bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of symptoms, severity and diagnosis of asthma in adolescents in the Province of Salamanca, Spain: Global Asthma Network (GAN) Phase I
Authors: Marín-Cassinello Ana , Vega-Hernández María Concepción , Lumbreras-Lacarra Blanca , Arriba-Méndez Sonia De , Pellegrini-Belinchón J.
Published: September 2021

Introduction and objectives: Asthma is the most prevalent chronic disease in childhood. However, the latest data on its prevalence in Spain are from Phase III of the International Study of Asthma and Allergies in Childhood (ISAAC), 2004. The objective of our study was to assess the prevalence of asthma symptoms, severity and diagnosis in the paediatric population aged between 13 and 14 years in the province of Salamanca.
Material and methods: Cross-sectional multicentre study carried out in 2017–2018 in 13- and 14-year-old school children in the province of Salamanca as a centre participating in of the Global Asthma Network(GAN) Phase I. The standardised validated written questionnaire and that directed by a video was administered; it was self-completed by the pupils.
Results: A total of 3485 questionnaires were completed, and the pupils’ participation rate was 95.01%. Among the total, 25.7% indicated having had wheeze ever (20.7% in the video questionnaire); 14.7% indicated having had wheeze in the past 12 months (11.3% in the video questionnaire). The prevalence of current wheeze that limited speech was 3.9% (7.5% in the video questionnaire) and the current prevalence of severe wheeze was 6.5%. Regarding asthma diagnosis, 19.7% of the sample answered that they had had asthma ever, whilst 14.0% referred to having physician-diagnosed asthma. The agreement between the written questionnaire and that directed by video was acceptable for the questions of wheeze ever (Cohen Kappa index [k] = 0.53) and current wheeze (k = 0.42).
Conclusions: The prevalence of current asthma (wheeze in the past 12 months) in the population aged 13 and 14 years in the province of Salamanca is higher compared with that presented in 2004 in Spain, but similar to that described at the world level (low-moderate level), according to the ISAAC Phase III studies.
Maternal smoking during pregnancy and asthma during the first year of life: a comparative study between smokers and non-smoker mothers
Authors: Rivas Juesas Cristina , Fernández Monge Lucia , Delgado Vicente Ana , Ledo García Ana , Giner Crespo Maribel , Coret Sinisterra Alicia
Published: September 2021

Background: We investigated the relationship between maternal smoking in pregnancy and health care problems in the offspring during the first year of life, particularly asthma.Material and Methods: A cohort of 648 consecutive infants born at term and alive at Hospitalde Sagunto (Valencia, Spain) over one year period was followed for 12 months. Clinical data of the infants were prospectively collected from the database of ambulatory medical records (ABUCASIS) of the Valencia health system.Results: Smoking during pregnancy was recorded in 164 (25.3%) mothers. During the first year of life, asthma was diagnosed in 101 infants, with an incidence of 15.7%. The diagnoses of asthma (25.6% vs. 12.3%; P < 0.0001) and bronchiolitis (44.5% vs. 28.6%; P = 0.0002) during the first year of life were more common among infants in the smoking group. The probability of developing asthma during the first year of life was two-fold higher for the male gender and 2.5 times higher when mothers smoked while pregnant. Up to 52% of asthma cases could have been avoided in infants born to smoking mothers if they did not smoke during pregnancy. Infants in the smoking group showed a lower weight and length at birth than infants in the nonsmoking group differences almost disappeared at 6 months and 12 months.Conclusion: Asthma during the first year of life showed a clear relationship with maternal smoking in pregnancy. Pregnant women should be advised of the significant perinatal risk for respiratory diseases associated with tobacco use.
Laboratory screening test with inhalant and food allergens in atopic Brazilian children and adolescents: a performance
Authors: Rozalem Reali Ana Carolina , Faria Pierotti Felipe , Sanchez Aranda Carolina , Rodrigues Cocco Renata , Cavalcanti Sarinho Emanuel S , Sano Flávio , Porto Neto Arnaldo , Rosário Nelson A , Chong Neto Herberto J , Simões Goudouris Ekaterini , Sanches Moraes Lilian , Falbo Wandalsen Neusa , Carlos Pastorino Antônio , Motta Franco Jackeline , Letícia Chavarria Maria , P Borres Magnus , Solé Dirceu
Published: September 2021

The Phadiatop Infant® (PhInf) is a panel developed to assess allergic sensitization (immunoglobulin E [IgE]) in children aged <5 years and combines inhalant and food allergens. The test has not been evaluated outside Europe. This is a cross-sectional study conducted at 11 pediatric allergy centers to evaluate PhInf as an allergic disease screening method in Brazilian children. Children as controls and patients (aged 6 months–18 years) were grouped according to their primary disease and age group. PhInf and specific serum IgE (sIgE) screening was performed for Dermatophagoides pteronyssinus (DP), cat and dog epithelia, a mix of grasses and pollens, eggs, cow’s milk, peanuts, and shrimp. Values ≥ 0.35 kUA/L (or PAU/L) were considered positive. A total of 470 children and adolescents, which included 385 patients and 85 controls, participated in the study (47.7% boys, average age: 6.3 years). In all, 72.6% of the participants had positive PhInf test (n = 341), with a higher proportion of those having food allergy (92.6%), atopic dermatitis (91.9%), and those aged >13 years having allergy (95%). The PhInf and sIgE agreement between patients (Kappa = 0.94, P < 0.001) and controls (Kappa = 0.84, P < 0.001) was high. PhInf and DP agreement in patients aged >13 years was excellent (Kappa = 0.936, P < 0.001). Compared with sIgE dosage, PhInf had high sensitivity (97%) and specificity (93%). Positivity of PhInf test in this population was high and had an excellent correlation with the allergens comprising the panel. It is a useful method for screening children suspected of having allergic diseases in a non-European country.
METTL3 promotes inflammation and cell apoptosis in a pediatric pneumonia model by regulating EZH2
Authors: Yang Xiaoqing , Yang Ying , Wu Yi , Fu Meijiao
Published: September 2021

Background: In recent times, the medical science has developed by leaps and bounds, however, the molecular mechanism of pediatric pneumonia is still unclear. Although prior researches have shown that methyltransferase-like 3 (METTL3) is up-regulated in a variety of inflammatory diseases, its role and mechanism has been rarely studied in pediatric pneumonia, and need to be defined elaborately.Objective: In this study, the related molecular mechanism of METTL3 on inflammation and cell apoptosis in a pediatric pneumonia was investigated. Materials and methods: Quantitative real-time polymerase chain reaction (qPCR) and western blot assays were employed to examine the mRNA and protein expression level of METTL3 and EZH2 in peripheral blood monocytes from pediatric pneumonia patients or cell model (WI-38). Then, qPCR and ELISA assay were applied to verify the inflammatory response in LPS-treated WI-38 cell lines after knockdown of METTL3. Besides, MTT cell viability assays, flow cytometry, and western blot assays were applied to examine the cell viability and cell apoptosis of LPS-treated WI-38 cell after knockdown of METTL3. Further, the western blot assays were employed to examine the protein expression levels of p-JAK2, JAK2, p-STAT3, STAT3, and EZH2 in LPS-treated WI-38 cell after knockdown of METTL3. Finally, ELISA and western blot were applied to verify the inflammatory response and cell apoptosis of LPS-treated WI-38 cell after knockdown of METTL3 and overexpression of EZH2.Results: In this study, the results showed that METTL3 and EZH2 were highly expressed in pediatric pneumonia patients and cell models (WI-38), respectively. Besides, downregulation of METTL3 inhibited LPS-induced inflammatory response and cell apoptosis. Then, the fact that METTL3 regulates the JAK2/STAT3 signaling pathway through EZH was proved. Furthermore, downregulation of METTL3 inhibits inflammation and apoptosis through EZH2.
Conclusion: This study found that METTL3 promotes inflammation and cell apoptosis in a pediatricpneumonia model by regulating EZH2.
Frequency of self-reported allergies at a high-complexity referral hospital in Colombia, a tropical Latin American country
Authors: Pérez-Herrera Lucía C. , Moreno-López Sergio , Peñaranda Daniel , García Elizabeth , Chapman Edgardo , Peñaranda Augusto
Published: September 2021

Background: The frequency of allergic diseases in tropical Latin American populations is poorly understood, and certain particularities can impact their natural history and risk factors.
Objective: The study aimed to determine the frequency of self-reported allergies (allergic diseases, drug, and food allergies) in patients who attended the Hospital Universitario Fundación Santa Fe de Bogotá, Colombia.
Material and methods: A retrospective study was conducted to assess the frequency of self-reported allergies reported by all the patients who attended an allergy referral center between June and December 2019.
Results: A total of 60978 patients were included. Allergic rhinitis was reported by 1.51% (n= 921), asthma by 1.28% (n = 782), and atopic dermatitis by 0.41% (n = 250) of the study population. A higher frequency of self-reported allergic diseases (rhinitis, asthma, and dermatitis) was found in the younger populations, while drug allergies were more frequently reported in adults. The most frequently self-reported drug allergies were penicillin allergy (4.07%, n = 2479), and non-steroidal anti-inflammatory drug (NSAID) allergy (1.85%, n = 1116). The most commonly reported food allergens included shellfish (0.58%), fruits (0.54%), cow’s milk protein (0.37%), and eggs (0.21%).
Conclusion: The distribution of food allergens showed a higher frequency of shrimp and fruit allergies compared to previous studies on African, Asian, and Arabic tropical populations that describe a higher predominance of egg and milk allergies. Patients reporting allergic diseases should always be referred to the allergology department for confirmatory testing.
Downregulation of FPR1 abates lipopolysaccharide-induced inflammatory injury and apoptosis by upregulating MAPK signaling pathway in murine chondrogenic ATDC5 cells
Authors: Chen Hongtao , Zhang Li
Published: September 2021

Background and objective: Osteoarthritis is the most common chronic osteoarthrosis disease. There are complex factors that lead to osteoarthritis. Therefore, it is essential to investigate the molecular mechanism of osteoarthritis, especially the mechanism of articular cartilage degeneration. In this study, the mechanism of FPR1 (formyl peptide receptor 1) in LPS (lipopolysaccharide) induced chondrogenic cell ATDC5 was investigated.Materials and methods: We employed real-time quantitative polymerase chain reaction (RT-qPCR) and western blot assay to analyze the expression level of FPR1 in ATDC5 cell linesinduced by LPS at 0, 2.5, 5, and 10 μg/mL concentrations. Then we constructed the FPR1 knockdown plasmid to transfect the LPS-ATDC5. MTT assay was used to test cell viability in control, LPS, LPS+shNC and LPS+shFPR1 groups. ELISA and RT-qPCR assay were employed to examine the TNF-α (tumor necrosis factor-α)、IL-6 and IL-1β expression level. Flow cytometry and western blot assay were employed to analyze the apoptosis of LPS-ATDC5. Finally, we utilized the western blot assay to text related protein expression level of MAPK (mitogen-activated protein kinase) signaling pathway.Results: In this study, we found the expression level of FPR1 was increased in LPS-ATDC5, downregulation of FPR1 improves the survival rate and alleviates inflammatory response of LPS-ATDC5. Meanwhile, downregulation of FPR1 alleviates apoptosis of LPS-ATDC5. Finally, downregulation of FPR1 inhibits the MAPK signal pathway.Conclusion: Present study revealed that FPR1 was highly expressed in LPS-induced chondrocytes ATDC5, and the downregulation of FPR1 abated the inflammatory response and apoptosis of LPS-ATDC5 cells by regulating the MAPK signaling pathway.
MiR-216a-5p alleviates LPS-induced inflammation in the human bronchial epithelial cell by inhibition of TGF-β1 signaling via down-regulating TGFBR2
Authors: Liu Shan , Li Jianjun , Hu Liya
Published: September 2021

Objective: Bronchopneumonia is a common respiratory infection disease and is the leading cause of hospitalization in children under 5 years of age. Inflammation is the primary response caused by bronchopneumonia. But the detailed underlying mechanism of inflammation in bronchopneumonia remains unclear. Therefore, this study focused on studying the effect of miR-216a-5p on inflammation induced by bronchopneumonia and investigate the potential mechanism underlying it.Methods: Human bronchial epithelial cells (BEAS-2B) were stimulated using lipopolysaccha-rides (LPS) to trigger bronchopneumonia in vitro. The production of interleukin (IL)-1β, IL-6, and Tumor necrosis factor (TNF)-α was measured using the enzyme-linked immunosorbent assay. The luciferase assay was conducted to explore the relationship between miR-216a-5p and TGFBR2. Quantitative real-time polymerase chain reaction and western blot were used to detect the gene expression.Results: miR-216a-5p gene expression decreased in BEAS-2B cells stimulated by LPS. Overexpression of miR-216a-5p suppressed the elevated levels of IL-1β, IL-6, and TNF-α induced by LPS. Transforming growth factor-beta receptor 2 (TGFBR2) proved to be a direct target of miR-216a-5p, and they negatively modulated TGFBR2 expression. In addition, overexpression of miR-216a-5p inhibited LPS-induced protein levels of TGFBR2,transforming growth factor (TGF)-β1, and phosphorylation of SMAD family member 2 (smad2),. This ectopic expression of miR-216a-5p was restored by overexpressed TGFBR2.Conclusion: miR-216a-5p was decreased in LPS-stimulated BEAS-2B cells. Overexpressed miR-216a-5p suppressed LPS-induced inflammation in BEAS-2B cells by inhibition of TGF-β1 signal-ing via down-regulating TGFBR2. miR-216a-5p may be a valuable target for anti-inflammation treatment in bronchopneumonia.
Pachymic acid inhibits inflammation and cell apoptosis in lipopolysaccharide (LPS)-induced rat model with pneumonia by regulating NF-κB and MAPK pathways
Authors: Gui Yanjun , Sun Lijuan , Liu Rui , Luo Jinzhu
Published: September 2021

Pneumonia is a common infectious disease with high morbidity and mortality. It is caused by a variety of pathogenic microorganisms that infect the lung parenchyma. Anti-infective drugs are one of the preferred choices for the treatment of pneumonia. Pachymic acid (PA) is a lanolin triterpene compound from Poria cocos, which has antiemetic, anti-inflammatory, and anticancer properties. Although PA inhibits inflammatory response in a variety of diseases, its role in pneumonia is not clear. In this study, we established that PA improved histopathological changes in the lungs of rats with pneumonia. PA inhibited the expression of inflammatory cytokines in the serum of rats having pneumonia. In addition, PA inhibited the apoptosis of cells from rat lung tissues. Mechanically, PA inhibited inflammation and cell apoptosis via NF-κB and MAPK pathways. Therefore, PA could serve as a promising drug for treating pneumonia.
MEF2C alleviates acute lung injury in cecal ligation and puncture (CLP)-induced sepsis rats by up-regulating AQP1
Authors: Liang Wenmei , Guo Li , Liu Tonghua , Qin Song
Published: September 2021

Background: Sepsis is a systemic inflammatory response syndrome and leads to patient’s death. Objective: To investigate the effect of myocyte enhancer factor 2 (MEF2C) on acute lung injury (ALI) with sepsis and its possible mechanism.Material and Methods: The cecal ligation and puncture (CLP)-induced sepsis rat model was established. The lung injury was determined by lung wet–dry weight ratio, the concentration of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), Interlukin (IL)-6, IL-1β, and IL-10, were measured by the enzyme-linked-immunosorbent serologic assay kit. The cell apoptosis was detected by TUNEL staining assay.Results: Interestingly, MEF2C was down-regulated in this model. Moreover, adeno-associated virus (AAV)-MEF2C treatment markedly suppressed TNF-α, IL-1β, and IL-6 concentrations but promoted IL-10 concentration in serum in CLP-challenged rats. Besides, overexpression of MEF2C alleviates CLP-induced lung injury. Interestingly, AAV-MEF2C treatment was confirmed to suppress apoptosis in CLP-induced sepsis rats as well as promote aquaporin APQ1 expres-sion. Mechanistically, the rescue experiments indicated that MEF2C alleviated CLP-induced lung inflammatory response and apoptosis via up-regulating AQP1.Conclusion: In summary, overexpression of MEF2C suppressed CLP-induced lung inflamma-tory response and apoptosis via up-regulating AQP1, providing a novel therapeutic target for sepsis-induced ALI.
Using evolutionary algorithms for continuous simulation of long-term reservoir inflows
Authors: Sedighkia Mahdi , Datta Bithin
Published: September 2021

Data-driven models provide upgradeable environments to simulate inflow to reservoirs. An important aspect in the development of an adaptive neural fuzzy inference system (ANFIS) model is to choose the correct procedure (i.e. efficient and effective) to train the model. In this study, a daily timescale ANFIS-based model was developed to simulate aggregated monthly long-term inflow to the Ross River reservoir in northern Queensland, Australia. The suitability of different evolutionary algorithms (EAs) was evaluated to train an ANFIS-based model, including a genetic algorithm (GA), particle swarm optimisation (PSO), shuffled frog leaping algorithm, biogeography-based optimisation, harmony search algorithm, differential evolution algorithm, invasive weed optimisation (IWO) and a cultural algorithm. Four indices – the root mean square error, Nash–Sutcliffe efficiency, reliability index and vulnerability index – were used to measure the performance of the model. It was found that application of either IWO, GA or PSO provided accurate simulated inflow time series. The outputs obtained using the other EAs were not sufficiently accurate. Use of a coupled EA–ANFIS-based model was found to improve the accuracy of simulating long-term monthly inflow compared with other models used in a few previous recent studies.
Multilayer modelling as a support tool for flood diagnosis and drainage system design
Authors: Radesca Fernanda Dias , Sousa Matheus Martins de , Veról Aline Pires , Oliveira Laurent Feu Grancer Silva , Fraga João Paulo Rebechi , Magalhães Paulo Canedo de , Miguez Marcelo Gomes
Published: September 2021

The use of dual-drainage models has increased in recent decades, mostly because of advances in mathematical methods and geographic information system tools. This type of modelling provides precise diagnoses and can be applied to evaluate stormwater infrastructure and diverse drainage alternatives. Minor and major drainage relations in the Dona Eugênia watershed, Brazil, were analysed in this study. Three alternatives simulated the quasi-two-dimensional hydrodynamic model Modcel. The first alternative considered only the terrain surface layer, representing the current situation. In the second alternative, a layer containing a designed storm sewer was introduced. Considering the previous alternatives as a way to diagnose the main minor and major deficiencies, a third alternative was formulated considering previous projects to solve floods in the watershed, incorporating them in the second alternative with functional minor drainage. The results identified the locations of minor drainage gaps, thus helping to direct efforts to the most critical regions, optimise operations and minimise maintenance costs. The results could also be used to evaluate the effects of storm sewer network discharges in major drainage systems, leading to better design of the drainage infrastructure. The major drainage itself can be optimised, once its responsibility in flooding is identified.
'Modernism and the Machinery of Madness: Psychosis, Technology, and Narrative Worlds' (2017), by Andrew Gaedtke
Authors: Schiff Noam
Published: September 2021

Review of Andrew Gaedtke, Modernism and the Machinery of Madness: Psychosis, Technology, and Narrative Worlds (Cambridge: Cambridge University Press, 2017).
THINKING DEVELOPMENT IN MATHEMATICS TEACHING
Authors:   Kyrgyz State University named after Arabaev, Sadieva Manarkan , Калдыбаев Salidin ,   Alatoo International University
Published: September 2021

The article considers the need for the development of thinking in the process of teaching mathematics at school. The documents adopted on improving education in the republic, as well as the republican test, emphasize the importance of students mastering mathematical knowledge. As stated in the article, favorable conditions for the development of thinking are created in teaching mathematics. Mathematics itself is a subject that studies the numerical and spatial relationships of reality. The student’s thinking is closely connected with the process of assimilation of educational material. The article also notes that the process of thinking occurs in parallel with the perception of knowledge, the preservation in memory and the application of knowledge in practice.
EFFECT OF 5 WT.% WPCB POWDER AS REDUCTANT ON INDONESIAN NICKELIFEROUS ORE PROCESSING
Authors: Ulum Reza Miftahul , Radhitya Dimas Ananda , Madsuha Alfian Ferdiansyah , Abidin Faizinal
Published: September 2021

EFFECT OF 5 WT.% WPCB POWDER AS REDUCTANT ON INDONESIAN LIMONITICNICKEL ORE PROCESSING. Waste printed circuit boards (WPCB) are among the most valuable parts of electronic waste with one of the fastest-growing waste streams in the world. The purpose of this study is to investigate the possibility of WPCB powder as an alternative reducing agent for the carbothermic process in nickel lateritic ore processing. WPCB waste was mixed with nickel ore at 1100ÚC in inert atmosphere. In addition, a conventional reductant of coal is also utilized for comparison. Both reductant are varied in concentration of 5 wt% and 15 wt%. Based on thermogravimetric analysis (TGA) and differential thermogravimetric analysis (DTA) investigation, it is observed that there exists a difference between WPCB powder, nickeliferous ore powder, and the mixture in their decomposed levels. The decomposed gasses of WPCB produced by thermal degradation in the TGA instrument are mainly composed of reduction gas, which plays a critical role in reducing the nickeliferous ore. This study shows that WPCB powder performs comparably to sub-bituminous coal in the pyrometallurgical processing of nickel ore, which is proved by X-Ray Diffraction (XRD) test results that the carbothermic products consists of FeNi, magnetite, wustite and fayalite. It can be concluded than WPCB powder has potential to be utilize as an alternative reductant.
AUTOCLAVE-ASSISTED DEACETYLATION: A RAPID METHOD TO RECYCLING CIGARETTE BUTTS TO CELLULOSE
Authors: Amanda Putri , Putri Anisyah , Masruchin Nanang , Kusumaningrum Wida Banar , Ningrum Riska Surya , Ismadi Ismadi
Published: September 2021

AUTOCLAVE-ASSISTED DEACETYLATION: A RAPID METHOD TO RECYCLINGCIGARETTE BUTTS INTO CELLULOSE. Cellulose acetate (CA)-based materials, like cigarette butts (CBs), become one of the most com-mon types of litter in the world. The toxic substances that are contained make this waste carry a hazardous risk for the environment and living organisms. Herein we report a rapid method for recycling cigarettes butts into more environmen-tally material. Cellulose was fabricated by deacetylation of cigarette butts with NaOH solution at various times 15, 30, 45, and 60 minutes in autoclave. Cellulose was optimized by a degree of deacetylation (DD%) and was further charac-terized by FTIR, SEM, TGA, and DSC analysis. The DD% and FTIR results confirmed the complete conversion of cellu-lose acetate from cigarette butts to cellulose within 15 minutes. Cellulose morphology under SEM showed the surface became rougher and textured after autoclave treatment. The results of autoclave-assisted deacetylation are comparable with the conventional deacetylation. Our rapid method offers substantially reduced deacetylation from 24 hours to just 15 minutes. This study has shown that the new and straightforward method for deacetylation cellulose acetate and it is potential as an alternative method for recycling cigarette butts waste in the future.
EFFECT OF AGAR AND KAPPA-CARRAGEENAN ON THERMAL AND MECHANICAL PROPERTIES OF THERMO PLASTIC STARCH (TPS)
Authors: Abdullah Akbar Hanif Dawam , Asri Putri Putih Puspa , Nissa Rossy Choerun , Fransiska Dina , Nurhayati Nurhayati , Agusman Agusman , Irianto Hari Eko , Priambudi Panji , Marliah Sri
Published: September 2021

Effect of Agar and kappa-Carrageenan on Thermal and Mechanical Properties of Thermo Plastic Starch (TPS) Thermoplastic starch needs to be blend with other materials because of its limited properties. In order to gain the desired properties, the interaction between the added component (filler, plasticizer, second polymer, etc) must be concerned as it can affect the performances of the final product. Thermoplastic starch has been successfully made with mixing and compression molding with the addition of agar and kappa-carrageenan. The effect of the added material has been studied with FTIR, Tensile, DSC, and TGA to investigate their mechanical and thermal properties. The FTIR spectrum has shown the shifting around the methyl group (C-H), a carboxyl group (C-O), and a hydroxyl group (O-H). Thermal analysis results presented different patterns in melting and degradation behavior. The tensile strength value did not show significant improvement with the presence of agar and kappa-carrageenan. It is shown that the addition of agar and kappa-carrageenan affects thermal behavior but does not significantly impact the mechanical properties.
Purification of the Recombinant RNA Chaperone CspA v1
Authors: Menendez-Gil Pilar , J. Caballero Carlos , Solano Cristina , Toledo-Arana Alejandro
Published: September 2021

This is part 3.1 of the "Fluorescent Molecular Beacons Mimicking RNA Secondary Structures to Study RNA Chaperone Activity" collection of protocols. Collection Abstract: Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5' and 3' ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases.
Identifying Genetic Dependencies in Cancer by Analyzing siRNA Screens in Tumor Cell Line Panels v1
Authors: Campbell James , J Ryan Colm , J. Lord Christopher
Published: September 2021

Loss-of-function screening using RNA interference or CRISPR approaches can be used to identify genes that specific tumor cell lines depend upon for survival. By integrating the results from screens in multiple cell lines with molecular profiling data, it is possible to associate the dependence upon specific genes with particular molecular features (e.g., the mutation of a cancer driver gene, or transcriptional or proteomic signature). Here, using a panel of kinome-wide siRNA screens in osteosarcoma cell lines as an example, we describe a computational protocol for analyzing loss-of-function screens to identify genetic dependencies associated with particular molecular features. We describe the steps required to process the siRNA screen data, integrate the results with genotypic information to identify genetic dependencies, and finally the integration of protein-protein interaction data to interpret these dependencies.
Perseus: A Bioinformatics Platform for Integrative Analysis of Proteomics Data in Cancer Research v1
Authors: Cox Juergen , Tyanova Stefka
Published: September 2021

Mass spectrometry-based proteomics is a continuously growing field marked by technological and methodological improvements. Cancer proteomics is aimed at pursuing goals such as accurate diagnosis, patient stratification, and biomarker discovery, relying on the richness of information of quantitative proteome profiles. Translating these high-dimensional data into biological findings of clinical importance necessitates the use of robust and powerful computational tools and methods. In this chapter, we provide a detailed description of standard analysis steps for a clinical proteomics dataset performed in Perseus, a software for functional analysis of large-scale quantitative omics data.
Microinjection Techniques in Fly Embryos to Study the Function and Dynamics of SMC Complexes v1
Authors: Carmo Catarina , Araújo Margarida , Oliveira Raquel
Published: September 2021

Structural maintenance of chromosomes (SMC) proteins are critical to maintain mitotic fidelity in all organisms. Over the last decades, acute inactivation of these complexes, together with the analysis of their dynamic binding to mitotic chromatin, has provided important insights on the molecular mechanism of these complexes as well as into the consequences of their failure at different stages of mitosis. Here, we describe a methodology to study both SMC function and dynamics using Drosophila melanogaster syncytial embryos. This system presents several advantages over canonical inactivation or imaging approaches. Efficient and fast inactivation of SMC complexes can be achieved by the use of tobacco etch virus (TEV) protease in vivo to cleave engineered versions of the SMC complexes. In contrast to genetically encoded TEV protease expression, Drosophila embryos enable prompt delivery of the protease by microinjection techniques, as detailed here, thereby allowing inactivation of the complexes within few minutes. Such an acute inactivation approach, when coupled with real-time imaging, allows for the analysis of the immediate consequences upon protein inactivation. As described here, this system also presents unique advantages to follow the kinetics of the loading of SMC complexes onto mitotic chromatin. We describe the use of Drosophila embryos to study localization and turnover of these molecules through live imaging and fluorescence recovery after photobleaching (FRAP) approaches.
Standard CD Measurements v1
Authors: Hussain Rohanah , S. Hughes Charlotte , Siligardi Giuliano
Published: September 2021

CD spectroscopy is the essential tool to quickly ascertain in the far-UV region the global conformational changes, the secondary structure content, and protein folding and in the near-UV region the local tertiary structure changes probed by the local environment of the aromatic side chains, prosthetic groups (hemes, flavones, carotenoids), the dihedral angle of disulfide bonds, and the ligand chromophore moieties, the latter occurring as a result of protein–ligand binding interaction. Qualitative and quantitative investigations into ligand-binding interactions in both the far- and near-UV regions using CD spectroscopy provide unique and direct information whether induced conformational changes upon ligand binding occur and of what nature that are unattainable with other techniques such as fluorescence, ITC, SPR, and AUC. This chapter provides an overview of how to perform circular dichroism (CD) experiments, detailing methods, hints and tips for successful CD measurements. Descriptions of different experimental designs are discussed using CD to investigate ligand-binding interactions. This includes standard qualitative CD measurements conducted in both single-measurement mode and high-throughput 96-well plate mode, CD titrations, and UV protein denaturation assays with and without ligand. The highly collimated micro-beam available at B23 beamline for synchrotron radiation circular dichroism (SRCD) at Diamond Light Source (DLS) offers many advantages to benchtop instruments. The synchrotron light source is ten times brighter than a standard xenon arc light source of benchtop instruments. The small diameter of the synchrotron beam can be up to 160 times smaller than that of benchtop light beams; this has enabled the use of small aperture cuvette cells and flat capillary tubes reducing substantially the amount of volume sample to be investigated. Methods, hints and tips, and golden rules tomeasure good quality, artifact-free SRCD and CD data will be described in this chapter in particular for the study of protein–ligand interactions and protein photostability.
Optogenetic Control of Subcellular Protein Location and Signaling in Vertebrate Embryos v1
Authors: E. Buckley Clare
Published: September 2021

This chapter describes the use of optogenetic heterodimerization in single cells within whole-vertebrate embryos. This method allows the use of light to reversibly bind together an “anchor” protein and a “bait” protein. Proteins can therefore be directed to specific subcellular compartments, altering biological processes such as cell polarity and signaling. I detail methods for achieving transient expression of fusion proteins encoding the phytochrome heterodimerization system in early zebrafish embryos (Buckley et al., Dev Cell 36(1):117–126, 2016) and describe the imaging parameters used to achieve subcellular light patterning.
Western Blotting and RNA Isolation from Membrane v1
Authors: L. Van Nostrand Eric , B. Nguyen Thai , Gelboin-Burkhart Chelsea , Wang Ruth , M. Blue Steven , A. Pratt Gabriel , L. Louie Ashley , W. Yeo Gene
Published: September 2021

Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.
3' RNA Adapter Ligation to Input RNA v1
Authors: L. Van Nostrand Eric , B. Nguyen Thai , Gelboin-Burkhart Chelsea , Wang Ruth , M. Blue Steven , A. Pratt Gabriel , L. Louie Ashley , W. Yeo Gene
Published: September 2021

Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.
Robust, Cost-Effective Profiling of RNA Binding Protein Targets with Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP) v1
Authors: L. Van Nostrand Eric , B. Nguyen Thai , Gelboin-Burkhart Chelsea , Wang Ruth , M. Blue Steven , A. Pratt Gabriel , L. Louie Ashley , W. Yeo Gene
Published: September 2021

Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.
Capture RBP -RNA Complexes on Beads v1
Authors: L. Van Nostrand Eric , B. Nguyen Thai , Gelboin-Burkhart Chelsea , Wang Ruth , M. Blue Steven , A. Pratt Gabriel , L. Louie Ashley , W. Yeo Gene
Published: September 2021

Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.
Biotinylation of Membrane Proteins for Binder Selections v1
Authors: T. Kuhn Benedikt , Zimmermann Iwan , Egloff Pascal , M. Hürlimann Lea , A.J. Hutter Cedric , Miscenic Christian , J.P. Dawson Roger , A. Seeger Markus , R. Geertsma Eric
Published: September 2021

The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.
Drug Sensitivity Assays of Human Cancer Organoid Cultures v1
Authors: E. Francies Hayley , Barthorpe Andrew , McLaren-Douglas Anne , J. Barendt William , J. Garnett Mathew
Published: September 2021

Drug sensitivity testing utilizing preclinical disease models such as cancer cell lines is an important and widely used tool for drug development. Importantly, when combined with molecular data such as gene copy number variation or somatic coding mutations, associations between drug sensitivity and molecular data can be used to develop markers to guide patient therapies. The use of organoids as a preclinical cancer model has become possible following recent work demonstrating that organoid cultures can be derived from patient tumors with a high rate of success. A genetic analysis of colon cancer organoids found that these models encompassed the majority of the somatic variants present within the tumor from which it was derived, and capture much of the genetic diversity of colon cancer observed in patients. Importantly, the systematic sensitivity testing of organoid cultures to anticancer drugs identified clinical gene–drug interactions, suggestive of their potential as preclinical models for testing anticancer drug sensitivity. In this chapter, we describe how to perform medium/high-throughput drug sensitivity screens using 3D organoid cell cultures.
Measuring the Action of Oligonucleotide Therapeutics in the Lung at the Cell Type-Specific Level by Tissue Disruption and Cell Sorting (TDCS) v1
Authors: Graves Helen , Evans Steven , Fauler Michael , Frick Manfred , A. Moschos Sterghios
Published: September 2021

The clinical potential of DNA and RNA-targeting therapeutics for airways disease has been hampered by the poor translation of promising drug candidates from cell culture to in vivo models and the clinic. For example, classical preclinical approaches routinely report 20–60% target knockdown effects in the lung, where 1 or 2 log effects are observed in isolated cell cultures in vitro. Preparation of monocellular suspensions of tissues by mechanoenzymatic disruption followed by cell sorting (TDCS) after in vivo drug dosing, however, can offer pharmacokinetic and pharmacodynamic insights on the effects of drugs to precise cell subpopulations. Moreover, this can be reliably achieved with up to 66% fewer animals than standard in vivo pharmacology approaches due to lower data variance afforded through analytics on defined, viable cell numbers. Here we describe the TDCS methodology for the isolation of total lung epithelia, lung macrophages, and epithelium/macrophage-depleted cell fractions from mouse lungs using a two-stage sorting process of immunomagnetic bead separation followed by flow cytometric sorting using fluorescent antibodies against well-established surface markers such as F4/80, CD11b, and CD326. Validated antibodies for additional cell types and markers are also provided.
3.6 Coculturing with Astrocytes v1
Authors: J. Sauter Evelyn , K. Kutsche Lisa , D. Klapper Simon , Busskamp Volker
Published: September 2021

This is part 3.4 of the "Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders" collection of protocols. Collection Abstract: Patient-derived or genomically modified human induced pluripotent stem cell s (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.
Generating Mutant Renal Cell Lines Using CRISPR Technologies v1
Authors: Perretta-Tejedor Nuria , Freke Grace , Seda Marian , A. Long David. , Jenkins Dagan
Published: September 2021

Gene editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalized cell lines. This procedure begins with a straightforward cloning step to generate a single plasmid encoding the Cas9 enzyme as well as a synthetic guide RNA (sgRNA) which is selected to target specific sites within the genome. This plasmid is transfected into cells either alone, in order to generate random insertion-deletion alleles (“indels”) at the desired locus via the nonhomologous end-joining pathway, or in conjunction with a homology-directed repair template oligonucleotide to generate a specific point mutation. Here we describe a procedure to perform gene editing in IMCD3 and HEK293 cells and to subsequently isolate clonal cell lines carrying mutations of interest.
TOOLS FOR ENSURING THE STRATEGIC SUSTAINABILITY OF NETWORK TRADE ORGANIZATIONS
Authors:   Siberian Federal University, Chaplina Albina
Published: September 2021

The development of crisis situations on a global scale and their deepening actualizes the problems of sustainable development of organizations operating in a competitive environment. The transformational changes taking place in the Russian economy today put forward new requirements for the theoretical and methodological support of the activities of commercial firms. This is especially true for large network enterprises, including retail businesses, which have a significant scale of activity and complex network interactions. In the works of both foreign and Russian scientists, various aspects of network business are considered, mechanisms and tools for strategic sustainability are proposed. However, in a turbulent environment, there is a constant search for new forms of network interaction and appropriate tools for effective activity. The purpose of this study is to develop a mechanism for managing the strategic stability of a network trade organization based on the formation of an optimal set of tools that provide the necessary level of each type of enterprise stability. The article considers the concept of "tool", offers modern tools for ensuring strategic stability in the context of various types of sustainability. This paper reveals the content of the most relevant modern tools of strategic sustainability in the context of digitalization of the network organization. The authors have developed a mechanism for strategic stability of a network trade organization, including the principles and methods of management, which allows optimizing the composition of stability tools depending on the factors of the external and internal environment. On the example of a specific organization, a list of strategic sustainability tools was formed on the basis of a comprehensive analysis and assessment of the level of each type of sustainability. The proposed methodology allows you to take into account the specifics of network trade organizations and achieve your goals more effectively using modern management tools.
SCIENTIFIC AND METHODOLOGICAL APPROACHES TO ASSESSING THE EFFECTIVENESS OF THE COMPANY'S EMPLOYEES' WORK
Authors:   Siberian Federal University, Demchenko Svetlana
Published: September 2021

The information base of the study was the works of economists in the field of approaches to assessing the effectiveness of worker’s work. The article is devoted to the development of an algorithm for evaluating labor efficiency, taking into account the improved methodology. The hypothesis of the study is the assumption of increasing the influence of the concept of sustainable economic development on the choice of methods for evaluating the effectiveness of labor resources. Within the framework of this study, the authors consider various concepts for the study of labor efficiency, highlight the advantages and disadvantages of existing approaches and offer their own concept of evaluating labor efficiency from the point of view of the concept of sustainable development. The approbation of the author's methodology is presented on the example of a specific company.