bluuebunny/crossref_metadata_2025 huggingface dataset:
An Experimental Study of the Cryopreserved Placenta Extract Effect On the Sodium Diclofenac Anti-Inflammatory Activity
Authors: Hladkykh Fedir
Published: October 2021

Background. As a means of correcting the ulcerogenic effect of nonsteroidal anti-inflammatory drugs, our attention was attracted by a cryopreserved extract of the human placenta, which has a multivector spectrum of biological activity. To date, there is no information about its effect on the specific activity of this class of medicines (anti-inflammatory, analgesic, etc.) in published sources.
Objective. We are aimed to characterize the effect of cryopreserved placenta extract on the anti-inflammatory activity of diclofenac sodium when administered separately in a model of acute exudative inflammation.
Methods. Experimental studies in vivo were conducted on 28 nonlinear male laboratory rats. The model of acute exudative inflammation was reproduced by subplantar injection of 0.1 ml of 1.0% aqueous solution of λ-karagenin into the right hind limb of rats. Cryopreserved placenta extract was administered intramuscularly at a dose of 0.16 ml/kg 60 minutes before diclofenac sodium (8 mg/kg).
Results. Preventive administration of diclofenac sodium caused an antiexudative effect as early as 30 minutes after administration of λ-karagenin – its anti-inflammatory activity was 11.0%, which is 4.6 times higher than similar indicators at the same time in rats injected with placental cryoextract. At 60 minutes of observation, diclofenac sodium was comparable in anti-inflammatory activity with cryopreserved placenta extract: 28.6% and 22.2%, respectively, but at 120 and 180 minutes, diclofenac sodium exceeded the studied cryoextract in antiphlogistic effect by 1.6 times in both periods of observation. The anti-inflammatory effect of the combined separate administration of placenta cryoextract and diclofenac sodium before λ-karagenin for 30 and 60 minutes was 12.7% and 32.3%, respectively, which is comparable with analogous indicators against the background of diclofenac sodium monotherapy. However, at 120 minutes of observation, the group of combined use of placenta cryoextract and diclofenac sodium showed the greatest anti-inflammatory effect among rats of all the studied groups – 52.6%, which was 2.2 times higher than the indicators of the placenta cryoextract monotherapy group and 1.4 times lower than the indicators of the rats of the diclofenac sodium monotherapy group.
Conclusions. 4 hours after administration, placental cryoextract had a suppressive effect on kinins like diclofenac sodium, and in the prostaglandin period of caragenin-induced inflammation against the background of combined use of the studied cryoextract and diclofenac sodium, the anti-inflammatory activity was 46.4 %. This suggests a suppressive effect on the production of prostaglandins as a possible mechanism of anti-exudative action of cryopreserved placenta extract.
The Role of Cord Blood in the Regulation of the Cellular and Humoral Link of Immunity in Experimental Atopic Dermatitis
Authors: Koval Hanna , Lutsenko Olena , Bondarovych Mykola , Ostankov Maksym , Goltsev Anatoliy
Published: October 2021

Background. Atopic dermatitis (AD) as one of the most common diseases of autoimmune genesis in the structure of dermatological practice, is characterized by itching, dryness, thickening of the skin, characteristic rashes. The drugs of choice in the treatment of AD are steroidal anti-inflammatory drugs. However, the development of unwanted side effects is a serious problem attributed to using hormone therapy. The search for effective methods of treating AD is an urgent task of medicine and in particular dermatology. At the same time, there is an obvious need for the participation in the solution of this problem also of specialists-immunologists working in the field of application of cell therapy drugs, acting on various pathogenetic links of the disease. The development of new or optimization of existing methods of treating AD is the urgent task facing them.
Objective. Evaluation of the immunocorrective effect of lyophilized (lHCBL) and cryopreserved human cord blood leucoconcetrate (cHCBL) on a AD model.
Methods. The experiments were carried out on 6-month-old Wistar rats. Upon induction of AD, the inflammation focus was formed on the rat's back (9–10 cm2) by daily rubbing in a 5% alcohol-acetone solution of dinitrochlorobenzene (DNCB) for 21 days. cHCBL and lHCBL were injected intraperitoneally, 0.5 ml at a dose of 5´106 cells in one day after the final DNCB treatment. The adhesive and phagocytic activity of the cells of the peritoneal cavity, the level of circulating immune complexes, the population and subpopulation of lymphocytes (CD3+, CD4+, CD8+, CD16+, CD4+CD25+), the immunoregulatory index of lymphocytes, the concentration of immunoglobulins in the blood serum were determined.
Results. For AD induced by DNCB, systemic changes in the immune status are characteristic, which is expressed by changes in the parameters of cellular and humoral immunity. The most fundamental changes in cell subpopulations in spleen of rats with AD were revealed: a decrease in the number of total T-lymphocytes and their two main subpopulations (CD4+ and CD8+ cells). Against this background, changes were noted in the monocytic-phagocytic and humoral systems of immunity. The paper shows the effectiveness of the use of cHCBL and lHCBL in the correction of pathological manifestations of experimental AD. On the background of treatment, the features of the immunocorrective effect of each of the drugs were noted. Thus, when assessing intergroup values, a more pronounced increase in T-reg was revealed in rats of the 5th group – 3.9 [3.8; 4.0] versus 3.2 [3.0; 3.3] in the 4th group (P < 0.01); IgA level – 1.6 [1.5; 1.7] versus 1.3 [1.2; 1.4] (P < 0.01).
Conclusions. Thus, lHCBL exhibits immunocorrective activity in the treatment of experimental AD, surpassing in some parameters the activity of сHCBL, which is promising for its use in clinical practice.
Efficiency of Combined Use of Fullerene C60 and Bovine Serum Albumin for Rehabilitation of Vitrified Fragments of Rat Immature Seminiferous Tubules
Authors: Volkova Nataliia , Yuhta Maria , Sokil Larisa , Chernyshenko Lyudmyla , Stepanuk Ludmila , Goltsev Anatoliy
Published: October 2021

Background. Today, cryopreserved reproductive tissues are used to treat some forms of male infertility. However, after long-term preservation of fragments of seminiferous tubules of testes (FSTT) in a low-temperature bank (-196 °С) their morphological and functional characteristics decrease reversibly. To solve this problem after freezing-thawing, the use of rehabilitation media with special additives is promising. Due to the fact that serum albumin and fullerene C60 have powerful protective and antioxidant properties, their use allows to stabilize the plasma membrane, osmotic pressure, and reduce free radicals that make them promising candidates to use in the development of rehabilitation media for biological objects after cryopreservation.
Objective. The efficacy of fullerene C60, bovine serum albumin (BSA), and their combination as components of rehabilitation medium of vitrified FSTT of immature rats was studied.
Methods. Vitrified-thawed samples of FSTT were incubated (22 °C) for 30 minutes in Leibovitz's medium with addition of 15 mg/mL C60, 5 g/L BSA or their combination. Control samples were incubated in the medium without C60 or BSA addition. Metabolic activity (MTT test), histomorphological data, total antioxidant status (TAS), reactive oxygen species (ROS) production, activity of g-glutamyltransferase (gGGT), and glucose-6-phosphate dehydrogenase were determined in the samples after rehabilitation in the investigated media.
Results. The use of C60 led to the increase of metabolic (by 1.26 times) and TAS (by 1.74 times) activities, to the decrease in the number of ROS+ cells (by 1.35 times) and to the improvement of the spermatogenic epithelium binding to the basement membrane versus control sample. Application of BSA did not significantly affect the studied biochemical indices but decreased the number of tubules with desquamation of spermatogenic epithelium in histological sections. The combined use of BSA and C60 had the best effect among investigated rehabilitation media that led to the increase of metabolic activity (by 1.51 times), TAS activity (by 1.78 times), gGGT activity (by 1.59 times), histostructure restoration and the decrease in the number of ROS+ cells (by 1.45 times) compared to the control samples.
Conclusions. The use of C60 and BSA combination increases the metabolic and antioxidant activity of vitrified FSTT and also has a positive effect on their histostructural characteristics compared to control samples. It should be noted that the effect of С60 and BSA addition to rehabilitation medium exceeds the results of using the investigated additives separately (by the metabolic and gGGT activity as well as architectonics of vitrified FSTT). These data relate to reproductive medicine and can be used to develop an effective rehabilitation protocol for vitrified FSTT.
La educación agroambiental en la escuela pedagógica
Authors: Smith Franciss Brown , Abad Amparo Osorio , Yanes Enrique Cecilio Cejas
Published: October 2021

La educación agroambiental es un enfoque para alcanzar el desarrollo sostenible o sustentable. En la actualidad existen diferentes concepciones sobre los principios que deben guiar las políticas agroambientales, también hay una gran variedad de contextos, escalas espaciales y temporales en los que se han implementado programas agroambientales. Las actividades agroambientales son diseñadas a nivel nacional, regional o local, con el fin de que puedan adaptarse a los diferentes sistemas agrícolas y condiciones ambientales. Desde el punto de vista educativo y formativo este enfoque es utilizado en la escuela pedagógica como herramienta para la educación ambiental. El trabajo tiene como objetivo: sistematizar los fundamentos teóricos de la educación agroambiental en la escuela pedagógica y que contribuya con la formación integral de los estudiantes y la preparación pedagógica agroambiental de los profesores. Se presenta como resultado la sistematización teórica de los fundamentos de la educación agroambiental en la escuela pedagógica y su importancia. Se empleó: la sistematización, el análisis documental, análisis y síntesis y la revisión bibliográfica que posibilitó la consulta de tesis, documentos rectores, libros entre otros. La sistematización teórica realizada constituye fundamento ya que se argumentan las diferentes posiciones en relación con las políticas agroambientales, la problemática del medio ambiente y la necesidad de la educación ambiental, así como los contenidos que en este sentido deben ser abordados en la preparación de profesores y estudiantes como centros en la ejecución de actividades agroambientales.
Monitoring the Nonconforming Fraction with a Dynamic Scheme When Sample Sizes are Time-varying
Authors: Morales Víctor Hugo , Panza Carlos Arturo , Blanco Javier
Published: October 2021

In many practical applications, it is more convenient to characterize the quality of production processes or service operations throughout the count of nonconformities. In the context of SPC, nonconformities are usually assumed to appear according to the binomial probability model. The conventional way for monitoring nonconformities involves Shewhart-type control procedures based on both constant and time-varying sample sizes. In this article, an EWMA scheme is proposed for monitoring the fraction of nonconforming items with time-varying sample sizes. The proposed control chart is referred to as the EWMAG-B and can be easily adapted to work with a constant sample size by fixing it at a needed value. By means of simulation, it was found out that the EWMAG-B chart outperforms the conventional p control chart in Phase II while detecting changes in the process level is wanted.
Abstract P-14: Molecular Modeling of the Transmembrane Domain of the SARS Cov-2 S-Protein and its Interaction with the Membrane
Authors: Novoseletsky Valery , Bozdaganyan Marine , Litvinov Daniil , Sokolova Olga
Published: October 2021

Background: The spike glycoprotein of SARS-coronavirus mediates the early events leading to infection of cells, including fusion of the viral and cellular membranes. The spike is a type I membrane glycoprotein that possesses a conserved transmembrane anchor and an unusual cysteine-rich domain that bridges the putative junction of the anchor and the cytoplasmic tail. In this study, we examined the role of these carboxyl-terminal domains in S-protein interaction with membrane. Methods: Structural model of the trimeric TM domain and adjacent fragments of ecto- and endo domains (residues 1157-1256) of the S-protein was built by homology basing on the solution structure of the SARS-coronavirus S-protein HR2 domain (pdb-code 2fxp), the structure of the transmembrane domain of HIV-1 gp41 in bicelle (5jyn), and assumption of generally coiled-coil fold of the considered domain. C-terminus of the domain was left unstructured but fully palmitoylated. Molecular dynamics simulation in heterogeneous lipid bilayer was prepared with CHARMM-GUI and performed with Gromacs during 100 ns. Results: 1. Ectodomain fragment (residues 1157-1212) demonstrates a tilt by the angle of 40-60 degrees from the axis of the TM domain (residues 1213-1237). This tilt is facilitated by glycine residues in position 1204. 2. Cholesterol molecules of the bottom layer tend to localize around protein due to interaction with palmitoyl tails while lipids in the upper layer do not show such tendency. Conclusion: Performed molecular simulations show that both palmitoylation and a large cluster of aromatic residues provide high stability of the S-protein TM domain.
Abstract P-20: Sub 3 Resolution Cryo-EM Structure of Eukaryotic Small (40S) Ribosomal Subunit
Authors: Baymukhametov Timur , Kravchenko Olesya , Afonina Zhanna , Vassilenko Konstantin
Published: October 2021

Background: The ribosome is a molecular machine that translates mRNAs into proteins. In eukaryotes, ribosome consists of small (40S) and large (60S) subunits. Translation in eukaryotes is a complicated molecular process that involves the formation of various molecular complexes consisting of ribosomal subunits and protein factors. Cryo-EM approaches such as single particle analysis are widely used for structural analysis of components and intermediates of the translation machinery. However, the process of translation in plants is still poorly characterized at a structural level. Here, we present the structure of Triticum aestium small ribosomal subunit obtained at sub 3 resolution that can be used for further structural studies of the translation process in plants. Methods: The structures of the 40S subunits purified from wheat germ extract were obtained using high-resolution single particle cryo-EM. For cryo-EM sample preparation were used Quantifoil R 1.2/1.3 grids coated with an additional 2 nm amorphous carbon film were glow-discharged for 30 seconds at 15 mA using PELCO easiGlow (Ted Pella). 3 µL of the sample were applied onto the grids, blotted for 3 sec at 10°C and 100% humidity, and plunge-frozen in liquid ethane using Vitrobot Mark IV (Thermo Fisher). Cryo-EM data were collected using a Cs-corrected Titan Krios (Thermo Fisher) transmission electron microscope, equipped with a Falcon II direct electron detector. Data were acquired with defocus range of -0.6 to -2.0 at a nominal magnification of 75,000x, giving a calibrated pixel size of 0.86 Å/pixel. The micrographs were recorded as movie stacks. The exposure time for each stack was 1.6 s, corresponding to a total electron dose of ~84 e-/Å2 fractionated into 32 frames (~2.6 e-/Å2 per single frame). A total of 5521 movie stacks was collected. Raw cryo-EM data preprocessing was performed with Warp software (Tegunov et al., 2019). All further data processing steps were performed using the cryoSPARC v3.2.0 software (Punjani et al., 2017). Results: For final cryo-EM map refinement, 140,000 particles were used resulting in 2.7 Å resolution estimated using an FSC=0.143 gold-standard threshold. The obtained structural data clearly demonstrate the peculiarities of the spatial organization of the 40S ribosomal subunit, like the motility of the head relative to the body revealed by 3D variability analysis. Conclusion: The resulting structure was solved at a significantly higher resolution compared to the previously published structure of a plant ribosome (Armache et al., 2010) and will be used as a reference for further studies of translation initiation in plants.
Abstract P-28: Structural Dynamics of DNA-Associated Chaperon Facilitates Chromatine Transcription
Authors: Volokh Olesya , Sivkina Anastasia , Karlova Maria , Kotova Elena , Studitsky Vasily , Sokolova Olga
Published: October 2021

Background: Histone chaperon FACT (“FAcilitates Chromatin Transcription”) is a multifunctional and conserved eukaryotic protein involved in DNA transcription, replication and repair; which can reversibly unfold nucleosomes in presence of ATP. FACT is necessary for the viability and growth of breast tumor cells meanwhile in normal cells it can be knocked out without loss of vitality. Human FACT (hFACT) is a target for promising anticancer drug curaxins, which causes FACT trapping in chromatin of cancer cells and destabilizes the nucleosome. The nucleosome-unfolding activity is an important function of hFACT in vivo; however, the mechanism of FACT-dependent nucleosome unfolding remains unknown. Methods: Here, we studied negative stained hFACT structure using single particle electron microscopy using JEOL 2100 TEM. Micrographs were captured with 25k magnification, and 4.1 Å pixel size. EM images pre-processing and single particles collection were performed in EMAN2.3, followed by 2D-particles analysis in RELION2.0. Final 2D-classes included ~70 000 single particles images. Results: Based on 2D-classess data analysis we evaluated several states of hFACT reflecting its conformational flexibility: the “closed” complex is characterized by four domains localized close to each other and forming a compact structure; “intermediate” state represented by classes with identified three domains having compact structure and more disordered fourth domain, and the “open” complex, represented by three domains forming almost linear structure. The “closed” and “open” states are present in comparable amounts and significantly outnumber the “intermediate” state. It has been shown that hFACT domains are connected through flexible linkers and SPT16 and SSRP1 dimerization domains (DDs) form the “joint”-like connection between the two subunits. In the “сlosed” conformation, the DNA-binding surface of FACT is covered by its two C-terminal and middle domains (MDs). The N-terminal domain (NTD) of SPT16 was not resolved previously, but it is the best candidate for the forth domain that is clearly visible only in the “closed” conformation of hFACT, based on its dimensions and the longest linker length. Conclusion: We propose that during conversion to the “open” complexes SPT16 NTD is moving away from the other subunits leading to formation of the first intermediate state with the NTD domain poorly resolved or not resolved, while less mobile DDs and MDs maintain more compact structure and the DNA-binding site is still protected by the CTDs. In the “open” state SPT16/SSRP1 visible MDs and DDs form almost linear structure, unmasking the DNA-binding sites and making them accessible for the interaction with a nucleosome.
Abstract P-29: Cryoem Study of the Inhibition of Bacterial Ribosomes by Madumycin II
Authors: Yakusheva Alena , Shulenina Olga , Pichkur Evgeny , Paleskava Alena , Myasnikov Alexander , Konevega Andrey
Published: October 2021

Background: The efficiency of widely used antibiotics is limited by continuous improvement of resistance mechanisms. Thus, the research of poorly studied drugs that have not received practical use until now becomes relevant again. Protein translation is one of the major targets for antibiotics. Madumycin II (MADU) is an antibiotic of the streptogramin A class that binds to the peptidyl transferase center of the initiated bacterial 70S ribosome inhibiting the ﬁrst cycle of peptide bond formation (I.A. Osterman et al. Nucleic Acids Res., 2017). The ability of MADU to interfere with translating ribosome is an open question that we address by investigation of high-resolution cryo-EM structures of MADU bound 70S ribosome complexes from Escherichia coli. Methods: Purified initiated and translating ribosome complexes preincubated with MADU were applied onto freshly glow discharged carbon-coated grids (Quantifoil R 1.2/1.3) and flash-frozen in the liquid ethane pre-cooled by liquid nitrogen in the Vitrobot Mark IV. Frozen grids were transferred into an in-house Titan Krios microscope. Data were collected using EPU software. Movie stacks were preprocessed in Warp software. For image processing, we have used several software packages: Relion 3.1, CryoSPARC, and CisTEM. The model was built in Coot. Results: We have obtained high-resolution cryo-EM structures of two ribosomal complexes with MADU before and after the ﬁrst cycle of peptide bond formation with an average resolution of 2.3 Å. Preliminary analysis of the structures shows no major differences in the MADU binding mode to the ribosomal complexes under study suggesting that the quantity of amino acid residues attached to the P-site tRNA does not impact MADU bonding. Moreover, in both cases, we observed similar destabilization of the CCA-ends of A- and P-site tRNAs underlining the comparable influence of MADU on the ribosomal complexes. Conclusion: Our results suggest that although MADU binding site is located in the peptidyl transferase center, the presence of the second amino acid residue on the P-site tRNA does not preclude antibiotic binding. We assume that further elongation of the polypeptide chain would not have any impact either. High conformational lability of the CCA-ends of tRNA at the A and P sites upon binding of MADU obviously plays an important role in the inhibition mechanism of the bacterial ribosome. The further structural and biochemical analysis will be necessary to shed more light on the detailed mechanism of MADU action.
Abstract P-30: Structure of the 80S Ribosome From Candida Albicans Revealed by Integrative Structural Biology Approach
Authors: Zgadzay Yuriy , Kolosova Olga , Stetsenko Artem , Usachev Konstantin , Validov Shamil , Guskov Albert , Yusupov Mara
Published: October 2021

Background: The fungus Candida albicans is one of the most common fungal human pathogens, causing numerous mucocutaneous infections. About 80% of the world population is colonized with this pathogen, and in many cases, it does not reveal its pathogenic properties. However in the events when the suppression of normal bacterial flora occurs, e.g. as a consequence of chemotherapy or antibiotics treatment, multi-resistant strains of Candida albicans can rapidly colonize the host organism and prevent the normal flora recovery, which in turn can trigger many other diseases especially in the case of immunocompromised patients. Methods: We used an integrated structural biology approach based on the single-particle cryo-EM reconstruction and macromolecule X-ray crystallography. Results: One of the most promising targets for antibiotic action is a protein synthesis apparatus and ribosomes in the cell. In this study, we characterized structurally the protein synthesis machinery of Candida albicans using the state-of-the-art techniques of single-particle Cryo-electron microscopy and macromolecular X-ray crystallography. Conclusion: We obtained the 2.4 Å resolution structure of the 80S ribosome from Candida albicans and the 4.2 Å resolution structure of the vacant C. albicans ribosome by X-ray crystallography. We believe that this study will shed light on mechanisms of antimicrobial resistance in C. albicans and improve candidiasis treatment in the future.
Abstract P-32: Lung Structural Changes in Covid-19 Patients
Authors: Kartashev Valeriy , Mardonov Djamshid , Mamirov Bahshillo , Butaev Azamat
Published: October 2021

Background: The main causes of death from Covid-19 are lung lesions with the development of respiratory failure. However, structural changes in the lung tissue in this pathology are poorly studied. We examined autopsy material from patients with Covid-19. The severe condition of patients, the manifestation of pulmonary symptoms of damage (cough, dyspnea) and the high probability of viral pneumonia at COVID-19 led to the widespread use of CT diagnostics in this group of patients, which allowed to identify of the primary signs of the disease, their subsequent transformation as well as the most adverse radiation symptoms corresponding to the severe course of the process (Speranskaya, 2020; Pan et al., 2019). In the cases studied by us, the detection of typical symptoms revealed by radiation diagnostics of COVID-19 was subsequently confirmed by PCR data, which may indicate a high information content and specificity of detecting CT symptoms of a lesion as a method of primary diagnostics. The aim of our study is to confirm at the microscopic level the correspondence of MSCT changes. Methods: The material was taken at autopsy of deceased patients, fixed in 10% formalin solution in phosphate buffer, paraffin sections were stained with hematoxylin and eosin. The autopsy material was examined using a Carl Zeiss light microscope, Axioskop 40. Results: Studies have shown that most of the alveoli of the lung tissue kept their airiness. However, their lumens were significantly reduced due to a significant thickening of the interalveolar septa caused by pronounced inflammatory infiltration mainly by lymphocytes. The most significant changes were revealed from the side of the microvasculature. There are numerous blood clots of various sizes in the lumen of most micro-vessels. The walls of microvessels have been significantly thickened with pronounced inflammatory infiltration and significant edema. The lumens of microvessels have been characterized by significant polymorphism. Intraluminal clots have been also characterized by pronounced polymorphism. The parietal pleura has been thickened. This has been reflected in the MSCT images and corresponds to the processes of perivascular infiltration. Conclusion: The primary CT pattern of COVID-19 is a picture of infiltration of individual secondary pulmonary lobules of the "ground glass" type, followed by a decrease in the lesion volume at a favorable course of the disease, or their increase, the addition of a CT picture of a "cobblestone pavement" and the appearance of alveolar infiltration in the area of "ground glass" at the unfavorable course of the disease.
Abstract P-39: Fluorescent Silver Nanoclusters with Immunoglobulins and Albumins
Authors: Morozova Olga , Shevlyagina Nataliya , Zhukhovitsky Vladimir
Published: October 2021

Background: Multiplex biomedical assays including molecular genetic tests and immunoanalysis require multiple fluorophores with a wide excitation range and different emission spectra. In comparison with organic fluorophores and quantum dots, the metal nanoclusters (NC) consisting of a few to hundred atoms have the following advantages: small size, large Stokes shift, prolonged fluorescence lifetime and biocompatibility. Our research was aimed at construction of fluorescent AgNC with the main blood proteins and transmission electron microscopy (TEM). Methods: AgNC were synthesized from AgNO3 in the presence of albumins and immunoglobulins (Ig) of different classes and origin at pH 9-11 with NaBH4 recovery. The resulting AgNC with proteins were loaded to "Formvar/Carbon 200 Mesh Copper" copper grids (Ted Pella, USA) and examined using TEM system JEM 2100 Plus (JEOL, Japan) without contrast. Fluorescence excitation/emission spectra were measured in quartz cuvette using the FluoroMax + spectrofluorometer (Horiba Scientific, Japan). Results: Recovery of Ag+ ions did not occur in the presence of IgG and albumins without NaBH4 at different temperatures, pH, and incubation time. Broad excitation spectra of AgNC were in a range 340-540 nm. Their emission spectra correlated with the original AgNO3 concentration and did not depend on protein and pH. NC stabilized with IgG or albumin with blue fluorescence and emission maximum at 420 nm contained NC from 0.6 nm and higher. Green AgNC with proteins had bright fluorescence at 430-470 nm and red NC showed emission maximum at 650 nm. TEM revealed discrete AgNC and their numerous aggregates in each sample of fluorescent NC in spite of different fluorescent emission spectra. According to the MTT test, AgNC with human IgG and BSA with protein concentrations up to 3 mg/ml were not toxic for human larynx carcinoma HEp-2 cells despite cytotoxicity of silver nanoparticles covered with IgG or albumin envelopes as well as Cd and AuNC with BSA. Conclusion: AgNC with antibodies and albumin with a broad size range and aggregation possess tunable fluorescence emission spectra with broad excitation at 340-540 nm. Different emission spectra permit AgNC to be used in multiplex assays. AgNC were not toxic for human tissue culture and may be applied for bioimaging.
Abstract P-4: Robust Method for Background Subtraction in Serial X-ray Diffraction Data
Authors: Marin Egor , Vakhrameev Daniil , Gusach Anastasiia , Luginina Aleksandra , Kovalev Kirill , Liu Wei , Weierstall Uwe , Park Jaehun , Nam Ki Hyun , Yunje Cho , Mishin Alexey , Cherezov Vadim , Borshchevskiy Valentin
Published: October 2021

Background: Membrane receptors play an important role in signal transduction across the cell membrane in all living organisms. Their structural studies have been enabled by multiple technological breakthroughs in their heterologous expression, stabilization, crystallization, and crystallographic data collection as well as in cryogenic electron microscopy (cryoEM). During the last decade, serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has enabled structure determination of previously inaccessible proteins, including several G-protein-coupled receptors (GPCR), that produce only micrometer-sized crystals, thus paving the way towards understanding their activation mechanism and rational drug discovery. In addition to experimental difficulties, membrane protein structure determination is also often accompanied by data processing challenges. In particular, the lipidic cubic phase that serves as a carrier for membrane protein microcrystals, as well as various XFEL beam-shaping devices may generate substantial background scattering that could complicate the structure factor extraction from the diffraction images. Methods: In this work, we tested an adaptation of the denoising algorithm via matrix decomposition to XFEL-SFX data. We benchmarked its performance using high-background data from PAL-XFEL and established its applicability to serial crystallography image denoising, as well as compared it to the CrystFEL-based image denoising algorithm. Results: We find that, although the decomposition-based image denoising does not outperform CrystFEL median subtraction, it performs better than the integration without any additional subtraction. We find the non-negative matrix factorization performing better than more traditional singular-value decomposition methods, both in terms of visual interpretability and final data quality. Conclusion: We hope that this work will draw attention to background subtraction methods in structural biology, and will pave the way towards processing of most challenging datasets in structural biology, in particularly, those collected from membrane proteins.
Abstract P-40: The Shape and Size of the Recombinant Virus-like Particles Were Checked by Means of Electron Microscope
Authors: Okhrimenko Ivan , Zagryadskaya Yulia , Tsarenko Alexey , Borshchevskiy Valentin , Rogachev Andrey , Shevchenko Marina
Published: October 2021

Background: Nucleocapsid protein of hepatitis-B virus (HBcAg) recombinantly synthesized in prokaryotic and eukaryotic cells is known to be capable to self-assemble into highly immunogenic stable viral-like particles (VLP) of icosahedral shape with a characteristic size of 32 nm (Schödel et al., 1994; Murray and Shiau, 1999). The VLP formation is tolerant to the insertion of some artificial epitopes to N- and C-termini of HBcAg monomer and also into major insertion region (MIR), forming a spike on the surface of VLP (Tordjeman et al., 1993, Peyret et al., 2015). Methods: We have investigated the possibility of heterologous expression of de novo designed gene coding the first 148 amino acid residues of HBcAg (Pumpens and Grens, 1999). The gene was specially designed to be suitable for the insertions of genes coding fluorescent proteins, which are desired for the studies of VLP distribution in tissues by confocal microscopy. Gene was optimized for overexpression in E. coli producer strains and special attention was taken to obtain a simple purification scheme, which reliably reduces the amount of pyrogens in purified VLP. The MIPT scientific platform of electron microscopy equipped with the transmission electron microscope Tecnai Polara G2 (Thermo Scientific (FEI)) was used. Carbon-coated (Lacey Carbon and 10 nm thin carbon) copper 200 mesh grids were treated with glow-discharge and coated with VLP suspension in deionized water. The samples were stained with uranyl acetate solution, air-dried, and inspected at the accelerating voltage of 300 kV. Results: The 32 nm size of heterologously synthesized VLP was successfully proved, and spherical shape was seen using negative contrasting.
Abstract P-43: Effect of Glucocerebrosidase Dysfunction on the Pool of Plasma Exosomes of Patients with Gaucher Disease
Authors: Garaeva Luiza , Kamyshinsky Roman , Kulabukhova Darya , Landa Sergey , Varfolomeeva Elena , Pchelina Sofya , Shtam Tatiana
Published: October 2021

Background: Extracellular vesicles (EVs) are small membrane vesicles released from different types of cells. EVs are found in many human biological fluids. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies with the plasma membrane. This type of vesicles is characterized by specific exosomal markers. Exosomes extracted from peripheral body liquids could have specific properties associated with different physiological conditions as well as human disorders, including neurodegenerative diseases. Gaucher disease (GD) – is the most common form of lysosomal storage disorders caused by mutations in the glucocerebrosidase (GBA) gene. Lysosome functionality is critical for the regulation of extracellular vesicle secretion and content. In model animals, the inhibition of glucocerebrosidase has been shown to increase the secretion of extracellular vesicles in brain tissues. Amount evaluation of EVs and their size in the biological fluids of patients with GD has not been early performed; therefore, it is unknown whether lysosomal dysfunction found in GD patients influences the plasma pool of EVs. The aim of this study was to evaluate the amount of blood plasma EVs in patients with GD and their characterization for morphology and size. Methods: EVs were isolated from the blood plasma of 8 GD patients and 8 controls by ultracentrifugation, and were characterized using cryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). Also, the presence of exosomal markers CD9, CD63, CD81, and HSP70 was analyzed by flow cytometry and western blot. Results: Here, it was first shown an increased proportion of exosome fraction in EVs from plasma of GD patients compared to controls by DLS and cryo-EM (p<0.001) that was confirmed by mode size detected by NTA (p<0.02). Moreover, an increased number of double and multilayer vesicles in plasma EVs from GD patients was demonstrated by cryo-EM. We also detected an increase in the expression of exosomal markers on the surface of vesicles from the blood plasma of patients with GD compared to controls. Conclusion: Here, we firstly report that the exosomes obtained from the blood plasma of GD patients have a larger size and altered morphology. Thus, we have shown that lysosomal dysfunction in GD patients leads to a striking alteration of blood plasma extracellular vesicle pool.
Análise da Implementação das Diretrizes do Programa Mais Médicos (PMM) por Municípios Brasileiros
Authors: Cezar Pinto Aderne Gomes Bruno , Molina Queiroz Juliana , Filgueiras Sauerbronn Fernanda , Loreiro Lourenço Rosenery
Published: October 2021

Resumo

Objetivo da pesquisa: Analisar as lacunas de implementação do Programa Mais Médicos (PMM) ao avaliar relatórios de auditoria da Controladoria Geral da União (CGU) e discutir características e disparidades regionais que impactam os resultados do PMM nos municípios brasileiros.

Enquadramento teórico: Esta pesquisa se enquadra na necessidade de analisar a implementação do PMM, destacada por Lima, Nunes, Santos, Silva e Melo (2017) e Alencar et al. (2016).

Metodologia: Foram analisados 149 relatórios de auditoria da CGU, de 2016, por meio da Análise de Conteúdo. Os municípios também foram avaliados a partir da Análise de Correspondência Múltipla e do Teste Qui-quadrado para compreender os aspectos que envolvem a atuação profissional, tais como moradia, alimentação, transporte e estrutura.

Resultados: Os resultados mostram que quando os municípios oferecem condições de moradia, alimentação, transporte e estrutura de forma adequada, a atuação do médico no PMM é considerada satisfatória, independentemente de o município apresentar maior/menor índice de desigualdade social. Esses elementos estão entre aqueles que contribuem para a fixação do médico nas regiões menos desenvolvidas.

Originalidade: A análise do PMM se mostra original, pois não foram encontrados trabalhos que analisassem as diferenças municipais relacionadas ao PMM, política pública que reúne um conjunto de ações do Governo Federal para melhorar o atendimento médico aos usuários do Sistema Único de Saúde nos municípios brasileiros.

Contribuições teóricas e práticas: Diante dos achados, é possível indicar contribuições teóricas para a literatura sobre a PMM. Também é possível indicar que para que se possa melhorar a PMM em qualidade, é necessário que os médicos recebam melhores condições.
Gestão Fiscal Municipal: uma análise sob a ótica do federalismo fiscal e dos ciclos políticos nos governos locais
Authors: Oliveira Tomas Matheus Giacomel de , Dall'Asta Denis , Zonatto Vinícius Costa da Silva , Martins Vinicius Abilio
Published: October 2021

Objetivo da Pesquisa: Analisar os fatores que influenciam a gestão fiscal municipal sob a ótica da teoria do federalismo fiscal e da teoria dos ciclos políticos nos governos locais. Enquadramento Teórico: Este estudo aborda a gestão fiscal municipal, o federalismo fiscal e os ciclos políticos. Metodologia: Utilizou-se de modelos de regressão para dados em painéis, nos governos locais representados pelos municípios do Paraná, no período de 2013 a 2019. Resultados: As variáveis representativas da autonomia arrecadatória influenciam positivamente os índices da dívida consolidada líquida e os gastos com pessoal. Também se identificou que a condição financeira auxilia na explicação dos indicadores da gestão fiscal. Por fim, com base nos ciclos políticos, observa-se uma tendência ao comportamento oportunista nos períodos eleitorais, já que ambos os indicadores tendem a ser maiores nos períodos de eleições locais. Originalidade: Explorar a gestão fiscal nos governos locais, sob as lentes teóricas do federalismo e dos ciclos políticos em diferentes cenários é relevante tendo em vista as particularidades federativas, fiscais e políticas, em especial nos governos subnacionais. Contribuições teóricas e práticas: A pesquisa apresenta contribuições teóricas/práticas ao retratar os fatores que impactam a gestão fiscal nos governos locais, sob a lente das abordagens teóricas do federalismo fiscal e dos ciclos políticos, demonstrando os elementos que podem afetar o equilíbrio das contas públicas.
HEDGEMONY: A Wargame to Evaluate Senior Joint Professional Military Education Learning Objectives
Authors: Cole Brian W.
Published: October 2021

The Officer Professional Military Education Policy directs Joint professional military education institutions to develop officers who demonstrate critical and creative thinking skills. The chairman of the Joint Chiefs of Staff ’s intent is to develop strategically minded officers who will “creatively apply military power to inform national strategy, conduct globally integrated operations, and fight under conditions of disruptive change.”1 The wargame Hedgemony is unlike most other wargames. Its focus is on teaching defense professionals how strategies are a complex interaction between force development, force posture, and force employment. Hedgemony also provides a way in which the Marine Corps War College measures its program outcomes.
Análisis cinemático de movimientos realizados en dispositivos de acondicionamiento físico tipo Five Minutes Shaper
Authors: Guerrero-Méndez Cristian David , Moreno-Arévalo Brayan Sneider , Ramírez-Ruiz Valery , Duarte-González Mario Enrique , Ruiz-Olaya Andrés Felipe , Jaramillo-Isaza Sebastián
Published: October 2021

Debido al aumento en el uso de dispositivos de acondicionamiento físico por parte de sujetos sin entrenamiento, la falta de evidencia científica de los dispositivos y los efectos que puede tener la mala ejecución de los movimientos en cuanto a lesiones osteomusculares, en este artículo se presenta un estudio de la actividad física realizada en el dispositivo de entrenamiento físico Five Minutes Shaper por medio de un análisis cinemático, en usuarios sin acondicionamiento físico. Se realizó una caracterización de las variables cinemáticas de velocidad, aceleración y amplitud articular relacionadas con el movimiento, de tres de los seis niveles del dispositivo —fácil, medio, extremo— en las articulaciones de la rodilla y la cadera. Además, se realizó una evaluación de significancia estadística para evaluar el efecto de los diferentes niveles analizados del dispositivo sobre las variables cinemáticas, en hombres y mujeres. La digitalización del movimiento se realizó mediante un sistema óptico de captura de movimiento, el análisis cinemático mediante el software Kinovea y Matlab, y el análisis estadístico por medio del software SPSS. En los experimentos han participado un grupo de cinco hombres y cinco mujeres, quienes cumplieron con los criterios de inclusión. Como resultados se obtiene una caracterización cinemática entre géneros que no cuentan con acondicionamiento físico para el movimiento de las dos articulaciones, así como para cada nivel analizado en el dispositivo Five Minutes Shaper. El estudio presenta un enfoque en la articulación de la rodilla y la cadera, dado que son estas los ejes principales en el movimiento.
MICROBIAL EXAMINATION OF MOULD AND YEAST IN FRUIT JUICES
Authors: Alam Ayesha
Published: October 2021

Fruit juices consists of water, carbohydrates, protein, lipids, acids, phenolics, dietary fibre and pigments that play a vivacious part in human nutrition, as they supply vitamins and essential minerals which are necessary growth factors in human diet and that can assist in maintenance of a good and healthy life. However due to their short shelf life these juices either glass bottles, plastic bottles, paper cups and plastic cups or unpacked can be contaminated by micro flora (especially mould and yeast) and can become a source of health hazard. Hence current study was focused to examine the diversity of mould and yeast contaminants in different types of packed and unpacked fruit juices (pH ranges from 3.35 to 4) of six different localities of Lahore such as Chouburji, Govt College of Science, Mozang, Muslim Town, Shadman and Iqbal town. The fungal contaminants (mould and yeast) isolated from their respective juices were cultured on malt extract agar as well as on potato dextrose agar under sterilized conditions. Serial dilution technique was used to compose the replicates in order to get viable count of micro flora in juices samples. Results indicated that in case of packed juices, the maximum fungal viable count was found in samples of Shadman (10x10-4 to 14x10-5 cfu/ml) while minimum was found in Chouburji samples (2x10-4 to 5x10-3 cfu/ml). The highest mould (803000 cfu/ml) in packed juices was found in locality of Mozang, but lowest value was observed in samples obtained from Muslim town i.e., 16000 cfu/ml. However, yeast contaminants were found in great concentrations in Shadman samples (71000 cfu/ml) and least count was found in locality of Iqbal town i.e., 200000 cfu/ml. For unpacked (fresh) juices the peak value of fungal viable count was observed in Shadman (8x10-5 to 14x10-5 and 18x10-5 ) while minimum was found in GCS (4x10-4 to 7x10-3 ). The uppermost value of mould count (1800000 cfu/ml was also observed in locality of Shadman and least mould (25000 cfu/ml) in unpacked juices was found in locality of GCS. Whereas maximum yeast i.e., 400000 cfu/ml was detected in locality of Shadman and least one 11000 cfu/ml was reported in Chouburji. Reported fungal species were Aspergillus niger, Aspergillus terreus, Penicilium digitatum, Curvularia americanna, Fusarium moniliforme, Alterneria alternate and Saccharomyces cerevisae. Results indicated that mould and yeast (fungal) count is much more standard tolerable range (102 to 103 cfu/ml) hence this negligence and unawareness can lead to serious health damage of consumers
EVALUATION OF NEW PROMISING RICE HYBRID AND ITS PARENTAL LINES FOR FLORAL, AGRONOMIC TRAITS AND GENETIC PURITY ASSESSMENT
Authors: Anis Galal
Published: October 2021

Development of new rice hybrids with high-yielding and superior in grain quality is the most important goal to enhance food security as well as to achieve consumer requirements. High seed costs and poor grain quality are the major limiting factors for adoption of hybrid rice. A practical way to improve hybrid seed quality and to increase production is possible to increase the seed setting rate through improving the floral traits for parental lines. In the context, the study was conducted at the Rice Farm and Laboratories of Sakha Agricultural Research Station, Sakha, Kafr El-Sheikh, Egypt in consecutive three rice growing seasons in 2015, 2016 and 2017. The study focused mainly on characterization of the new promising hybrid ‘G46A/GZ9399R’ and its parental lines. The cytoplasmic male sterile (CMS) line and restorer line were evaluated for twenty-three floral traits that influence outcrossing rate in hybrid rice under two seasons and there was a marked stability for most of these traits. As compared with commercial check variety ‘Giza 178’, the new promising hybrid ‘G46A/GZ9399R’ showed its superiority in all agro-morphological traits except plant height. This hybrid was very early, adapted widely and recorded 20.19% more yield than commercial check variety ‘Giza 178’. Instead, this hybrid produced superiority in all grain quality properties except grain length was medium grain and amylose content was equal for both studied genotypes compared to the check variety. In parallel, the assessment of genetic purity established the SSR markers RM154, RM331, and RM297, amplified alleles specific to differentiate parental lines of hybrid ‘G46A/GZ9399R’.