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Authors: Gary Cline, John Stack
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure used to measure the activity of Aspartate Amino Transferase (AST). AST activity is measured by the enzymatically coupled reactions of AST (to form oxaloacetate from aspartate and α-ketoglutarate) and malate dehydrogenase (conversion of oxaloacetate to malate with oxdiation of NADH to NAD). The rate of NAD formation is monitored by the change in absorbance at 340 nm.
Proper citation: Gary Cline, John Stack 2019. Yale - Aspartate Amino Transferase. protocols.io dx.doi.org/10.17504/protocols.io.yz9fx96 Copy
Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads than with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.
Proper citation: Sam Li 2019. MojoSort™ Mouse anti-APC Nanobeads Column Protocol. protocols.io dx.doi.org/10.17504/protocols.io.699hh96 Copy
Authors: Eftychis Frangedakis, Marta Tomaselli, Marius Rebmann, Susana Sauret-Gueto
Group: OpenPlant Project
Proper citation: Eftychis Frangedakis, Marta Tomaselli, Marius Rebmann, Susana Sauret-Gueto 2019. Domestication of L0 parts for Loop type IIS (BsaI and SapI). protocols.io dx.doi.org/10.17504/protocols.io.4whgxb6 Copy
Authors: Hasheemah Afaneh , Gabrielle Gonzalez, Olivia Sugarman, Edward Trapido, Susanne Straif-Bourgeois, Evrim Oral, Ashley Wennerstrom
Group: Louisiana State University Health Sciences Center - New Orleans
Summary: As of 2017, there were more than three million individuals in the United States infected with Hepatitis C virus (HCV) [1]. Because most cases are asymptomatic, leading to a higher rate of unreported cases, this number is suspected to be much higher. Furthermore, there is a rise of HCV cases, and this increase documented since 2013 is primarily due to injection drug transmission alongside the rise of the opioid epidemic [2]. The Centers for Disease Control and Prevention (CDC) encourages HCV testing for all adults at least once in their lifetime, and those that test positive for HCV should be linked to treatment [3]. In 2013, only 13%-18% of patients with HCV received treatment in that year, indicating that there are barriers to treatment, such as lack of treatment acceptance, co-existing conditions, extensive treatment, side-effects, and access to treatment[4]. Thus, health disparities may ensue and the eradication of HCV as a public health issue becomes even more challenging. Edlin and Winkelstein (2014) suggested that to achieve optimal HCV eradication, social determinants of health, such as homelessness, need to be addressed [5]. Because there is room for improvement in linkage-to-care opportunities, it is important to explore whether linkage-to-care interventions address social determinants of health, and if so, which ones, to obtain a better understanding of what may help improve patient outcomes.References: U.S. Department of Health and Human Services. 2019.Retrieved June 11, 2020, fromhttps://www.hhs.gov/opa/reproductive-health/fact-sheets/sexually-transmitted-diseases/hepatitis-c/index.htmlCentersfor Disease Control and Prevention(CDC). 2019. Commentary portion of U.S. 2017 Surveillance Data for Viral Hepatitis. Retrieved June 11, 2020, fromhttps://www.cdc.gov/hepatitis/statistics/2017surveillance/index.htmSchillie, S., Wester, C., Osborne, M., Wesolowski, L., & Ryerson, A.B. (2020). CDC Recommendations for Hepatitis C Screening Among Adults – United States, 2020. MMWR Recommendations & Reports, 69(2), 1-17.https://www.cdc.gov/mmwr/volumes/69/rr/rr6902a1.htmInfectious Disease Society of America. 2019. HCV Testing and Linkage to Care: HCV Guidance. Retrieved June 23, 2020, fromhttps://www.hcvguidelines.org/evaluate/testing-and-linkageEdlin, B. & Winkelstein, E. (2014). “Can Hepatitis C be eradicated in the U.S.?”Antiviral Research.110: 79-93.https://dx.doi.org/10.1016/j.antiviral.2014.07.015
Proper citation: Hasheemah Afaneh , Gabrielle Gonzalez, Olivia Sugarman, Edward Trapido, Susanne Straif-Bourgeois, Evrim Oral, Ashley Wennerstrom 2020. Addressing Social Determinants of Health in Linkage-to-Care Interventions for Hepatitis C: A systematic review . protocols.io dx.doi.org/10.17504/protocols.io.bka6kshe Copy
Authors: Ales Mishchenko, Herve Glotin
Summary: The method described here is from the PLoS ONE paper entitled "Joint time delay and Doppler passive acoustic 3D tracking, applied to studies of bats in natural habitats".
This method detects acoustic events (possible bat calls) and filters them according to their
consistency with each other as well as with extracted kinematics of the bat. This allows to filter out false detections, related to echoes and noise, improves the reconstruction of bats trajectories and allows to study bats behavior.This method (with another parameters) can be also potentially applied to the extraction of the trajectory of any fast moving objects, in particular, sound-emitting machinery, such as trajectories of cars before and after accident, and many others.This protocol (together with bat-related parameters, such as frequency ranges, intervals between calls, etc) is designed to reproduce the method of Joint time delay and Doppler passive acoustic 3D tracking in a case of tracking bats by an array of 4 ultrasonic microphones.
Proper citation: Ales Mishchenko, Herve Glotin 2017. Joint time delay and Doppler passive acoustic 3D tracking of bats.. protocols.io dx.doi.org/10.17504/protocols.io.iyjcfun Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol outlines some of the steps that are required to align a TissueCyte 1000 serial two-photon imaging system and install commonly replaced parts.
Proper citation: Allen Institute for Brain Science 2020. TissueCyte Installation And Alignment Guide. protocols.io dx.doi.org/10.17504/protocols.io.bbx9ipr6 Copy
Authors: Ayush Alag
Summary: Current laboratory tests have a less than 50% accuracy in distinguishing between people who have food allergies (FA) and those who are merely sensitized to foods, resulting in the use of expensive and potentially dangerous Oral Food Challenges. Our study presents a purely-computational machine learning approach, conducted using DNA Methylation (DNAm) data, to accurately diagnose food allergies and find genes that are strong biomarkers of the disease. We built two deep learning classifiers with twelve CpG-input features each that achieved perfect accuracy and an AUROC of 1 on the completely hidden cross-validation cohort. In addition, 24 additional classifiers were created that each had an average cross-validation accuracy of 98.35%. These 26 classifiers yielded a total of 18 unique CpGs, which mapped to 13 genes that are strong epigenetic biomarkers of FA.Biological enrichment on the 13-gene signature yielded new insights. Notably, our FA-discriminating genes were strongly associated with the immune system, which helps validate our findings. Seven of the 13 genes overlapped with previous food-allergy and DNAm studies.Previous studies have also created a perfect classifier for this dataset, but they used a 96-CpG input feature set built on both data-driven and a priori biological insights. Our study is an improvement on previous work because it maintains a perfect classification accuracy using only 18 highly discriminating CpGs (0.005% of the total available features). In machine learning, simpler models, as used in our study, are preferred over more complex ones (all other things being equal).In addition, our completely data-driven approach eliminates the need for \textit{a priori} information and allows for generalizability to DNAm classification problems in other disease areas, which may result in novel gene associations or accurate diagnostic tests for those diseases.
Proper citation: Ayush Alag 2019. Machine learning approach yields epigenetic biomarkers of food allergy: A novel 13-gene signature to diagnose clinical reactivity. protocols.io dx.doi.org/10.17504/protocols.io.x7pfrmn Copy
Authors: B.R. Cunningham, J.R. Brum, S.M. Schwenck, M.B. Sullivan, S.G. John
Group: VERVE Net, Sullivan Lab
Summary: Purpose: This method for the enumeration of aquatic viruses is a low-cost alternative to the commonly used filter-mount method. Briefly, fluorescently-stained samples are wetmounted directly onto slides for epifluorescence microscopy after an optional chemical flocculation concentration step used for samples with anticipated virus concentrations of 7 viruses mL-1 (samples with >5×107 viruses mL-1 do not require this concentration step prior to analysis). Virus concentration in the wet-mounted sample is determined from the ratio of viruses to microsphere beads, which are added at a known concentration. This wet-mount method for enumerating viruses is significantly less expensive than the filter-mount method (i.e., the cost of microsphere beads per sample is ~500-fold lower than the cost of one filter per sample), and is appropriate for rapid, precise and accurate enumeration of aquatic viruses over a wide range of viral concentrations encountered in field and laboratory samples. The only limitation of this method is that samples with virus concentrations ≤1×106 viruses mL-1 cannot be enumerated, as the abundance of viruses is too low for efficient enumeration.Figure 1. Overview of the wet-mount method for enumeration of aquatic viruses.
Proper citation: B.R. Cunningham, J.R. Brum, S.M. Schwenck, M.B. Sullivan, S.G. John 2016. Wet-mount Method for Enumeration of Aquatic Viruses. protocols.io dx.doi.org/10.17504/protocols.io.c8pzvm Copy
Authors: Szymon T Calus, Umer Zeeshan Ijaz, Ameet Pinto
Group: Pinto Lab
Proper citation: Szymon T Calus, Umer Zeeshan Ijaz, Ameet Pinto 2018. NanoAmpli-Seq - Bioinformatics Workflow. protocols.io dx.doi.org/10.17504/protocols.io.u25eyg6 Copy
Authors: Sam Li
Group: BioLegend
Summary: Immunoprecipitation is a procedure by which proteins or peptides that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics. Immunoprecipitation can also be used to “enrich” a protein population prior to Western Blotting. For example, one can perform immunoprecipitation with a pan-specific antibody against a protein of interest followed by Western blotting with a modification-specific antibody (such as a phospho-specific antibody or an acetylation-specific antibody).
Proper citation: Sam Li 2019. Immunoprecipitation Protocol. protocols.io dx.doi.org/10.17504/protocols.io.98qh9vw Copy
Authors: Chantal Vogels, Joseph Fauver, Isabel Ott, Nathan Grubaugh
Group: Coronavirus Method Development Community
Summary: The protocol describes how to generate high-quality single-stranded RNA transcript standards (starting from a virus RNA stock) targeting the nsp10, RdRp, nsp14, envelope (E), and nucleocapsid (N) coding regions for use with China CDC, Hong Kong University (HKU), Corman et al. (Berlin), and US CDC SARS-CoV-2 primer and probe sets for qRT-PCR. (Sequences for transcripts generated, along with their corresponding assays, are provided under 'Guidelines.')
Proper citation: Chantal Vogels, Joseph Fauver, Isabel Ott, Nathan Grubaugh 2020. Generation of SARS-COV-2 RNA transcript standards for qRT-PCR detection assays. protocols.io dx.doi.org/10.17504/protocols.io.bdv6i69e Copy
Authors: Ekaterina V. Stepanova, Anatoly V. Kondrashin, Vladimir P. Sergiev, Lola F. Morozova, Natalia A. Turbabina, Maria S.Maksimova, Alexey I. Brazhnikov, Sergei B. Shevchenko, Evgeny N. Morozov
Summary: ObjectivesThe objectives of our studies were to determine:a) the prevalence of chronic toxoplasmosis in population of Moscow city and Moscow region; b) to determine a probable role of the disease in the epidemiology of the RTA in Russian Federation.
Proper citation: Ekaterina V. Stepanova, Anatoly V. Kondrashin, Vladimir P. Sergiev, Lola F. Morozova, Natalia A. Turbabina, Maria S.Maksimova, Alexey I. Brazhnikov, Sergei B. Shevchenko, Evgeny N. Morozov 2017. Significance of chronic toxoplasmosis in epidemiology of road traffic accidents in Russian Federation. protocols.io dx.doi.org/10.17504/protocols.io.iyucfww Copy
Authors: Meta Herdiana Hanindita
Summary: Surgical intervention in infants is associated with postoperative sepsis and severe outcomes due to immature immune system1. Gastrointestinal surgery induces excessive cytokine secretion, which may lead to increased postoperative mortality and morbidity2. Parenteral nutrition plays a crucial role in pediatric patients who undergo gastrointestinal surgery3. Intravenous lipid emulsion is an integral part of parenteral nutrition because it contains high energy density and low osmolarity, hence becoming the main source of energy and essential fatty acids4,5. Fatty acid determines structural integrity and fluidity of cell membrane, and it has been proven that fatty acid helps to regulate expression of various genes and modulate cell-signaling pathway, which occurs during inflammation6,7. The current standard type of IVFE is a 50:50 mixture of medium chain triglyceride (MCT) and long chain triglyceride (LCT)8. This type of emulsion is rich in ω-6 and contains high levels of linoleic acid (LA, C18:2 ω -6) and alpha-linolenic acid (ALA, C18:3 ω-3). According to several studies, ω-6 is associated with impaired cell-mediated immunity and higher potential risk of elevated proinflammatory markers and severe inflammatory response. This mechanisms may lead to the increase in mortality, morbidity, duration of treatment, and recovery time in patients who undergo gastrointestinal surgery9-11.Calder (2010) showed that the structure modification of fatty acids may alter their functions7. Some studies have shown that the addition of ω-3 in soy oil-based fat emulsion may improve patients’ outcome by modulating inflammatory response3,4,12. ω-3, particularly eicosapentaenoic acid (EPA) and doxohexanoic acid (DHA), is a competitive enzyme inhibitor of arachidonic acid (AA). ω-3 has potential anti-inflammatory properties by inhibiting AA pathway and generating inflammatory eicosanoids, such as prostaglandine E3, thromboxane A3 and leukotriene B5, which are considered less inflammatory. Up to this time, the effect of ω-3-enriched intravenous fat emulsion compared to standard intravenous fat emulsion on the IL-1b ,IL-8 levels and plasma fatty acid composition in infants who undergo gastrointestinal surgery has yet to be elucidated. The purpose of this study is aimed to investigate the effect of ω-3-enriched intravenous fat emulsion compared to standard intravenous fat emulsion on the IL-1b ,IL-8 levels and plasma fatty acid composition in infants who undergo gastrointestinal surgeryTrial Design:Type: Parallel randomized controlled trialAllocation ratio:1:1Framework: SuperiorityAim of the study:The purpose of this study is to investigate the effect of ω-3-enriched intravenous fat emulsion compared to standard MCT/LCT intravenous fat emulsion on the IL-1b ,IL-8 levels and plasma fatty acid composition in infants who undergo gastrointestinal surgery.Research QuestionWill the Intravenous omega-3 enriched-fat emulsion make difference in IL-1β, IL-8 and fatty acid composition when compared to standard intravenous MCT/LCT fat emulsion?PICO approach:P: Patients underwent gastrointestinal surgery. I: ω-3-enriched intravenous fat emulsionC: MCT/LCT standard intravenous fat emulsionO: ABCD1OutcomeTool for MeasurementUnit of Measurement2PrimaryInflammatory Response: IL-1b, IL-8Bloodpg/ml3Fatty Acid CompositionBlood% Total Fatty Acid4SecondaryLaboratory parameters: HemoglobinBloodg/dL5Laboratory parameters: LeukocyteBlood/microL6Laboratory parameters: AlbuminBloodg/L7Laboratory parameters: CRPBloodmg/L
Proper citation: Meta Herdiana Hanindita 2020. Comparison of Two Lipid Emulsions on Interleukin-1β, Interleukin-8 and Plasma Fatty Acid Composition in Infants Post Gastrointestinal Surgery: A Randomized Trial. protocols.io dx.doi.org/10.17504/protocols.io.bknmkvc6 Copy
Authors: Natalie Solonenko
Group: Sullivan Lab
Summary: Now includes media used for phosphate limitation experiments.
Proper citation: Natalie Solonenko 2016. Pseudoalteromonas Media Recipes. protocols.io dx.doi.org/10.17504/protocols.io.fz4bp8w Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol is used to produce crude preps of AAV of any serotype.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Proper citation: Allen Institute for Brain Science 2020. Production of Crude AAV Virus Extract. protocols.io dx.doi.org/10.17504/protocols.io.bdvwi67e Copy
Authors: Stephen Floor
Group: Stephen Floor Lab
Summary: How to precipitate RNA with ethanol and resuspend it! Two separate protocols - one for small and one for large volumes. The procedure for large volume precipitation is similar, but begins with an initial precipitation and transfers to a small tube for the final steps to avoid loss in the large tube. Strongly recommended to move to a smaller tube. Recommended reading: Walker & Lorsch RNA purification--precipitation methods.
Proper citation: Stephen Floor 2019. Ethanol precipitation of RNA from small or large volumes. protocols.io dx.doi.org/10.17504/protocols.io.36fgrbn Copy
Authors: Cristian Riccio
Summary: Count worms, e.g. arrested L1s after an overnight starvation in M9 on a rotating wheel.
Proper citation: Cristian Riccio 2019. Counting worms / C. elegans / nematodes. protocols.io dx.doi.org/10.17504/protocols.io.5ung6ve Copy
Authors: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino
Summary: Published work using this protocol:- Kustrimovic N., Comi C., Magistrelli L., Rasini E., Legnaro M., Bombelli R., Aleksic I., Blandini F., Minafra B., Riboldazzi G., Struchio A., Mauri M., Bono G., Marino F., Cosentino M. Parkinson’s disease patients have a complex phenotypic and functional Th1 bias: cross-sectional studies of CD4+ Th1/Th2/T17 and Treg in drug-naïve and drug-treated patients (2018). Journal of neuroinflammation, 15(1), 205. https://doi.org/10.1186/s12974-018-1248-8- Kustrimovic, N., Rasini, E., Legnaro, M., Bombelli, R., Aleksic, I., Blandini, F., Comi, C., Mauri, M., Minafra, B., Riboldazzi, G., Sanchez-Guajardo, V., Marino, F., & Cosentino, M. (2016). Dopaminergic Receptors on CD4+ T Naive and Memory Lymphocytes Correlate with Motor Impairment in Patients with Parkinson's Disease. Scientific reports, 6, 33738. https://doi.org/10.1038/srep33738- Cosentino M., Ferrari M., Kustrimovic N., Rasini E., Marino F. (2015). Influence of dopamine receptor gene polymorphisms on circulating T lymphocytes: A pilot study in healthy subjects. Human immunology, 76, 10, 747-752. https://doi.org/10.1016/j.humimm.2015.09.032- Boydum A.Isolation of mononuclear cells and granulocytes from human blood. Scand.J.Clin.Lab. Invest. 21 (Suppl.97): 77-89, 1968- Alex Mabou Tagne, Franca Marino, Massimiliano Legnaro, Alessandra Luini, Barbara Pacchetti and Marco Cosentino. A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions. Int J Mol Sci2019 Apr; 20(8): 1833. Published online 2019 Apr 13. doi: 10.3390/ijms20081833.- Angela Scanzano, Laura Schembri, Emanuela Rasini, Alessandra Luini, Jessica Dallatorre, Massimiliano Legnaro, Raffaella Bombelli, Terenzio Congiu, Marco Cosentino, Franca Marino. Adrenergic Modulation of Migration, CD11b and CD18 Expression, ROS and interleukin-8 Production by Human Polymorphonuclear Leukocytes. Inflamm Res. 2015 Feb;64(2):127-35. doi: 10.1007/s00011-014-0791-8. Epub 2015 Jan 6.
Proper citation: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino 2020. CELL COUNT- 02 - Manual cell count with Türk Solution. protocols.io dx.doi.org/10.17504/protocols.io.bpxsmpne Copy
Authors: Oscar Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium
Group: Coronavirus Method Development Community
Summary: This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.
Proper citation: Oscar Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium 2020. V.4 - Direct wastewater RNA capture and purification via the "Sewage, Salt, Silica and SARS-CoV-2 (4S)" method. protocols.io dx.doi.org/10.17504/protocols.io.bpdfmi3n Copy
Authors: pooyan.eshkevari , riley.sumner , Robert Leon Flint, David Seligson
Group: Project Tibia
Summary: Background: The maxillofacial as well as orthopedic community have known proximal tibia bone as an abundant, reliable, and safe autogenous bone donor for reconstructive purposes. There have been reports of uncommon complications, mostly resolving seromas and hematomas, and rarely bone fractures following such procedures. Objectives: The authors aim to locate, review, and summarize the evidence concerning the biomechanics of the proximal tibial donor site following graft harvestation.Methods and analysis: Our study will follow the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist. With the help of a librarian, we will design a search strategy using Boolean features through PubMed, Embase, and Google Scholar using terms like tibia, graft, mechanical, shear, etc, restricted to English language. We will upload the results in EndNote, eliminate the duplicates, and scan the titles for potential inclusion. We will export the chosen titles into Rayyan QCRI website for abstract review and selection of articles for full text review. A summary and a critical appraisal of the extracted evidence will follow. Thestudy protocol is registered in Open Science Framework, https://osf.io/mequ6. Article summary: this study maps and summarizes the available evidence on the biomechanics of donor site following proximal tibial autogenous bone graft harvest.Strengths and limitations of this studyThe strength of this study relies on its systematic review of the evidence based on gold standard methods and protocol registrations. This study suggests there is need for further investigation into the biomechanics of proximal tibia autogenous bone harvest.This study is a scoping review and harbors the inherent limitations of such studies.
Proper citation: pooyan.eshkevari , riley.sumner , Robert Leon Flint, David Seligson 2020. Protocol: A Scoping Review of the Biomechanics of the Autogenous Bone Graft Harvest from Proximal Tibia. protocols.io dx.doi.org/10.17504/protocols.io.bq3kmykw Copy
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