bluuebunny/crossref_metadata_2025 huggingface dataset:
FORMATION OF THE COMPOSITION OF INFLUENTIAL HARMFUL AND DANGEROUS FACTORS OF THE PRODUCTION SYSTEM
Authors: Kalinchyk V. , Kalinchyk V. , Pobigaylo V. , Melnyk D.
Published: October 2021

The article analyzes the production system and the composition of the influencing harmful and dangerous factors. Glass and glass products are analyzed as the object of research. The technology of glass and glass products production is presented. It is shown that the technology of glass production consists of two production cycles: the cycle of glass technology and the cycle of glass products technology. Glassmaking is carried out in

three stages: the first stage - heating in glass furnaces, when there is a chemical interaction and clarification of the viscous mass, the second stage - lighting, the removal of bubbles, as well as dissolving the remaining undissolved grains of sand and the third stage - cooling glass to temperature convenient for making products from it. In such technology it is possible to allocate the following characteristic production processes which are characterized by existence of dangerous and harmful to the person factors. Transportation of raw materials is characterized by the influence of noise, the impact on the respiratory tract of aerosols, the influence of ergonomic factors, occupational injuries. Burning and melting is characterized by the influence of combustion products, the influence of smoke and aerosols of raw materials by fires or explosions, the influence of infrared radiation of molten material, electrocution, noise. Manual operations in the process of production, processing, packaging and storage in the warehouse and restoration work and reconstruction is characterized by various injuries. These factors make up the factor field of dangerous human influences, which contains four groups of factors: mechanical - noise, ergonomic factors of production, occupational injuries; temperature - thermal radiation, exposure to combustion products, fires and explosions, heat stroke and burns; the effect of aerosols on the human respiratory tract, the effect of auxiliary chemicals; electric – electrical unjury, the influence of electromagnetic fields, as well as the influence of the microclimate of industrial premises, which is determined by the combinations of temperature, humidity and air velocity acting on the human body, the temperature of the surrounding surfaces. It is shown that the level of influence of dangerous and harmful factors can be expertly established using the known method of rank correlation. The obtained results can be used for preliminary expert assessment of the impact of hazardous and harmful factors on the personnel of the enterprise for the production of glass and glass products.
WASTEWATER TREATMENT FROM HEAVY METAL IONS
Authors: Zhukova N. , Kryuchkov A. , Zaichenko S. , Smoliar V.
Published: October 2021

The article considers the problem of wastewater treatment of industrial enterprises containing heavy metal ions. Because of insufficient treatment of wastewater from heavy metal ions, they enter ecological systems, accumulate in them and cause toxic effects on phyto and zoocenosis, even in small concentrations, which in turn leads to imbalance of ecosystems and their degradation. Therefore, the treatment of industrial effluents is becoming increasingly important over time. The application of existing methods and sorbents for purification of industrial effluents from heavy metal ions is considered and analyzed.However, existing combined wastewater treatment methods and sorbents have little effectiveness against heavy metals. The paper proposes the use of clay- based sorbents. The purpose of research is to substantiate the feasibility of using sorbents based on clays of deposits in Ukraine for wastewater treatment of industrial enterprises from heavy metal ions. Clays of domestic deposits are not scarce, inexpensive in comparison with existing sorbents. Samples of sorbents based on clay were studied, their composition was developed and analyzed.A method for obtaining clay-based sorbents has been developed. Materials such as peat, wood ash and dolomite were used as activating additives. The composition and structure of the developed sorbents, as well as their sorption capacity and ability to heavy metals have been studied. These sorbents have been shown to be an effective material for cleaning industrial effluents from heavy metal ions and can be recommended for use.
Оцінка якості виготовлення роз’ємних конструкцій паперово-білової продукції
Authors: Хмілярчук Ольга Іларіонівна , Чепурна Катерина Олександрівна , Клішина Марія Олександрівна
Published: October 2021

Комплексний показник якості видань з роз’ємними блоками — це характеристика, визначена у кількості декількох факторів, їх властивостей, які визначають якість продукції. Цей показник визначається відповідно до експлуатації та способів виготовлення.
Серед факторів, що впливають на якість виготовлення роз’ємних конструкцій паперово-білової продукції виділено наступні: виготовлення блоку, зошитів, сторінок; обладнання, матеріали, технологія, методи та засоби вимірювання, персонал (економічна зацікавленість, умови праці, кваліфікація та спеціалізація, дисципліна). Проаналізувавши вплив людських ресурсів, розглянуто ранжування факторів ризику. При розгляданні матеріалів як складової фактору виготовлення, було визначено, що на довготривалість виготовлення впливає тип конструкції, складність конструкції (кількість деталей, складність скріплень), час ручних та машинних операцій при брошурувально-палітурних процесах.
В результаті виконаних досліджень проведено загальний аналіз факторів впливу на якість роз’ємних конструкцій паперово-білової продукції, їх ранжування; представлено комплексний показник якості даного виду продукції,  розроблено діаграму Ісікави. Згідно із розробленою причинно-наслідковою діаграмою, систематизовано чинники впливу на якість виготовлення роз’ємних конструкцій паперово-білової продукції. Представлена діаграма дозволяє встановити можливі причини отримання браку на всіх етапах виготовлення продукції, провести аналіз причин появи браку та знайти шляхи їх усунення, що в цілому дозволить покращити якість.
За результатами опитування двох експертних груп з користувачів (група 1) та виробників (група 2) продукції встановлено, що дані не завжди корелюються між собою. Погляди користувача та виробника деколи відрізняються. Це спричинено тим, що при виробництві окремим фактором при оцінці виробу до уваги береться технологія виготовлення. Тобто в певних випадках використання того чи іншого матеріалу може бути недоцільним або неможливим. Відповідно продукція, що цікава споживачам, досить часто викликає складнощі в технологічних процесах виготовлення на виробництві.
За результатами проведених досліджень було розроблено алгоритм керування якістю виготовлення роз’ємних конструкцій паперово-білової продукції, чим було сформульовано вимоги до продукції та потреби або очікування користувачів. Якістю визначається ступінь, для якого сукупність характеристик задовольняє вимоги, що призводять до задоволення замовника та його сприйняття ступеня виконання вимог.
Розроблення пристрою для зрізування корінцевих фальців книжкових блоків дисковими ножами з планетарним приводом
Authors: Книш Олег Богданович
Published: October 2021

За результатами аналізу встановлено, що міцність незшивного клейового скріплення книжкових блоків залежить від якості обробки корінців та виду клею. Проаналізовано пристрої для зрізування корінцевих фальців книжкових блоків при незшивному клейовому скріпленні. Встановлено, що застосування циклових механізмів у приводі різальних інструментів має ряд недоліків. Це значні інерційні навантаження та складність конструкції. Розглянуті варіанти застосування дискових ножів для зрізування корінцевих фальців мають ряд обмежень.
З урахуванням проведеного аналізу запропоновано удосконалений пристрій для зрізування корінцевих фальців. Він складається із дискових ножів, що приводяться в рух від планетарного механізму. Для якісного функціонування такого пристрою застосовано нерухомий контрніж. Розглянуто будову та принцип роботи такого пристрою. Акцентовано увагу на перевагах пропонованого пристрою порівняно із аналогами. Виходячи із умови застосування пристрою на виробничих машинах незшивного скріплення запропоновано основні геометричні параметри: радіус дискових ножів, габарити планетарної передачі.
Проведено аналітичні розрахунки та побудовано траєкторію руху довільної точки ножа. Встановлено, що траєкторією руху ножа є епіциклоїда. Досліджено вплив геометричних параметрів планетарної передачі на траєкторію руху ножа. Обґрунтовано доцільність застосування для привода ножа планетарного механізму, що забезпечує його рух по вкороченій епіциклоїді. Запропонований пристрій забезпечить «ковзаюче» різання. Це надасть можливість зменшити технологічні зусилля і потужність привода різальної секції.
З урахуванням переміщення книжкового блока та траєкторії руху ножа виведено залежність для математичного опису сліду точки ножа на корінці. Побудовано сліди двох точок послідовно розміщених ножів. Визначено величину припуску, що зрізається одним ножем.
Можливості використання лазерних пристроїв при обробці друкованої паперової продукції
Authors: Бабич Віталій , Шостачук Юрій Олександрович
Published: October 2021

У процесі роботи проведено аналітичний огляд існуючих видів паперів, визначена їх структура, особливості поверхні і механічні характеристики та інші параметри паперових матеріалів, а також визначено особливості виконання окремих технологічних процесів з можливістю використання лазерних пристроїв.
Для оптимізації якості при обробці паперових матеріалів з використанням лазерів необхідно визначати певні параметри матеріалів та їх відповідність лазерному випромінювачу відповідно до зазначеного процесу, основними з яких є довжина хвилі випромінюваного світлового променю, потужність лазерного променю, швидкість обробки матеріалу, кількість аркушів у стопі, а також якість та обробка поверхні матеріалу).
Algorithm for controlling the process of buffer wastewater neutralization
Authors: Zhuchenko Anatolii , Osipa Ruslan , Osipa Liudmyla , Ladieva Lesia
Published: October 2021

In Ukraine, the condition of surface water near industrial enterprises is extremely critical. The operation of enterprises leads to intensive water pollution with industrial and domestic wastewater. Therefore, improving the quality of treatment facilities through the introduction of automated control systems is an urgent problem.

For the operation of automated control systems for typical cleaning processes, a software package is required, which is developed on the basis of appropriate algorithmic software and mathematical models of processes. To obtain them, methods of mathematical and simulation modeling and block diagram method of algorithmization were used.

In order to assess the quality of the developed algorithm for controlling the process of buffer wastewater neutralization during operation, a comparison of control system operation based on this algorithm with the most successful foreign variants of neutralization control systems was made. Simulation for the average values of operating parameters Q = 75 m3 / h, CP = 75 g / l, and Ck = 2 g / l at minimum b = 0.02 g / l pH and maximum buffer value b = 0.47 g / l pH, and also with  unidirectional extreme combination of parameters Q = 50 m3 / h, CP = 100 g / l, b = 0,02 g / l pH and Q = 100 m3 / h, CP= 50 g / l, for minimum b = 0.02 g / l pH and maximum buffering value  b = 0.47 g / l pH shows that the best quality of transient processes is for the control system operating on the basis of the developed algorithm. For any combination of parameters, the transients for this control system provide better quality transients. Studies have shown that the control system based on the developed algorithm in comparison with the previously proposed systems provides better process control by reducing the time of transients and reducing the dynamic deviation of the output parameters, which improves the quality of wastewater treatment.

Given the non-stationary process and high requirements for the cleaning parameters, manual control of this process is beyond the power of even an experienced operator. The developed mathematical model describing the dynamics of the wastewater neutralization reactor with buffer properties and the process control algorithm made it possible to proceed to the development of the control system software, which is necessary for the automated control of this process.
The Effect of Magnetite Nanoparticles on the Growth and Development of Nicotiana Tabacum Plants in Vivo and in Vitro Culture
Authors: Gorobets Svitlana , Ilchuk Nina , Demianenko Iryna , Bannikova Maria
Published: October 2021

Background. Nanomaterials are easily modified and have unique characteristics associated with a large reactive surface Due to these properties, nanomaterials are used in various branches of sciences and technology, such as pharmaceuticals, biotechnology, chemical technology, etc. Recently, the effect of magnetite nanoparticles on the morphological properties of plants has been actively studied for their further use as nanoadditives to increase yields and improve the properties of agricultural plants. Tobacco (Nicotiana tabacum) is a model object of plant biotechnology, it is used to study the effect of various factors on dicotyledonous plants, so it was chosen to study the effect of magnetite on the growth, development, and mass accumulation by plants.
Objective. We are aimed to study the effect of magnetite nanoparticles on the growth and development of Nicotiana tabacum in vivo and in vitro.
Methods. The ability of tobacco to produce biogenic magnetic nanoparticles by searching for mammal proteins homologues in theNicotiana tabacum proteome using the Blast NCBI program was studied using comparative genomics methods. The plants were divided into groups (control, magnetite nanoparticle concentration 0.1 mg/cm3, magnetite nanoparticle concentration 1 mg/cm3) for both in vivo and in vitro experiments. Analysis of plant parameters was performed every 14 days to study the dynamics of the effects of magnetite nanoparticles.
Results. It was determined that magnetite nanoparticles at a concentration of 0.1 mg/cm3 in culture in vitro and in vivo significantly affect the growth of the root system and sprouts of Nicotiana tabacum. On the 56th day of plant cultivation in vitro on a salivary medium supplemented with magnetite nanoparticles at a concentration of 0.1 mg/cm3, an increase in the shoot length by 13.3%, root length by 31.7%, and the mass of absolutely dry substances by 18.75% was observed compared to the control. Treatment of magnetite nanoparticles with a suspension at a concentration of 0.1 mg/cm3 led to more pronounced results when growing tobacco in vivo. So, on the56th day, the root length increased by 23.3%, the length of the shoot – by 19.2%, and the mass of absolutely dry substances – by2 times, the first leaves appeared 2 days earlier compared to the control. The addition of magnetite nanoparticles to the substrate on which the plants were grown in vivo at a concentration of 1 mg/cm3 inhibits the growth of tobacco.
Conclusions. Studies have shown the expediency of using magnetic nanoparticles at a concentration of 0.1 mg/cm3 as nanofertilizers in tobacco cultivation.
Ruralidades, Escuela y Comunidades: configuración de deseos en jóvenes de la Escuela “Las Aves” de Colombia
Authors: Moncada Jennifer Munireh Cortes , Nicoletti Felipe Navarro , Aguirre Jimena Isabel Petrona
Published: October 2021

Las juventudes rurales se encuentran en la disyuntiva –muchas veces– de decidir dejar sus comunidades en busca de un futuro mejor a causa de diferentes factores (psicosociales, económicos, culturales). Este trabajo surge a partir de una pasantía investigativa que promovió el diálogo interdisciplinario en el marco del recorrido de escuelas rurales de la norpatagonia argentina y, por otra parte, instituciones educativas colombianas. En este caso, el estudio se centra en la Escuela “Las Aves” en la comunidad El Águila, ubicada en el Norte del Cauca colombiano. Se problematizan categorías como territorio-comunidad, liderazgo educativo y social y memoria cultural. La metodología utilizada fue cualitativa con enfoque psicosocial comunitario, y se apeló a la técnica video-documental para el análisis del contenido narrativo discursivo. Asimismo, se realizaron entrevistas narrativas a referentes claves como líderes educativos, rector de escuela, y jóvenes que cursan séptimo año de la básica secundaria. El análisis propuesto (desde el prisma educativo, de la psicología comunitaria y la comunicación) fortaleció procesos investigativos que solidifican aportes –especialmente– desde las voces y deseos de los jóvenes para la visibilización de la conversación del derecho a condiciones de progreso e inclusión social.
2001-2021, duas décadas da Brigada Nacional de Teatro do MST Patativa do Assaré: ressonâncias, desafios e interfaces com o trabalho teatral da Educação do Campo
Authors: Villas-Boas Rafael Litvin
Published: October 2021

O artigo analisa a experiência de duas décadas de atuação da Brigada Nacional de Teatro do MST em comunidades camponesas de diversos estados brasileiros, por meio das metodologias da observação participante, da revisão bibliográfica e da pesquisa qualitativa O trabalho evidencia as tradições do teatro político e popular que ensejam a experiência teatral do MST. Um dos resultados da pesquisa é destacar que a peculiar dinâmica de produção e circulação da Brigada teve desdobramentos em universidades públicas, nas Licenciaturas em Educação do Campo, demonstrando o potencial de construção de uma cultura política contra-hegemônica.
Palavras-chave: teatro do oprimido, MST, cultura política, educação do campo.
 
2001-2021, two decades of the National Theater Brigade of the MST Patativa do Assaré: resonances, challenges and interfaces with the theatrical work  of Rural Education
ABSTRACT. The article analyzes the experience of two decades of work by the MST National Theater Brigade in peasant communities in several Brazilian states, through the methodologies of participant observation, literature review and qualitative research. The work highlights the traditions of popular and political theater that give rise to the theatrical experience of the MST. One of the results of the research is to highlight that the peculiar dynamics of production and circulation of the Brigade had consequences in public universities, in the Degrees in Rural Education, demonstrating the potential for building a counter-hegemonic political culture.
Keywords: theater of the oppressed, MST, political culture, rural education.
 
2001-2021, dos décadas de la Brigada Nacional de Teatro del MST Patativa do Assaré: resonancias, desafíos e interfaces con el trabajo teatral de Educación del Campo
RESUMEN. El artículo analiza la experiencia de dos décadas de trabajo de la Brigada Nacional de Teatro MST en comunidades campesinas de varios estados brasileños, a través de las metodologías de observación participante, revisión de literatura e investigación cualitativa. El trabajo destaca las tradiciones del teatro popular y político que dan origen a la experiencia teatral del MST. Uno de los resultados de la investigación es resaltar que la peculiar dinámica de producción y circulación de la Brigada tuvo consecuencias en las universidades públicas, en los Grados en Educación Rural, demostrando el potencial para construir una cultura política contra hegemónica.
Palabras clave: teatro del oprimido, MST, cultura política, educación del campo.
Investigating Benefits and Barriers of Distance Education during Coronavirus Pandemic
Authors: Alotaibi Khalid Abdullah
Published: October 2021

This study aims to identify benefits and barriers to distance education, particularly from the perspective of teachers in Saudi Arabia. As the applied data collection tool, a questionnaire was distributed to the general education teachers in three districts. The sample size of the study was 1076 teachers. The results revealed that despite several benefits gained from distance learning, there are also some barriers. Teachers found that the most important advantage in distance learning is the acquisition of technical skills during the online teaching processes, they learn more and use digital education platforms, they have sufficient time to prepare the scientific content, they were able to provide adequate technical solutions for their courses, and they have the opportunity to use multiple media to deliver their courses. With the introduction of distance learning, teachers have explored new ways to deliver course contents to students. It has fostered better ways to provide more interactive real-time and on-demand teaching and learning using modern technology, thus, helping teachers become familiar with the use of electronic resources. It seems that teachers invest in technical methods to enhance students’ performance. Also, teachers reported some obstacles that they face during remote teaching. Most of these problems are connection problems applied with devices and the internet, lack of students’ motivation to learn in distance, problems associated with urban learners.
Circular Economy Business Model and Purchase Intention for Recycled PET Products by Brazilian Consumers
Authors: Queiroz Fernanda Cristina Barbosa Pereira , Lima Nilton Cesar , Silva Christian Luiz da , Queiroz Jamerson Viegas , Souza Gustavo Henrique Silva de
Published: October 2021

Circular Economy involves structural changes in traditional business models and consumers’ behaviour toward recycled products. The recycling of PET products has been increasing but there is still a gap between consumption and demand for PET packaging in Brazil. In this research, the waste reduction was examined under a projective scenario to the current ecological purposes in Brazil for reducing environmental pollution. In that manner, this paper aimed to comprehend the intention to purchase recycled PET products of Brazilian consumers. With a non-probabilistic and convenience sample, the study counts 422 participants. The method employed was based on Structural Equation Modelling and Partial Least Squares, used to test the hypotheses of causality among the variables. Results showed the perception of low quality about recycled products reduced consumers’ intention to purchase. Also, the sustainability of recycled products positively influenced the intention to purchase. In addition, recycled products presented a negative impact on the perceived safety, which could influence the purchase of recycled products in Brazil. We conclude that the image that Brazilian consumers have about recycled products positively affects the intention to purchase.
PENGEMBANGAN TEPUNG KECAMBAH KACANG TUNGGAK (Vigna unguiculata) SEBAGAI SEREAL FUNGSIONAL KAYA SERAT PANGAN DAN BERPOTENSI ANTIOKSIDAN
Authors: Ariviani Setyaningrum , Sholihin Nurul Hidayati , Nastiti Galih Poetri
Published: October 2021

The average consumption of dietary fiber in Indonesian is still below the recommended daily intake. Low dietary fiber intake has an impact on the development of degenerative diseases and metabolic syndrome. One of the risk factors for the occurrence of the disease is oxidative stress. The most effective strategy to overcome oxidative stress is through dietary antioxidants intake. Cowpea is proven to be rich in bioactive compounds such as flavonoids, dietary fiber as well as antioxidants, and these compounds have been successfully increased by germination. This study aims to develop cowpea sprouts flour as a functional cereal high in dietary fiber and antioxidants. The cereal was prepared by the best formula based on two steps of sensory analysis. The first one, the sensory analysis conducted to select the formula based on the best sensory quality of the cereal made by the various proportions of cowpea flour (%b/b) (80, 85, 90, 95, 100). The selected formula was further used to prepare the cereal with different flavoring agents (5% b/b) (ginger, cinnamon, and ginger-cinnamon) to find the best formula based on the preference level. The cereals were further analyzed to determine the sensory quality compared to the commercial cereal, the dietary fiber content which includes the total, soluble, and insoluble dietary fibers, as well as the antioxidant potential with the total phenolic content (TPC) evaluation. Formula with 80%(b/b) cowpea flour and 5% (b/b) ginger as flavoring agents produce cereal with the highest preference level and stated as the best formula. The cowpea sprouts cereal exhibited a significantly higher quality of aroma, texture, and overall than the commercial ones. The cereal also showed the highest levels of total, soluble and insoluble dietary fiber as well as the TPC. These results have significant implications on the development of functional cereal using locally legumes sprouts flour.
The Manufacturing Planning of Installation Series-Parallel Combination Centrifugal Pump Testing Equipment
Authors: Widodo Edi , Dwi Jayanto Rizky
Published: October 2021

This study discusses the planning in making a centrifugal pump installation with a combination of two series, namely series and parallel. The concept of discussion refers more to the upgrading of the installation system and calculates the estimated processing time needed to test the performance of the results of the installation that has been made. From the results of the research, it was found that for the preparation of series series installation it takes ± 8.5 minutes including the cutting process of materials and component installation, while the time required in making the installation of parallel circuits ± 28 minutes so that the total manufacturing of the two types of series takes 36, 5 minutes. In terms of performance generated from each installation obtained at full aperture where for the series of head series the value obtained is 37.6 m with a flow capacity of 3.25 l / s while for the head value on the parallel pump obtained by 27.7 m with a capacity flow of 3.75 l / s and this pump operates at 2900 rpm. So it can be seen from the results of the head and flow capacity obtained values ​​indicate good pump performance and ready to be used as a practical tool in the laboratory.
Pengaruh Kecepatan Fluida Dan Jarakantar Zig Zag Filter Foam Filler Terhadap Efektifitas Cooling Tower
Authors: Setiawan Andrea , Fahruddin A’rasy
Published: October 2021

The purpose of this study was to determine the effect of fluid velocity and the distance between zig zagfilters on the cooling tower by varying the distance between zig zag foam bio filters. In this study using bio filterfoam material in the form of zig zg by using wire mesh where the tested distance is 5cm, 10cm, and also 15cm. Thenthe water is heated using a heater until it reaches a temperature of 40ºC and then the water is flowed up with a pipeusing a pump and released using a nozzle, then the hot water will go down to the reservoir by passing the bio filterfoam filter that has been installed with a distance of 5cm , 10cm and 15cm. Where there is also a disposal valve thatis done that is closed and faucets are also open ½ and fully open. Good cooling value is indicated by good results oncooling or also cooling faster which is 5cm which results in an average cooling value of 36.53 ºC when the exhaustvalve is fully open, while the value is not good or less optimal cooling it is shown when the drain valve is closed at azig zag distance of 10cm and also 15cm which yields 37.60 ºC data at a zig zag distance of 15cm.
Acoustic nondestructive testing and measurement of tension for steel reinforcing members
Authors: McInerney Michael , Brenner Matthew , Morefield Sean , Weber Robert , Carlyle John
Published: October 2021

Many concrete structures contain internal post-tensioned steel structural members that are subject to fracturing and corrosion. The major problem with conventional tension measurement techniques is that they use indirect and non-quantitative methods to determine whether there has been a loss of tension. This work developed an acoustics-based technology and method for making quantitative tension measurements of an embedded, tensioned steel member. The theory and model were verified in the laboratory using a variety of steel rods as test specimens. Field tests of the method were conducted at three Corps of Engineers dams. Measurements of the longitudinal and shear velocity were done on rods up to 50 ft long. Not all rods of this length were able to be measured and the quality and consistency of the signal varied. There were fewer problems measuring the longitudinal velocity than shear velocity. While the tension predictions worked in the laboratory tests, the tension could not be accurately calculated for any of the field sites because researchers could not obtain the longitudinal or shear velocities in an unstressed state, or precise measurements of the longitudinal and shear velocities due to the unknown precise length of the rods in the tensioned state.
Abstract OR-2: The Formation of Dps-DNA Complexes under Different Conditions According to Cryo-EM and SAXS
Authors: Kamyshinsky Roman , Chesnokov Yury , Dadinova Liubov , Mozhaev Andrey , Vasiliev Alexander , Shtykova Eleonora
Published: October 2021

Background: The effect of Dps-DNA co-crystals formation, which occurs in stressed Escherichia coli cells exposed to extreme conditions, is well described in the literature. However, the exact mechanisms of co-crystals formation are yet to be postulated remaining largely unknown. Here we summarize the results obtained by our group over the last few years using cryo-Electron Microscopy (cryo-EM) and Small Angle X-ray Scattering (SAXS). Methods: Samples for cryo-EM were plunge frozen in liquid ethane with Vitrobot Mark IV and studied with Titan Krios (ThermoFisher Scientific, US) cryo-EM, equipped with Falcon 2 direct electron detector, Image corrector (CEOS, Germany), and Volta phase plate. Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET) studies were conducted with 300 kV accelerating voltage in low dose mode using EPU and Tomography software (ThermoFisher Scientific, US). Cryo-EM data processing was conducted using Warp, CryoSPARC, IMOD, EMAN, and Relion software packages. SAXS measurements were performed at the EMBL on the P12 BioSAXS beam line at the PETRAIII storage ring (DESY, Hamburg). Results: In this work, Dps-DNA complex formation is thoroughly studied using complementary cryo-EM (including SPA, cryo-ET, and subtomogram averaging) and SAXS methods. The formation of individual complexes of Dps with small linear DNA fragments and the Dps-Dps interaction was visualized using cryo-EM. It was found that Dps-DNA complex remains stable under various conditions and while the addition of different ions leads to the disruption of co-crystals, the process is completely or partially reversible. Conclusion: Recent studies conducted by our group showed that Dps-DNA co-crystals adopt triclinic or cubic crystal lattice (FEBS Lett., 2019; Biomolecules, 2020). Here we present the results on the studies of Dps interaction with small linear DNA fragments, demonstrate the effects of MgCl2, FeSO4, and EDTA on the Dps-DNA complex and individual Dps protein structure, discuss the influence of the temperature and time on the co-crystals.
Abstract OR-6: Baseplate Structure of Bacteriophage Phi812 and Mechanism of Cell Wall Binding and Penetration
Authors: Bíňovský Ján , Šiborová Marta , Nováček Jiří , Van Raaij Mark , Plevka Pavel
Published: October 2021

Background: Antibiotic-resistant strains of Staphylococcus aureus cause human infections that are difficult to treat and can lead to death. Bacteriophage (phage) phi812K1/420 from the family Myoviridae infects 95% of clinical isolates of S. aureus and therefore is a promising candidate for a phage therapy agent. As the native phage particle approaches its host cell, phage receptor-binding proteins make a contact with the host cell wall. This interaction triggers a cascade of structural changes in the baseplate resulting in phage tail contraction and genome ejection. Mechanistic description of the baseplate re-organization, however, remains unknown. Methods: Using cryo-electron microscopy (cryo-EM), we studied the baseplate of the phage phi812K1/420. Also, selected proteins involved in the host cell wall binding and penetration were produced in recombinant form and their structures were solved using X-ray crystallography and cryo-EM single-particle reconstruction. Results: We reconstructed the phage baseplate in native and contracted states. The reconstruction of the native baseplate reaches a resolution of 4 Å, which enables us to discern individual protein structures. Solved protein structures will be fitted into the reconstruction of the contracted baseplate. Conclusion: Our results provide the first structural characterization of contractile phage infecting a Gram-positive bacterium. Comparison of the two distinct baseplate states will allow us to describe the molecular mechanism of the initial stage of phage infection in detail.
Abstract OR-8: Cryo-EM Structure of Mature Yellow Fever Virus
Authors: Samygina Valeria , Pichkur Eugene , Cherepanov Peter , Egorov Alexey , Ishmukhametov Aydar , Vorovich Michail
Published: October 2021

Background: Yellow fever virus (YFV) is the prototype virus of the genus Flavivirus. It is endemic to sub-Saharan Africa and tropical South America. YF disease ranges from asymptomatic to severe jaundice and hemorrhagic fever. The flavivirus virion core is enveloped by a lipid membrane with integrated membrane (M) proteins and envelope (E) proteins that form the outer surface of the virion. The Е protein provides stability to the viral particle and is responsible for early infection stages. Flaviviruses are heterogeneous in nature, which is related to their maturation process. Samples always contain mature, immature, half-mature, and damaged particles. Thus, cryo-EM is a method of choice for their structure determination. During the early stages of cryo-EM development, structures of flaviviruses were studied at 10–20 Å resolution. However, due to the progress of recent years, it became possible to determine flavivirus structures at a resolution of 5.6-2.6 Å. The cryo-EM method was used to obtain structural data of virions of dengue fever virus, Zika virus, TBE virus, etc. For YFV, only the cryo-EM structure of the immature virions at a low resolution of 25 Å was determined (Y. Zhang et al. 2003 doi:10.1093/emboj/cdg270). However, the structure of mature (most infectious) YFV particles is still unknown. Methods: Virus sample was produced in Vero cell culture. YFV-17D was inactivated and purified using ultracentrifugation. The concentration of viral particles in the target inactivated YFV-17D (iYFV-17D) was evaluated by spectrophotometry and by estimating the concentration of E protein determined by PAGE electrophoresis. Preliminary quality control of iYFV-17D sample was performed using negative staining TEM. Cryo-EM data were collected using cryo-TEM Krios (Thermo-Fisher, USA) at 300kV using DED Falcon II. Dataset was preprocessed using Warp. Further processing was performed in Relion 3.1 and CisTEM. Model building was carried out using Isolde, Phenix and Coot software. Results: A protocol of production and purification of highly concentrated (~2x1012) monodisperse inactivated iYFV-17D sample was developed. Cryo-EM structure of mature iYFV was solved at 4.1 Å resolution. The structure of YFV is similar to other known flavivirus structures, with 180 copies of protein E arranged in a herringbone pattern that makes up the icosahedral shell. Conclusion: The high-resolution structure of mature iYFV-17D allowed to elucidate special features of this flavivirus and may be useful for vaccine improvement and drug development.
Abstract P-10: Solvatochromic Fluorescent Dyes Tested for Spectroscopic Measurements of Protein Conformational Dynamics
Authors: Belousov Anatolii , Maslov Ivan , Khorn Polina , Mishin Alexander , Baranov Mikhail , Gensch Thomas , Borshchevskiy Valentin
Published: October 2021

Background: Recoverin is a 23 kDa protein, belonging to the superfamily of EF-hand Ca2+-binding proteins. One of the functions of recoverin is to regulate the activity of the rhodopsin kinase GRK1, which regulates the activity of rhodopsin. In dim ambient light, the level of calcium in the rod cells of the retina is high, so recoverin binds to and inhibits rhodopsin kinase, leaving rhodopsin very sensitive to photons to enable the eye to detect visual signals even under low-light conditions. Many biophysical methods have previously been used to study the conformational dynamics of recoverin, including NMR, SPR and fluorescence spectroscopy. Here we describe fluorescent solvatochromic dyes suitable for spectroscopic observation of conformational changes in recoverin. Methods: We tested four fluorescent dyes, which were covalently attached to Cys39 of recoverin via the thiol-maleimide interaction. Results: Two out of four labeled recoverin samples showed EGTA-induced changes in the fluorescence lifetime and excitation and emission spectra. Conclusion: Our experiments show solvatochromic fluorescent dyes that can be successfully used for spectroscopic observation of conformational dynamics in proteins.
Abstract P-11: Microscale Thermophoresis of Mycobacterial Cytochrome P450 with Azole Drugs
Authors: Kapranov Ivan , Bukhdruker S , Karpova M , Zagryadskaya Yulia , Okhrimenko Ivan , Gilep A , Strushkevich N , Borshchevskiy Valentin
Published: October 2021

Background: Cytochrome P450 family members are found in most organisms where they are involved in the metabolism and synthesis of steroids, bile acids, unsaturated fatty acids, phenolic metabolites as well as exogenic chemicals. Drugs targeting cytochrome P450 have been shown to inhibit the growth of Mycobacterium tuberculosis, the causative agent of one of the deadliest diseases – tuberculosis. Recently, we showed that CYP124, CYP125, and CYP142 can bind and metabolize a panel of human immunoactive oxysterols in vitro (Varaksa et al., 2021) and one of them (CYP124) can metabolize antituberculosis drugs (Bukhdruker et al., 2020). Thus, inhibition of cytochrome P450 is a promising strategy for the development of new anti-tubercular drugs. The existing methods used to assess protein-ligand interactions for cytochromes P450 (spectral titration and Surface Plasmon Resonance) have a number of limitations. In this regard, we used an alternative approach for this purposes – microscale thermophoresis (MST) which was not previously used for proteins of the cytochrome P450 superfamily Methods: Here we show that MST can be used to determine the micromolar-range dissociation constants (Kd) of membrane-associated mycobacterial cytochrome CYP124 with small-molecule azole drugs. CYP124 was fluorescently labeled with Cy3-NHS and MST curves were collected at Monolith NT.115 instrument (blue/green channel, NanoTemper Technologies) in presence of various concentrations of azole compounds: econazole, ketoconazole, itraconazole, and miconazole. The experimental results were approximated by the second-order bimolecular binding equation as well as by the Hill-Langmuir equation. Results: Therefore, MST is a valuable method for the assessment of cytochrome P450 binding to their ligands for cases when traditional approaches are not applicable. The binding regime of CYP124 with azole derivatives was characterized by the structure of the CYP124 complex with carbethoxyhexyl imidazole solved with ~1Å resolution.
Abstract P-19: 15-Microsecond Molecular Dynamics Simulations of Nucleosome Show Structural Heterogeneity and Plasticity
Authors: Armeev Grigoriy , Kniazeva Anastasiia , Komarova Galina , Kirpichnikov Mikhail , Shaytan Alexey
Published: October 2021

Background: Nucleosomes are basic units of chromatin organization, resembling spools with ~150 base pairs of DNA wrapped around the octamer of histone proteins. They play a crucial role in chromatin compactization and gene expression. Currently, there are more than 340 structures of nucleosomes and their complexes with proteins in the protein data bank, 159 of them are made with cryoEM, 60 of those in 2020 and later. It is clear that cryoEM will soon yield even more structures of nucleosomes with different histone variants, mutations, DNA sequences, and interacting proteins. Despite the variety, the majority of the aforementioned structures look very similar. This is due to the fact that most of the models are built on the basis of very similar crystal structures. However, the dynamics of nucleosomes are crucial for understanding the mechanisms that govern the chromatin functions. Computational methods can supplement experimental approaches and recreate the dynamic conformational landscape of nucleosomes from initial static structures. We present an all-atom molecular dynamics simulation of nucleosome core particles at a record timescale of 15 microseconds. Methods: All-atom MD simulations were performed using GROMACS 2018 with AMBER ff14SB force field with parmbsc1 DNA and CUFIX ion parameters. Crystal structures with PDB IDs 1KX5 and 3LZ0 were used. Analysis was performed with custom-developed python scripts based on MDAnalysis and 3DNA. Models of chromatin fibers were built by connecting random snapshots from MD trajectories with straight linker segments of B-DNA of different lengths. Results: We observed the inner dynamics of histone octamer, which covers the conformational space of the most deformed structures reported by cryoEM. We showed that histone dynamics play important role in DNA mobility, allowing for twist-defects propagation. Conclusion: We observed unprecedented unwrapping of nucleosomal DNA with truncated histone tails. Through multi-scale modeling, we showed that such unwrapping alone is crucial for nucleosomal fibers geometry and elastic properties.
Abstract P-21: The Investigation of S.aureus Ribosome-Binding Factor A Localization on the 30S Ribosomal Subunit by Cryo-Electron Microscopy
Authors: Bikmullin Aydar , Stetsenko Artem , Golubev Alexander , Nurullina Liliia , Khusainov Iskander , Klochkova Evelina , Garaeva Natalia , Usachev Konstantin
Published: October 2021

Background: Ribosome biogenesis is a complex process of ribosomal RNA and protein binding. Bacterial ribosome maturation and components involved in it are especially interesting, because they are widespread targets for antibiotics. A number of special protein factors facilitating the maturation of the 30S small ribosomal subunit are known. One of them is a ribosome-binding factor A (RbfA). This is a small (~14 kDa) protein with KH-domain organization distinguishing RNA binding proteins. Recent cryo-EM reconstruction of E.coli 30S-RbfA complex indicates that RbfA binds to 30S subunit on the central decoding region and promotes the switch from the immature state of h28 (neck) to mature state. RbfA interacts with 3`-end of 16S rRNA on mRNA exit channel and stabilizes the conformation of the region between h28, h44/h45 linker and 3`-end. Methods: Pure S.aureus RbfA was obtained by homologous expression in E.coli BL21 strain followed by Ni-NTA and gel filtration. The 30S subunits were obtained by dissociation of the S.aureus 70S ribosomes in a sucrose gradient (0-30%). We performed 30S subunit and RbfA complex reconstitution, sample and grid preparation. Data was collected on Talos Arctica, Falcon 2 detector (FEI Company/Thermo Fisher). Results: The 30S-RbfA complex density map with average resolution ~ 3.5 Å (FSC=0.143) was obtained. In comparison with the free subunit map (EMD 23052) we observed an extra density on the neck region near the decoding center region. Conclusion: Obtained data is correlated with recent structural results of the homologous E.coli RbfA. We consider that S.aureus RbfA binds to the 30S subunit at the same region. The next step of our structural research is building the model of S.aureus 30S-RbfA complex.
Abstract P-22: Enhanced Crosslinking and Immunoprecipitation (Eclip) Data Reveal Interactions of RNA Binding Proteins with the Human Ribosome
Authors: Buyan Andrey , Kulakovskiy Ivan , Dmitriev Sergey
Published: October 2021

Background: The ribosome is a protein-synthesizing molecular machine composed of four ribosomal RNAs (rRNAs) and dozens of ribosomal proteins. In mammals, the ribosome has a complicated structure with an additional outer layer of rRNA, including large tentacle-like extensions. A number of RNA binding proteins (RBPs) interact with this layer to assist ribosome biogenesis, nuclear export and decay, or to modulate translation. Plenty of methods have been developed in the last decade in order to study such protein-RNA interactions, including RNA pulldown and crosslinking-immunoprecipitation (CLIP) assays. Methods: In the current study, using publicly available data of the enhanced CLIP (eCLIP) experiments for 223 proteins studied in the ENCODE project, we found a number of RBPs that bind rRNAs in human cells. To locate their binding sites in rRNAs, we used a newly developed computational protocol for mapping and evaluation of the eCLIP data with the respect to the repetitive sequences. Results: For two proteins with known ribosomal localization, uS3/RPS3 and uS17/RPS11, the identified sites were in good agreement with structural data, thus validating our approach. Then, we identified rRNA contacts of overall 22 RBPs involved in rRNA processing and ribosome maturation (DDX21, DDX51, DDX52, NIP7, SBDS, UTP18, UTP3, WDR3, and WDR43), translational control during stress (SERBP1, G3BP1, SND1), IRES activity (PCBP1/hnRNPE1), and other translation-related functions. In many cases, the identified proteins interact with the rRNA expansion segments (ES) of the human ribosome pointing to their important role in protein synthesis. Conclusion: Our study identifies a number of RBPs as interacting partners of the human ribosome and sheds light on the role of rRNA expansion segments in translation.