bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating the association between allergic diseases and COVID-19 in 400 Iranian patients
Authors: Darabi Amirhossein , Dehghanfard Mohammadrezad , Jozan Sara , Tahmasebi Rahim , Movahed Ali , Zamani Marjan , Farrokhi Shokrollah
Published: September 2021

Introduction: Allergic diseases could play a role of a predisposing factor for coronavirus dis-ease 2019 (COVID-19). The aim of this study was to investigate allergic comorbidity and its association in COVID-19 patients.
Methods: Demographic data, clinical manifestations, laboratory reports, and radiologic findings, together with underlying comorbidity of patients, were studies. Allergic diseases were identified by using the standard GA2LEN questionnaire. The severity of COVID-19 was assessed by a visual analog scale (VAS) and an intensive care unit (ICU) report.
Results: Out of 400 COVID-19 patients admitted in the hospital, 158 (39.5%) presented with different allergic diseases, and a reverse association was observed between having allergic comorbidity and severity of COVID-19 infection (P = 0.005, relative risk = 0.96; 95% Confidence Interval (95% CI): 0.77–1.19). The respective frequency of asthma, allergic rhinitis (AR), chronic rhinosinusitis (CRS), atopic dermatitis, chronic urticaria, and food or drug allergy was 7.3%, 16%, 1.8%, 5%, 10% and 13.3%. Significantly, only AR was reversely associated with the severity of COVID-19 (P = 0.02, relative risk = 0.45; 95% CI: 0.77–1.19). Additionally, 43% of the patients presented hypoxemia, and 93.5% had chest CT scan involvement. Interestingly, patients with allergic diseases had significantly lower hypoxemia and chest CT involvement as compared with non-allergic patients (P = 0.002 and 0.003, respectively).
Conclusion: The results of this study established that allergic diseases were not determined to be a predisposing factor for the severe acute respiratory syndrome (SARS) due to coronavirus 2 (SARS-CoV-2) infection. Significantly, AR patients developed mild clinical manifestations of COVID-19 and admitted to ICU as compared to non-AR patients.
Efficacy of air purifier therapy for patients with allergic asthma
Authors: Jia-ying Luo , Li-li Ou , Jing Ma , Xin-yuan Lin , Li-min Fan , Hai-cheng Liu , Bao-qing Sun
Published: September 2021

Objectives: This study aimed to evaluate the efficacy of air purifier therapy for patients with allergic asthma.
Methods: Thirty-eight subjects were categorized under two groups namely treatment group and control group. All subjects were under 18 years of age and they had been clinically diagnosed with allergic asthma. The treatment group used high efficiency particulate air (HEPA) purifiers for six consecutive months, and the control group did not use the air filters. Particulate matter (PM) data and dust samples (from bedding and a static point) were collected from the subjects’ bedrooms before they started using the air purifiers and each month thereafter. Simultaneously, the subjects were asked to complete a questionnaire for the Asthma Control Test (ACT) or Childhood Asthma Control Test (C-ACT). Fractional exhaled nitric oxide (FENO) tests were performed at the start and end of the study. The concentrations of Der p1 and Der f1 were measured in the dust samples.
Results: (1) After utilizing the air purifier, the concentrations of house dust mite (HDM) allergens (Der p1+ Der f1) in the dust samples decreased. In addition, the PM indoor/outdoor values significantly decreased. (2) The ACT and C-ACT scores in the treatment group maintained a steady significant upward trend. (3) At the end of the study, the FENO levels in both groups were lower, although the differences were not significant.
Conclusions: It is witnessed that HEPA air purifiers can decrease indoor HDM allergen and PM levels and improve the quality of life for allergic asthma patients.
Rhinomanometry: point of care test (POCT) for allergic rhinitis in children?
Authors: Malizia Velia , Ferrante Guiliana , Cilluffo Giovanna , Fasola Salvatore , Montalbano Laura , La Grutta Stefania
Published: September 2021

Allergic rhinitis (AR) is a global health problem: its prevalence is 23% in Europe, although it is underestimated because as many as 45% of the cases remain undiagnosed. Globally, almost 500 million people suffer from AR, which shows its increasing incidences. The diagnostic course of AR is based on clinical history, supported by anterior rhinoscopy. This inspects the anterior part of the nasal cavity accompanied by allergic sensitivity tests (cutaneous allergic skin tests or specific immunoglobulin E levels). The availability of standardised diagnostic proceduresis able to provide objective evaluations of inflammatory situation, and the degree of nasal obstruction may give an advantage in reducing the risk of underestimating the diagnosis of AR. Diagnostic tests with a high level of accuracy are able to provide immediate results, which can sustain the doctor in diagnostic–therapeutic framework. The development of Point of Care Tests (POCTs) could be a useful tool. Considering that nasal obstruction is the mostcommon symptom in patients with AR, the rhinomanometry (RM) test is the most indicated objective evaluation for nasal obstruction. Several studies have also shown the practicability of such diagnostic techniques applied in children. So far, no study has evaluated whether all the applicable requirements are fulfilled by RM in order to be considered as a POCT. The purpose of this perspective was to assess whether all the POCT requirements are fulfilled by RM by conducting a narrative review of the existing literature in which RM has been used in the diagnosis and management of AR in children. A few but encouraging results of studies on children supported the potential use of RM in the area of POCT. However, costs of instruments and the training of personnel involved remain to be explored. The studies support the potential use of RM in POCTs.
Echinococcus multilocularis induces surface high expression of inhibitory killer immunoglobulin‐like receptor on natural killer cells
Authors: Bayindala  , Huang He , Gao Song , Xu Xinjian
Published: September 2021

Alveolar echinococcosis (AE) is a malignant and fatal parasitic disease caused by the larvae of Echinococcus multilocularis (E. multilocularis), which inhibits the activity and proliferation of natural killer (NK) cells. In this study, the functional alteration of hepatic NK cells and their related molecules were studied. The AE-infected patient’s tissue was fixed with formalin, embedded in paraffin, and stained with Masson’s trichrome or hematoxylin and eosin (H&E). Single cells from AE-infected patient or E. multilocularis-infected mice were blocked with Fc-receptor (FcR), and stained with monoclonal antibodies, including CD16, CD56, CD3, KIR2DL1, granzyme B, perforin, Interferon gamma (IFN-γ), and tumor necrosis factor-α (TNF-α) or isotype control, to measure molecules and cytokines of NK cells and analyzed by flow cytometry. The Sirius red staining was used to quantitate hepatic fibrosis by calculating quantitative collagen deposition. AE can adjust both the number of hepatic CD56+ NK cells and its KIR2DL1 expression processes. Moreover, the overexpression of KIR2DL1 in NK cells could downregulate the functioning of immune cells in the liver area close to parasitic lesions. The number and dysfunction of NK cells in E. multilocularis infection could be related to the molecule dynamics of cell surface inhibitory receptor Ly49A, leading to hepatic damage and progression of fibrosis. This study illustrated significant increase in hepatic fibrogenesis and apparent upregulation of hepatic CD56+ NK cell population and its KIR2DL1 expression in AE-infected patients. This opposite variation might be related to the impaired NK cells functioning, such as granzyme B, IFN-γ, and TNF-α secretion. In addition, the cell surface inhibitory receptor Ly49A was related to the intracellular cytokine secretion functions of NK cells.
Steroid-sparing effect of mepolizumab in children with severe eosinophilic nonallergic asthma
Authors: Tosca Maria Angela , Girosi Donata , Sacco Oliviero , Bernardini Roberto , Ciprandi Giorgio
Published: September 2021

Background: Asthma is characterized by a chronic airway inflammation, usually sustained by type 2 immunity. Bronchial and peripheral eosinophilia are biomarkers for type 2 asthma. Biologicals are the most effective treatment for severe asthma at present. Mepolizumab is an antagonist of interleukin-5 (IL-5), the most relevant cytokine involved in eosinophilia.Objective: This case report evaluated the effectiveness of mepolizumab in two girls with severe eosinophilic non-allergic asthma.Materials and methods: Two female children with severe eosinophilic nonallergic asthma were treated with mepolizumab for two years. Clinical findings, lung function, peripheral eosinophils, asthma control, and bronchial endoscopy were performed.Results: Biologicals reduced the eosinophilia, asthma exacerbations, and improved lung function in both patients. The treatment was also safe and well-tolerated.Conclusion: Mepolizumab represents an effective therapeutic option in the management of severe pediatric asthma.
Arabian undergraduates perceptions regarding barriers among the geriatric patients for failing to keep dental appointments: A cross-sectional study
Authors: Shaikh Saquib Ahmed , Aldhuwayhi Sami , Joseph Angel Mary , Varadharaju Vinutha kumari , Khan Abdul Rehman Ahmed , Thakare Amar Ashok , Manva Mohnish Zulfikar , Desai Farheena , Saleem Shaikh  , Mustafa Mohammed Ziauddeen , Mallineni Sreekanth Kumar
Published: September 2021

Abstract:

Background: Regular visits to the health care providers can develop a relationship that can extend beyond the physical health alone as the patient is transiting towards older age, adapting to changes in physical health, emotional health, and social connections. Apart from limiting access to health care services, the attitudes, beliefs, comfort level of the treating doctors towards the geriatric patients can motivate or demotivate them to access dental care.

Aims:  To explore the Saudi Arabian undergraduate student’s perception of geriatric patients' and identify potential barriers that prevent the utilization of their dental appointment.

Methods: A close-ended questionnaire with one question and eight reasons was administered to the 5th year clinical students. The students were requested to specify their agreement with each question on 5 points Likert scale. Among the barriers presented, each reason's approval was expressed as the percentage of the total number of responses. In addition, the gender comparison of mean scores was made, and an independent sample t-test was used to analyze the statements agreed by the students. All analyses were performed using SPSS (Statistical Package of Social Sciences) version 21.0 (IBM, USA) with the probability of statistical significance at 0.05 level.

Results: Fifty-one students recorded their perceptions on the questionnaire administered during their clinical posting in the fifth year of the geriatric dental education program. It was concluded that students believed that geriatric patients give overwhelming importance to other problems with minor importance to oral health care. In addition, gender comparison was more evident as the percentage expressed was more in females.

Conclusion: There is a need for more clinical exposure of geriatric patients during their clinical postings. Student’s acquaintance with didactic and clinical settings appears to be a critical element towards positive knowledge and attitude towards the geriatric population.

Keywords: Geriatric dentistry, perception about geriatric, dental students, geriatric patients
Impact of chemical weathering on microstructures and mechanical properties of karstic limestone
Authors: Niazi F. S. , Piñán-Llamas A. , Fekadu F.
Published: September 2021

Dissolution and chemical weathering of carbonate rocks have complex effects on their mechanical properties, accelerating their rate of deterioration that can lead to early failure under external stresses. To investigate the effects of acidic solutions on the mechanical properties of inherently low-porosity karstic limestone, compression tests were performed on sound and weathered samples exposed to different corrosive environments and for different durations. The tested samples exhibited a general increase in porosity and decrease in peak strength and Young's modulus for higher acidity and exposure duration. To establish links between these mechanical changes and those at the micro-scale, microstructures and mechanisms of deformation were also studied. As indicators of the sample degradation, the distribution, orientation and intensity of the fractures in the tested samples and their twinning density were used. Based on these parameters, it was found that chemical erosion influences microcrack propagation, orientation, distribution and intensity, due to modification of the competence contrast between the constituents of the rock. These effects significantly influence the semi-brittle behaviour of the samples, including the distribution and intensity of microcracking and crystal plasticity.
REQUIREMENTS FOR THE DEVELOPMENT OF ELECTRONIC EDUCATIONAL MATERIALS
Authors:   Kyrgyz Academy of Education, Ibirayim kyzy Aizhan , Solpubashova Askerbүbү ,   Kyrgyz Academy of Education, Kaldybaev Salidin ,   Alatoo International University
Published: September 2021

In the system of school education of the Kyrgyz Republic, the problem of developing electronic educational material and introducing it into the educational process of schools is becoming urgent. This goal allows the further implementation of the issue of computerization of school education. This article focuses on the quality of education. To achieve high-quality modern training, the use of electronic materials is required. Electronic materials must be designed in accordance with the rules. Therefore, the content of this article is aimed at revealing the importance of e-learning, at developing electronic materials, at characterizing the requirements for compiling electronic materials and the requirements for using electronic educational materials.
PROBLEM LEARNING AS AN ACTIVE LEARNING METHOD
Authors:   Kyrgyz State University named after Arabaev, Kaldybaeva Aichuruk , Dzhumagulova Gulnur ,   Bishkek Humanitarian University named after Karasaev
Published: September 2021

Active learning technologies include problem-based learning. It is based on the solution of a problem or task. In a broad sense, a problem is a complex theoretical and practical issue that requires study and resolution; in science, it is a contradictory situation that appears as opposite positions in the explanation of any phenomena, objects, or processes and requires an adequate theory to resolve it. The advantages of problem learning are, first of all, great opportunities for the development of attention, observation, activation of thinking, activation of cognitive activity of students; it develops independence, responsibility, criticism and self-criticism, non-standard thinking.
PROPERTIES OF EDIBLE FILM FROM MODIFIED SAGO STARCH PRECIPITATED BY BUTANOL
Authors: Ningrum Riska Surya , Sondari Dewi , Amanda Putri , Widyaningrum Bernadeta Ayu , Burhani Dian , Akbar Fazhar , Sampora Yulianti
Published: September 2021

ROPERTIES OF EDIBLE FILM FROM MODIFIED SAGO STARCH PRECIPITATED BY BUTANOL. The edible film is a thin layer that can be used as food packaging and safe for consumption. Starch is a material that can be used as an edible film because it is biodegradable, non-toxic, able to form a strong and transparent film. In this research, sago starch has been precipitated using butanol for 1, 2, 3, 4, and 5 hours. Precipitated sago is reacted with 3% glycerol and then formed into a film by printing it on a petri dish. Characterizations of edible films are FTIR, contact angle, water solubility, swelling power, WVTR, and mechanical properties (thickness, tensile strength, and elongation). Edible film (B2) has the best mechanical properties, that is high hydrophobicity (contact angle is 60,351 degree), tensile strength (6,8843 N/mm2), and elongation (49,5081 %), also low water solubility (0,3352 %), moisture content (18,6005 %), and WVTR (0,02986 g s-1 m-2).
PICKERING EMULSION TECHNOLOGY IN FABRICATE CELLULOSE FOAM FROM OIL PALM EMPTY FRUIT BUNCH WASTE
Authors: Amanda Putri , Nabila Syarifah , Ismadi Ismadi , Purnomo Deni , Masruchin Nanang
Published: September 2021

PICKERING EMULSION TECHNOLOGY IN FABRICATE CELLULOSE FOAM FROM OILPALM EMPTY FRUIT BUNCH WASTE. Cellulose from the oil palm empty fruit bunch (OPEFB) waste can make a porous material. This study aims to make cellulose foam with Pickering emulsion technology used cellulose nanofiber as a Pickering agent. The mechanism of Pickering emulsion is learned from foamability and stability of foam in the presence of various concentrations of surfactant. The result showed that using Pickering emulsion technology only needed surfactant with a small concentration to improve foamability and stability. The addition of CNF indeed improved the stability and foamability with the Pickering effect. The stability test shows that the foam stabilized with CNF appeared to be relatively stable. In contrast to the CNF free system, the foams were collapse in three days tested. Structures of foam was characterized using an optical microscope and showed that the foam was composed into two- or three dimensional microstructures formed by gas bubble of wet foam in random orientations. This process generated the lightweight Cellulose foam from OPEFB waste, with a density of 0.07 g/cm3. Using Pickering emulsion technology to make cellulose foam can be one way to overcome OPEFB waste and this foam is potential for various applications.
SYNTHESIS AND CHARACTERIZATION: COMPOSITE OF GRAPHENE OXIDE BASED PALM KERNEL SHELL WASTE WITH FE3O4
Authors: Widyaningrum Bernadeta Ayu , Apriani Dita , Amanda Putri , Ismadi Ismadi , Sutanto Sutanto
Published: September 2021

SYNTHESIS AND CHARACTERIZATION: COMPOSITE OF GRAPHENE OXIDE BASED PALM KERNEL SHELL WASTE WITH Fe3O4. In this study, GO-Fe3O4 were fabricated by co-precipitation technique and the graphene oxide (GO) were synthesized from an agricultural biomass, palm kernel shell, via Hummer’s method. Field Emission Scanning Electron Microscopy and Energy Dispersive Spectrum (FESEM-EDS), Fourier Transform Infra-Red (FT-IR) spectroscopy, X-Ray Diffractometer (XRD), and Raman spectroscopy were used to analysis the successful attachment of Fe3O4 onto the surface of GO. Morphology observation showed that Fe3O4 were heterogeneously deposited on the surface of GO. FT-IR spectra shows peak that incorporated to oxygenated functional groups and sharp peak at 586 cm-1 confirmed to lattice absorption of Fe3O4. The percentage of composition of GO-Fe3O4 was characterized by energy dispersive spectroscopy and the results also confirmed in XRD exhibits similar properties with JCPDS 19-0629 for magnetite more dominant than GO. From Raman spectroscopy analysis shows that 1343.82 cm-1 (D-band) and 1584.62 cm-1 (G-band) and 2698 cm-1 (2D-band) indicates GO and GO-Fe3O4 were successfully synthesized.
Fluorescent Molecular Beacons Mimicking RNA Secondary Structures to Study RNA Chaperone Activity v1
Authors: Menendez-Gil Pilar , J. Caballero Carlos , Solano Cristina , Toledo-Arana Alejandro
Published: September 2021

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.
Isolation and Phenotyping of Adult Mouse Microglial Cells v1
Authors: Grabert Kathleen , W. McColl Barry
Published: September 2021

Microglia are the resident macrophages of the central nervous system parenchyma and fulfill crucial roles in brain development, homeostasis, and inflammation. The isolation of a pure microglia population from brain tissue enables the examination of microglial phenotypes without the interference of other cell populations. Microglial extractions from the neonatal brain have been described in various protocols, yet the more established and complex adult mouse brainposes a greater challenge. Here we describe a refined protocol including enzymatic and mechanical dissociation of adult mouse brain tissue and removal of myelin by Percoll density gradient. Microglial cells were subsequently extracted by an immunomagnetic approach. This isolation procedure enables the use of functionally viable cells for various applications such as cell culture, flow cytometry, functional assays including bacteria- or bead-based phagocytosis, stimulation assays, and transcriptome profiling techniques such as qRT-PCR and microarray/RNA sequencing.
Bioinformatics Analysis of Estrogen-Responsive Genes v1
Authors: E. Handel Adam
Published: September 2021

Estrogen is a steroid hormone that plays critical roles in a myriad of intracellular pathways. The expression of many genes is regulated through the steroid hormone receptors ESR1 and ESR2. These bind to DNA and modulate the expression of target genes. Identification of estrogen target genes is greatly facilitated by the use of transcriptomic methods, such as RNA-seq and expression microarrays, and chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq). Combining transcriptomic and ChIP-seq data enables a distinction to be drawn between direct and indirect estrogen target genes. This chapter discusses some methods of identifying estrogen target genes that do not require any expertise in programming languages or complex bioinformatics.
Efficient Depletion of Fission Yeast Condensin by Combined Transcriptional Repression and Auxin-Induced Degradation v1
Authors: Kakui Yasutaka , Uhlmann Frank
Published: September 2021

Structural maintenance of chromosomes (SMC) complexes play pivotal roles in controlling chromatin organization. Condensin is an essential SMC complex that compacts chromatin to form condensed chromosomes in mitosis. Complete condensin inactivation is necessary to reveal how condensin converts interphase chromatin into mitotic chromosomes. Here, we have developed a condensin depletion system in fission yeast that combines transcriptional repression with auxin-inducible protein degradation. This achieves efficient condensin depletion without need for a temperature shift. Our system is useful when studying how condensin contributes to chromosome architecture and is applicable to the study of other SMC complexes.
Predicting Tankyrase Binders v1
Authors: Pollock Katie , Ranes Michael , Collins Ian , Guettler Sebastian
Published: September 2021

This protocol is part of a collection: Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach The poly(ADP-ribose)polymerase (PARP) enzyme tankyrase (TNKS/ARTD5, TNKS2/ARTD6) uses its ankyrin repeat clusters (ARCs) to recognize degenerate peptide motifs in a wide range of proteins, thereby recruiting such proteins and their complexes for scaffolding and/or poly(ADP-ribosyl)ation. Here, we provide guidance for predicting putative tankyrase-binding motifs, based on the previously delineated peptide sequence rules and existing structural information. We present a general method for the expression and purification of tankyrase ARCs from Escherichia coli and outline a fluorescence polarization assay to quantitatively assess direct ARC–TBM peptide interactions. We provide a basic protocol for evaluating binding and poly(ADP-ribosyl)ation of full-length candidate interacting proteins by full-length tankyrase in mammalian cells.
Validation of Tankyrase Binding and PARylation in Full-Length Protein Context v1
Authors: Pollock Katie , Ranes Michael , Collins Ian , Guettler Sebastian
Published: September 2021

This protocol is part of a collection: Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach The poly(ADP-ribose)polymerase (PARP) enzyme tankyrase (TNKS/ARTD5, TNKS2/ARTD6) uses its ankyrin repeat clusters (ARCs) to recognize degenerate peptide motifs in a wide range of proteins, thereby recruiting such proteins and their complexes for scaffolding and/or poly(ADP-ribosyl)ation. Here, we provide guidance for predicting putative tankyrase-binding motifs, based on the previously delineated peptide sequence rules and existing structural information. We present a general method for the expression and purification of tankyrase ARCs fromEscherichia coliand outline a fluorescence polarization assay to quantitatively assess direct ARC–TBM peptide interactions. We provide a basic protocol for evaluating binding and poly(ADP-ribosyl)ation of full-length candidate interacting proteins by full-length tankyrase in mammalian cells.
Enzyme–Ligand Interaction Monitored by Synchrotron Radiation Circular Dichroism 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.
Low-Volume Titrations for Ligand Binding Monitored by Circular Dichroism 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.
Processing of Circular Dichroism Data Collected at B23 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.
Versatile Cell-Free Protein Synthesis Systems Based on Chinese Hamster Ovary Cells v1
Authors: Thoring Lena , Kubick Stefan
Published: September 2021

We present an alternative production platform for the synthesis of complex proteins. Apart from conventionally applied protein production using engineered mammalian cell lines, this protocol describes the preparation and principle of cell-free protein synthesis systems based on CHO cell lysates. The CHO cell-free system contains endogenous microsomes derived from the endoplasmic reticulum, which enables a direct integration of membrane proteins into a nature like milieu and the introduction of posttranslational modifications. Different steps of system development are described including the cultivation of CHO cells, cell harvesting and cell disruption to prepare translationally active CHO cell lysates. The requirements for DNA templates and the generation of linear DNA templates suitable for the CHO cell-free reaction is further depicted to underline the opportunity to produce different protein variants in a short period. This experimental setup provides a basis for high-throughput applications. The productivity of the CHO cell-free systems is further increased by using a non-canonical translation initiation due to the attachment of an internal ribosomal entry site of the Cricket paralysis virus (CRPV IRES) to the 5´ UTR of the desired gene. In this way, a direct interaction of the IRES structure with the ribosome facilitates a translation factor independent initiation of translation. Cell-free reactions were performed in fast and efficient batch reactions leading to protein yields up to 40μg/mL. The reaction format was further adjusted to a continuous exchange CHO cell-free reaction (CHO CECF) to prolong reaction time and thereby increase the productivity of the cell-free systems. Finally, protein yields up to 1g/L were obtained. The CHO CECF system represents a sophisticated resource to address structural and functional aspects of difficult-to-express proteins in fundamental and applied research.
Immunoprecipitation Washes and RNA Dephosphorylation 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.
Ligate 3′ RNA Adapter (On-Bead) 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.
Dephosphorylation of 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.
Crosslinking of Cultured Cells 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.
Cell Lysis and RNA Fragmentation 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.
Purification of Cyclic GMP-AMP from Viruses and Measurement of Its Activity in Cell Culture v1
Authors: Mayer Alice , Maelfait Jonathan , Bridgeman Anne , Rehwinkel Jan
Published: September 2021

Sensing of cytoplasmic DNA by cGAS is essential for the initiation of immune responses against several viruses. cGAS also plays important roles in some autoinflammatory and autoimmune diseases and may be involved in immune responses targeting cancer cells. Once activated, cGAS catalyzes the formation of the di-nucleotide 2′-3′-cyclic GMP-AMP (cGAMP), which propagates a signaling cascade leading to the production of type I interferons (IFNs). Interestingly, cGAMP is incorporated into enveloped viruses and is transferred to newly infected cells by virions. In this article, we describe a method to purify cGAMP from viral particles and a bioassay to measure its activity. This assay takes advantage of a reporter cell line that expresses the genes encoding green fluorescent protein (GFP) and firefly luciferase under the control of the IFNß promoter, allowing the testing of several samples in a single experiment taking not more than 3 days.
Super-Resolution Single Molecule FISH at the Drosophila Neuromuscular Junction v1
Authors: S. Titlow Joshua , Yang Lu , M. Parton Richard , Palanca Ana , Davis Ilan
Published: September 2021

The lack of an effective, simple, and highly sensitive protocol for fluorescent in situ hybridization (FISH) at the Drosophila larval neuromuscular junction (NMJ) has hampered the study of mRNA biology. Here, we describe our modified single molecule FISH (smFISH) methods that work well in whole mount Drosophila NMJ preparations to quantify primary transcription and count individual cytoplasmic mRNA molecules in specimens while maintaining ultrastructural preservation. The smFISH method is suitable for high-throughput sample processing and 3D image acquisition using any conventional microscopy imaging modality and is compatible with the use of antibody colabeling and transgenic fluorescent protein tags in axons, glia, synapses, and muscle cells. These attributes make the method particularly amenable to super-resolution imaging. With 3D Structured Illumination Microscopy (3D-SIM), which increases spatial resolution by a factor of 2 in X, Y, and Z, we acquire super-resolution information about the distribution of single molecules of mRNA in relation to covisualized synaptic and cellular structures. Finally, we demonstrate the use of commercial and open source software for the quality control of single transcript expression analysis, 3D-SIM data acquisition and reconstruction as well as image archiving management and presentation. Our methods now allow the detailed mechanistic and functional analysis of sparse as well as abundant mRNAs at the NMJ in their appropriate cellular context.
Functional Analysis of Membrane Proteins Produced by Cell-Free Translation v1
Authors: Kumar Dondapati Srujan , A. Wüstenhagen Doreen , Kubick Stefan
Published: September 2021

Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20μL up to several mL, faster and suitable for the high-throughput protein production. Here, we present two cell-free systems derived from Escherichia coli (E. coli) and Spodoptera frugiperda (Sf21) lysates. In the case of the E. coli cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. In the case of the Sf21 system, endogenous microsomes with an active translocon complex are present within the lysates which facilitate the incorporation of the bacterial potassium channel KcsA within the microsomal membranes. Following cell-free synthesis, these microsomes are directly used for the functional analysis of membrane proteins.
3.1 Synthesis of Glutathione Beads v1
Authors: Simons Peter , Bondu Virginie , Wandinger-Ness Angela , Buranda Tione
Published: September 2021

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then beattached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borneGTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells. Section 3.1 'Synthesis of Glutathione Beads' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee
3.4 Assay Runtime v1
Authors: Simons Peter , Bondu Virginie , Wandinger-Ness Angela , Buranda Tione
Published: September 2021

Small, monomeric guanine triphosphate hydrolases (GTPases) are ubiquitous cellular integrators of signaling. A signal activates the GTPase, which then binds to an effector molecule to relay a signal inside the cell. The GTPase effector trap flow cytometry assay (G-Trap) utilizes bead-based protein immobilization and dual-color flow cytometry to rapidly and quantitatively measure GTPase activity status in cell or tissue lysates. Beginning with commercial cytoplex bead sets that are color-coded with graded fluorescence intensities of a red (700nm) wavelength, the bead sets are derivatized to display glutathione on the surface through a detailed protocol described here. A different glutathione-S-transferase-effector protein (GST-effector protein) can then beattached to the surface of each set. For the assay, users can incubate bead sets individually or in a multiplex format with lysates for rapid, selective capture of active, GTP-bound GTPases from a single sample. After that, flow cytometry is used to identify the bead-borneGTPase based on red bead intensity, and the amount of active GTPase per bead is detected using monoclonal antibodies conjugated to a green fluorophore or via labeled secondary antibodies. Three examples are provided to illustrate the efficacy of the effector-functionalized beads for measuring the activation of at least five GTPases in a single lysate from fewer than 50,000 cells. Section 3.4 'Assay Runtime' from 'Small-Volume Flow Cytometry-Based Multiplex Analysis of the Activity of Small GTPases' https://www.protocols.io/view/small-volume-flow-cytometry-based-multiplex-analys-bpssmnee