 15 pages, 14 figures, updated references, clarified discussion in intro section. Submitted to JCA

Биопсия сторожевого лимфатического узла при раке молочной железы с применением метода флуоресцентной визуализации красителя индоцианин зеленый

We presented the preliminary results of sentinel lymph node biopsy (SLN) using the method of fluorescent visualization with the indocyanine green. The study included 40 breast cancer patients with the prevalence of the cTis-2N0M0 process. 1 ml of Indocyanine Green (ICG) aqueous solution was administered periareolarly and subcutaneously from the tumor side with total drug dose of 5 mg. In the following few minutes the ICG trail along the lymphatic vessels was observed by its fluorescence in the infrared spectral range using a special camera with image transmitted to a computer screen. After the trail reached the axillary region and broke off, skin and subcutaneous tissue incision in axillary area was made, and the superficial fascia was dissected. The first contrasted lymph nodes were extracted. The incidence of SLN was 92.5%. Metastases were detected in 20% of cases. On average, it took 17.6 minutes to identify and remove the SLN. In 7 patients the use of the fluorescent SLN detection method was combined with radioisotope (Technefite 99m Tc colloid) – in all cases the same SLN was identified. The use of the ICG lymphotropic dye with the subsequent detection of SLN by the fluorescence method makes it possible to diagnose the status of SLN with a high degree of accuracy and can be comparable in effectiveness with the isotopic method. The specificities of lymphatic drainage were found out with the use of fluorescence lymphography: inverse dependence of the lymphatic drainage rate on the body mass index and breast size, and absence of dependence on degree of ptosis of breast.В работе представлены результаты исследования биопсии сторожевого лимфатического узла (СЛУ) с помощью метода флуоресцентной визуализации красителя индоцианин зеленый. В исследование были включены 40 больных раком молочной железы с распространенностью процесса сTis-2N0M0. 1 мл водного раствора красителя индоцианина зеленый вводили периареолярно внутрикожно и подкожно со стороны опухоли в дозе активного вещества 5 мг. В ближайшие минуты наблюдали движение красителя по лимфатическим путям в виде «дорожки» способом флуоресценции в инфракрасном спектре с помощью специальной камеры с передачей изображения на экран компьютера. После того, как дорожка достигала подмышечной области и обрывалась, производили разрез кожи и подкожной клетчатки в подмышечной области, рассекали поверхностную фасцию. Выделяли первые контрастиро ванные лимфатические узлы. Частота выявления СЛУ составила 92,5%, из них метастазы выявлены в 20% случаях. В среднем на этап идентификации и удаления СЛУ уходило 17,6 мин. У 7 больных комбинировали применение метода флуоресцентного (краситель ICG) определения СЛУ с радиоизотопным (коллоид Технефит 99m Tc) – во всех случаях идентифицировали одни и те же СЛУ. Применение лимфотропного красителя индоцианина зеленого с последующим выявлением СЛУ методом флуоресценции позволяет с высокой степенью точности диагностировать состояние СЛУ, а также сопоставимо по эффективности с изотопным методом. Выявлены осо бенности лимфооттока в молочной железе с помощью флуоресцентной лимфографии: обратная зависимость скорости лимфоотто ка от индекса массы тела и размера молочной железы, отсутствие зависимости от степени птоза железы

Divalent cation chelators citrate and EDTA unmask an intrinsic uncoupling pathway in isolated mitochondria.

We demonstrate a suppression of ROS production and uncoupling of mitochondria by exogenous citrate in Mg2+ free medium. Exogenous citrate suppressed H2O2 emission and depolarized mitochondria. The depolarization was paralleled by the stimulation of respiration of mitochondria. The uncoupling action of citrate was independent of the presence of sodium, potassium, or chlorine ions, and it was not mediated by the changes in permeability of the inner mitochondrial membrane to solutes. The citrate transporter was not involved in the citrate effect. Inhibitory analysis data indicated that several well described mitochondria carriers and channels (ATPase, IMAC, ADP/ATP translocase, mPTP, mKATP) were not involved in citrate's effect. Exogenous MgCl2 strongly inhibited citrate-induced depolarization. The uncoupling effect of citrate was demonstrated in rat brain, mouse brain, mouse liver, and human melanoma cells mitochondria. We interpreted the data as an evidence to the existence of a hitherto undescribed putative inner mitochondrial membrane channel that is regulated by extramitochondrial Mg2+ or other divalent cations

MESENTERIC LYMPH NODES IN SIMULATED RECTAL CANCER UNDER CONDITIONS OF CHEMOTHERAPY

The purpose of the study was to find out the typical morpho-functional changes in mesenteric lymph nodes of Wistar rats after modeling of rectal cancer and intraperitoneally injected cytostatic therapy with 5-fluorouracil and leucovorin. Malignant epithelial tumor (dimorphous cancer) invaded the wall of the rectum was detected 11 months after instillation of chemical carcinogens. Under conditions of tumor growth, significant changes in structural organization, activation of proliferative process and blast transformation in areas responsible for cellular and humoral immune response were found in mesenteric lymph nodes. Similar changes occurred against the background of intranodular redistribution of lymph to the cortical intermediate sinuses influencing on changes in cytoarchitectonics in areas responsible for detoxification processes and antitumor resistance. Under conditions of intraperitoneal injection of chemical agents, activity of proliferation and blast transformation in germinal centers and paracortical area was maintained, that, however, was followed by signs of suppression of immune responses in brain strands. Activation of indirect lymph flow and redistribution of lymph flow into cortical, intermediate and brain sinuses took place. Nonuniformity of structural transformations in 1 and 2 mesenteric lymph nodes related to different regions of lymph collection was noted. Increase in transport function of the mesenteric lymph nodes was observed

FUNCTIONAL ABILITY AND QUALITY OF LIFE IN PATIENTS WITH RHEUMATOID ARTHRITIS RECEIVING TOCILIZUMAB THERAPY

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease causing joint dysfunction; reduction of quality of life (QoL); loss of work ability, self-care ability, and executing daily routines in most patients 5–10 years after the disease onset.Objective. To study QoL and the functional status (FS) of Russian RA patients receiving tocilizumab (TCZ).Material and Methods. The study involved 42 patients with verified RA diagnosis (moderate or high activity) who had earlier undergone inefficient therapy with basic anti-inflammatory medications. The limitation of the FS of the RA patients was determined quantitatively using the Russian-language version of the HAQ questionnaire. QoL was evaluated using the EuroQol-5D (EQ-5D) Quality of Life questionnaire prior to treatment and after 4, 8, 12, 16, 20, and 24 weeks.Results. TCZ therapy demonstrated a rapid improvement of the FS of RA patients with a 64% decrease in the HAQ index (ΔHAQ=1.12), which corresponded to a 50% improvement of the health status of patients according to the ACR criteria.The median value [25th; 75th percentile] of the EQ-5D index was 0.52 [-0.02; 0.52]; 27.7% patients assessed their QoL as “worse than death”. The index reliably increased by week 8 of therapy; there were no patients with the negative EQ-5D index by week 24. Depending on QoL, all the patients were subdivided into two groups. Group 1 (n=12) comprised the patients with the EQ-5D no higher than 0; in group 2 patients (n=30), it was higher than 0. The groups were comparable in terms of disease duration, age, disease activity indices, and the previous treatment. The low QoL index in all 12 patients in group 1 was attributed to the infeasibility of performing daily activities and the reliably higher pain level (75.0 [61.0; 86.0] and 66.0 [48.0; 71.0] in groups 1 and 2, respectively; p=0.02). Improved QoL and reduced pain level were observed in both groups as early as after the first TCZ infusion. By week 24, the pain level decreased to 26.5 [11.0; 24.0] and 10.0 [2.0; 31.0] in groups 1 and 2, respectively. The EQ-5D index in group 1 became positive by week 8 of therapy.Conclusion. TCZ therapy quickly improves the FS and QoL of RA patients, including those with the most severe condition

Socially-Economic Behaviour of Students in the City of Kemerovo

Article is devoted to questions of social and economic behavior of students in regions, the city of Kemerovo. In view of dynamic changes of the public relations, the party of social and economic actions in behavior of students naturally changes, as defines rather high relevance of formation of the correct social and economic behavior. Priority activities on development of technology of increase in economic activity of youth are considered.Статья посвящена вопросам социально-экономического поведения студентов в регионах, в частности городе Кемерово. Ввиду динамических изменений общественных отношений естественным образом видоизменяется сторона социально-экономических действий в поведении студентов, что и определяет достаточно высокую актуальность формирования правильного социально-экономического поведения. Рассматриваются приоритетные направления деятельности по разработке технологии повышения экономической активности молодежи