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Authors: Rebecca Hufft, Margo Paces, Meghan McGill, Richard Levy
Group: Denver Botanic Gardens Research and Conservation
Summary: This protocol outlines the basic methods for measuring water quality and aquatic macroinvertebrate diversity. These measurements estimate the abundance and presence of aquatic macroinvertebrate species in an area, and link them to environmental conditions (plant community, water temperature and pH, water nutrients, etc.) that may affect their distributions. Together, these data can reveal patterns in aquatic community or ecosystem processes. The stream measurements can be done independently but are usually paired with plant monitoring plots. When a plant monitoring plot is next to a creek or stream, the aquatic sampling occurs in the body of water nearest to the origin of the plant plot. See vegetation monitoring protocol (Hufft et al 2019) for more details.

Proper citation: Rebecca Hufft, Margo Paces, Meghan McGill, Richard Levy 2019. Aquatic Monitoring Protocol for measuring and collecting ecological data. protocols.io https://dx.doi.org/10.17504/protocols.io.9j3h4qn Copy   

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Authors: Ligia Pinto, Troy Kemp
Summary: Luminex Milliplex Cardiovascular Disease 3-plex Panel 2 instructions per manufacturer.

Proper citation: Ligia Pinto, Troy Kemp 2017. Milliplex Cardiovascular Disease 3-plex Panel 2. protocols.io https://dx.doi.org/10.17504/protocols.io.hvcb62w Copy   

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Authors: Yin-Tse Huang

Proper citation: Yin-Tse Huang 2020. Brad-seq mRNA (for Shotgun or DGE). protocols.io https://dx.doi.org/10.17504/protocols.io.bqyymxxw Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: NEBExpress® Ni Resin is an affinity matrix for the isolation and purification of polyhistidine-tagged (His-tagged) fusion proteins. It is intended for use in gravity or pressure flow columns, and batch purifications. NEBExpress Ni Resin is comprised of a highly uniform and stable chemical-tolerant resin, pre-charged with nickel ions on the matrix surface.  It is resistant to a wide range of chemicals, including NaOH, EDTA, DTT and β-Mercaptoethanol.Purification of ≥10 mg His-tagged protein per 1 ml of resinIntended for use in gravity or pressure flow columns, and batch purificationsHigh specific binding of His-tagged proteins yielding purities of >95%Strong nickel ion binding provides excellent resistance to EDTA and reducing agents. Compatible with commercially available detergent-based cell lysis reagentsIsolation and purification of His-tagged fusion proteins under native or denaturing conditions

Proper citation: New England Biolabs 2020. NEBExpress Ni Resin Pressurized Column Typical Protocol (NEB #S1428). protocols.io https://dx.doi.org/10.17504/protocols.io.bfa4jigw Copy   

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Authors: DAVID DUNIGAN AND IRINA AGARKOVA
Group: VERVE Net
Summary: For use in CviJI Purification From IL-3A Virus Infected NC64A Chlorella.

Proper citation: DAVID DUNIGAN AND IRINA AGARKOVA 2016. 1X CviJI Assay Buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.espbedn Copy   

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Authors: Solomon Hailemariam1, 2, 3*, Yabibal Gebeyehu4, Eskinder Loha1, 5, Kjell Arne Johansson2, Bernt Lindtjørn1, 2
Summary: Abstract Background: Hypoxemia is common in children with pneumonia. The World Health Organization and United Nations Children’s Fund developed the Integrated Management of Childhood Illness strategy for diagnosing pneumonia. But the strategy has limitations to accurately detect hypoxemia in children with severe pneumonia. This study will assess whether pulse oximetry improves health workers’ performance in diagnosing severe childhood pneumonia and whether the pulse oximeter is a cost-effective tool to improve diagnoses of severe pneumonia at primary health care units in South Ethiopia.Methods: A cluster-randomized controlled trial will be conducted at 24 health centres in southern Ethiopia from September 2018 to February 2019. The trial has two arms: the first arm will examine the combined use of the Integrated Management of Childhood Illness (IMCI) strategy with pulse oximetry, and the second arm will examine the use of the IMCI strategy alone. The study population will include children between 2 months and 59 months of age who present with cough or difficult breathing. The primary outcome of this trial is severe childhood pneumonia, as diagnosed via the IMCI guideline and pulse oximetry. Secondary outcomes will be referred cases of severe pneumonia, and treatment failure on day 14 after enrolment. A cost effectiveness study will be conducted simultaneously.Discussion: This study will determine if using a pulse oximeter at health facilities in rural Southern Ethiopia improves diagnostic accuracy for paediatric severe pneumonia. Our secondary aim will be to evaluate if the benefit of pulse oximetry justifies its cost. Trial registration: PACTR201807164196402(14/06/2018). URL:https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=3466.

Proper citation: Solomon Hailemariam1, 2, 3*, Yabibal Gebeyehu4, Eskinder Loha1, 5, Kjell Arne Johansson2, Bernt Lindtjørn1, 2 2019. Improving pneumonia diagnoses using pulse oximetry at rural health institutions in southern Ethiopia: Protocol for a cluster-randomized controlled trial. protocols.io https://dx.doi.org/10.17504/protocols.io.88mhzu6 Copy   

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Authors: Andrew Baker
Summary: This solution is used in the coral genomic DNA extraction protocol.The following recipe is for 50 mL of CTAB mix. Store at -20°C.

Proper citation: Andrew Baker 2016. CTAB mix. protocols.io https://dx.doi.org/10.17504/protocols.io.dyt7wm Copy   

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Authors: Gladys Yinusa, Janet Scammell, Jane Murphy, Gráinne Ford
Summary: Objective: The review will examine the evidence and characteristics of multidisciplinary care interventions in food and nutritional care provision for adult in-patients.Introduction: Providing appropriate nutritional care is fundamental in patient-centred care. Nutritional care requires a coordinated approach to the delivery of food and fluids by different healthcare professionals and the wider hospital staff. Evidence demonstrates improved patients’ clinical outcomes by enhancing hospital food and providing nutritional care throughout a patient’s pathway. While there are good examples of nutritional care initiatives, implementing appropriate nutritional care remain a challenge. Managing malnutrition particularly with adult in-patients on admission or at risk of becoming malnourished requires multidisciplinary care; the hospital team plays a crucial role in ensuring these patients’ nutritional needs are met. The review aims to identify the evidence with specific focus on reported multidisciplinary coordination and collaboration excluding artificial nutritional support (enteral and parenteral nutrition)a domain where well-established evidence already exists.Inclusion criteria: The context is limited to the hospital setting with an adult in-patient population. Studies involving healthcare professionals or the wider hospital staff will be included. Methods: Primary evidence will be included from both published and unpublished sources. The search strategy will follow the three-step process recommended by Joanna Briggs Institute’ (JBI). Data will be extracted after screening from relevant papers by two or more independent reviewers and one or more verifier using a developed data extraction tool. A narrative description of findings will be reported following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) guidelines.IntroductionKey nutrition interventions and strategies to reduce malnutrition have been recommended and some integrated into standard guidelines and policies. For instance, the recommendations for nutrition screening in routine practice to identify people with (or at risk of) malnutrition using validated malnutrition screening tools by the European Society for Clinical Nutrition and Metabolism, British Association of Parenteral and Enteral Nutrition and the National Institute for Clinical Excellence in the UK.13,14 Nutrition screening as the first step enables appropriate interventions with subsequent monitoring and assessment to be implemented. Global consensus on core diagnostic criteria for malnutrition has also been proposed to enable comparison of malnutrition prevalence, interventions, and outcomes across the world.15 Other strategies include the Protected Mealtime policy (2004) implemented in the UK aimed at avoiding all non-urgent activity on the wards during mealtimes and allowing patients to have uninterrupted meals.16 Mealtime volunteers have been shown to provide valuable support on the ward to assist with eating and drinking processes.17 However, barriers to the effective implementation of this model into practice have been reported.18,19 While supportive interventions are reported to have a beneficial effect for patients, the specific features of collaborative working approaches remain unclear.In providing nutritional care, hospital staff play a significant role in ensuring that patients’ nutritional needs are met. Some studies, guidance documents, and reports have similarly highlighted the importance of implementing an multidisciplinary approach to address the problem with the emphasis on a person-centred approach.2,21,22 The use of the term ‘nutritional care’ in this paper refers to a coordinated approach to the delivery of food and fluids by different healthcare professionals and views the patient as an individual with needs and preferences.23In a systematic review, Feinberg et al.24 reported a lack of quality evidence that examined different forms of nutrition support including general support, fortified foods, and artificial nutrition support. Rasmussen et al.25 suggested from a systematic review and meta-analysis that providing multidisciplinary nutrition support for older patients may have a positive effect on patients’ clinical outcomes, mortality and quality of life. This review sought to assess the effectiveness of multidisciplinary nutrition support studies including randomised controlled trials and excluding heterogeneous studies on multidisciplinary interventions of interest to this review. Moreover, the nature of the multidisciplinary coordinated approach merits more exploration. Evidence on the impact of complex nutritional care is well documented, including enteral or parenteral nutrition as part of artificial nutritional support for some patients.26-28 In contrast, knowledge on the coordinated delivery of other forms of nutritional care such as support for patients in the meal environment including meal support and eating support, is sparse. The present review aims to map and identify the nature of the evidence for the provision of food and nutritional care with a specific focus on reported multidisciplinary coordination and collaboration. The present study aims to map the diverse literature about multidisciplinary care and to review what has been studied and how it has been studied.Review questionsThe scoping review will be guided by this research question and the following sub-questions:What are the features of multidisciplinary collaborative care approaches implemented to improve the provision of food and nutritional care to hospitalised adult inpatients?What disciplines are involved in the multidisciplinary collaborations and what component of nutritional care are implemented? What collaborative activities, processes and outcomes of multidisciplinary nutritional care are reported?

Proper citation: Gladys Yinusa, Janet Scammell, Jane Murphy, Gráinne Ford 2020. Multidisciplinary provision of food and nutritional care to hospitalised adult in-patients: A scoping review protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bgzajx2e Copy   

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Authors: Yusheng Yao
Summary: Male adult Sprague–Dawley rats, weighing 270–300 g, obtained from the Animal Center of the Fujian Medical University, were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg). After intravenous injection of heparinized (50 IU) the hearts were quickly excised and fixed on a Langendorff apparatus (Radnoti, Monrovia, CA, USA) and perfused with 37°C Krebs–Henseleit buffer (KHB) at constant pressure (75 mmHg). After equilibrated with KHB for 15 min, isolated hearts were randomly divided into five groups: 1. Sham group: hearts subjected to 195-min perfusion without I/R injury; 2. I/R group: hearts received 30- min ischemia followed by 120-min reperfusion; 3. SPC group: 2.5% sevoflurane preconditioning was gived for 15 min followed by 15-min washout before ischemia; 4. SPC+MF1 group: combined application of MF1(ImClone, Somerville, NJ, USA), rat anti-VEGFR-1 monoclonal antibody,10 μmol/L and 2.5% sevoflurane preconditioning for 15 min followed by 15-min washout before ischemia; 5. SPC+PIGF group: combined application of PIGF(Abcom, Cambridge, United Kingdom)10 μmol/L and 2.5% sevoflurane preconditioning for 15 min followed by 15-min washout before ischemia.  To implement sevoflurane preconditioning, the KHB was pre-equilibrated with sevoflurane under a separate reservoir with a Vapor 2000 (Dräger Medical AG & Co., Lübeck, Germany) using an air bubbler.

Proper citation: Yusheng Yao 2018. Up-regulation of vascular endothelial growth factor receptor-1 contributes to sevoflurane preconditioning mediated cardioprotection. protocols.io https://dx.doi.org/10.17504/protocols.io.mmrc456 Copy   

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Authors: Terry Powley, Zhenjun Tan, Matthew Ward
Group: SPARC
Summary: This protocol describes a process for the measurement of electrical stimulation-induced effects on duodenal motility in young adult Sprague-Dawley rats. Signals recorded from strain gauges attached to the proximal duodenal surface were used to measure the effect of stimulation by patch electrodes implanted at multiple sites across the rat stomach in an acute anesthetized preparation. The effect of stimulation was quantified as the ratio of various motility assessments during and after stimulation vs. before stimulation, and the data was used to create a functional map of duodenal motor response to localized gastric stimulation.

Proper citation: Terry Powley, Zhenjun Tan, Matthew Ward 2020. Measurement of duodenal motility using implanted strain gauges. protocols.io https://dx.doi.org/10.17504/protocols.io.2irgcd6 Copy   

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Authors: Adam Shepherd
Summary: Describes how to update files at the Woods Hole Open Access Server - the institutional repository used by BCO-DMO for dataset versioning.

Proper citation: Adam Shepherd 2016. Replacing a Bitstream at the Woods Hole Open Access Server. protocols.io https://dx.doi.org/10.17504/protocols.io.fypbpvn Copy   

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Authors: Lis Rocha
Summary: Many efforts have been made to understand the pathogenesis of bovine mastitis to reduce losses and promote animal welfare. Staphylococcus aureus may cause bovine clinical mastitis, but it is mainly associated with subclinical infection, which is usually persistent and can easily reoccur. Here, we conducted a comparative genomic analysis between four strains of S. aureus causing subclinical infection (Sau170, 302, 1269, 1364), previously sequenced by our group, and two well-characterized strains causing clinical mastitis (N305 and RF122) to find differences that could be linked to mastitis outcome. A total of 146 virulence-associated genes were compared and no appreciable differences were found between the bacteria. However, several nonsynonymous single nucleotide polymorphisms (SNPs) were identified in genes present in the subclinical strains when compared to RF122, especially in genes encoding host immune evasion and surface proteins. The comparison of orthologous genes using OrthoMCL identified a membrane transporter in the genomes of the bacteria belonging to the subclinical group, but this finding was not confirmed by polymerase chain reaction (PCR) on a collection of field isolates of S. aureus associated with clinical or subclinical mastits. The secreted and surface proteins predicted by different in silico tools were compared through multidimensional scaling analysis, revealing a high degree of similarity among the six strains. However, differences were seen in the nucleotide sequences of a gene that codes for a hypothetical protein (cl3309) and a lipoprotein (cl3700). These findings were also analyzed by PCR on DNA extracted from field isolates of S. aureus. The lipoprotein, but not the hypothetical protein, was able to separate the clinical isolates from the subclinical ones. These results show that sequence variation among bovine S. aureus, and not only the presence/absence of virulence factors, is an important aspect to consider when comparing isolates causing different mastitis outcomes

Proper citation: Lis Rocha 2019. Microorganisms and culture conditions - Comparative genomics of Staphylococcus aureus associated with subclinical and clinical bovine mastitis (Rocha et al., 2019). protocols.io https://dx.doi.org/10.17504/protocols.io.zhrf356 Copy   

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Authors: Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms, RUSTIS
Summary: Extraction of high quality DNA for long read sequencing e.g. PacBioOptimized for DNA extraction from wheat stripe rust spores and also tested on barley leaf rust.Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.Critical steps to obtain high quality DNA:Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.Do NOT incubate samples with KAc for prolonged time periodsPerform two steps of buffered Phenol:Chloroform:Isoamylalcohol purification to reduce co-purifying metabolites.DNA fragments were well above the 40kb mark based on Pippin Pulse Gels. The sequencing center performed a second AMPure purification step before library construction. Summary statistics of sequencing runs to follow.

Proper citation: Benjamin Schwessinger 2016. High quality DNA from Fungi for long read sequencing e.g. PacBio. protocols.io https://dx.doi.org/10.17504/protocols.io.ewtbfen Copy   

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Authors: Angel Justiz-Vaillant, Suzette E. Curtello
Group: University of the West Indies, [email protected]
Summary: Salmonella are motile, flagellated rod-shaped Zoonotic pathogens which may survive with or without oxygen and do not absorb crystal violet stain.They are decolourised by alcohol due to their outer lipopolysaccharide membrane and thin peptidoglycan layer.They belong to the family Enterobacteriaceae and is implicated with typhoid fever and food-borne illnesses.This pathogen is associated with intestinal disease which may become fatal and has negatively impact the health of individuals and various economies globally.The poultry industry is most impacted and vulnerable to the onslaught of this pernicious microbe.The Lipopolysaccharide Somatic O antigen, flagellar H and virulent V antigenic structure determines the serotype designate of Salmonella species [1]. A wide range of antisera is used in the location of the O antigen associated with the carbohydrate constituent of Lipopolysaccharide and H protein antigen linked with the flagella of the bacteria.Salmonellosis is an inflammation of the intestinal mucosal lining by Salmonella bacteria infiltration resulting in painful symptoms.Both man and animal are vulnerable.Salmonellosis is caused by ingestion of Salmonella with food (Wall et al., 1994).The main route of transmission of Salmonella microbe is fecal-oral. Ingestion of small concentration of Salmonella such as 6 cells with contaminated food or water will lead to salmonellosis infection [2].References1. Varma JK, Marcus R, Stenzel SA, et al. Highly resistant Salmonella Newport-MDRAmpC transmitted through the domestic US food supply: a FoodNet case-control study of sporadic Salmonella Newport infections, 2002-2003.J Infect Dis. 2006;194(2):222-230. doi:10.1086/505084.2. Chen W, Martinez G, Mulchandani A. Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella.Anal Biochem. 2000;280(1):166-172. doi:10.1006/abio.2000.4518

Proper citation: Angel Justiz-Vaillant, Suzette E. Curtello 2020. Salmonella Typhimurium isolation from specimens. protocols.io https://dx.doi.org/10.17504/protocols.io.bjjdkki6 Copy   

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Authors: Bonnie Hurwitz
Group: Hurwitz Lab, MetaFunc Course

Proper citation: Bonnie Hurwitz 2016. MG_HW1: Starting your class project. protocols.io https://dx.doi.org/10.17504/protocols.io.fs7bnhn Copy   

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Authors: Kotaro Takahagi, Komaki Inoue, Minami Shimizu, Yukiko Uehara-Yamaguchi, Yoshihiko Onda, Keiichi Mochida
Group: GigaScience Press
Summary: This protocol provides an useful technique for detecting allele frequencies in the cDNA sample

Proper citation: Kotaro Takahagi, Komaki Inoue, Minami Shimizu, Yukiko Uehara-Yamaguchi, Yoshihiko Onda, Keiichi Mochida 2018. Detection of allele frequencies in the cDNA sample. protocols.io https://dx.doi.org/10.17504/protocols.io.nafdabn Copy   

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Authors: Maigan Brusko
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: This standard operating procedure (SOP) provides instructions for staining and sorting live cells. This SOP applies to cryopreserved cells that are stained with Live/Dead and sorted in the BD ARIA III to remove dead cells prior to 10x sequencing.

Proper citation: Maigan Brusko 2020. HuBMAP UF TMC - FACS Sorting of Live Cells for 10x scRNASeq. protocols.io https://dx.doi.org/10.17504/protocols.io.bfqzjmx6 Copy   

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Authors: Kelsey Knight
Group: BioLegend

Proper citation: Kelsey Knight 2018. T Cell Activation with anti-CD3 Antibodies Protocol - Human. protocols.io https://dx.doi.org/10.17504/protocols.io.tkbeksn Copy   

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Authors: Rozana Rosli, Mohd Amin Ab Halim, Chan Kuang Lim, Jayanthi Nagappan, Norazah Azizi, Faizun Kadri, Nadzirah Amiruddin, Nik Shazana Nik Mohd Sanusi, Lee Pei Lee Angel, Priscilla E Morris, Leslie Low Eng Ti, Chan Pek-Lan, Meilina Ong-Abdullah, Ravigadevi Sambanthamurthi, Rajinder Singh, Denis J Murphy

Proper citation: Rozana Rosli, Mohd Amin Ab Halim, Chan Kuang Lim, Jayanthi Nagappan, Norazah Azizi, Faizun Kadri, Nadzirah Amiruddin, Nik Shazana Nik Mohd Sanusi, Lee Pei Lee Angel, Priscilla E Morris, Leslie Low Eng Ti, Chan Pek-Lan, Meilina Ong-Abdullah, Ravigadevi Sambanthamurthi, Rajinder Singh, Denis J Murphy 2018. Protocols to accompany paper entitled “Comparative genomic and transcriptomic analysis of selected fatty acid biosynthesis genes and CNL disease resistance genes in oil palm”. protocols.io https://dx.doi.org/10.17504/protocols.io.mwcc7aw Copy   

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Authors: Xingqi Chen, Ulrike Litzenburger, Yuning Wei, Alicia N. Schep, Edward L. LaGory, Hani Choudhry, Amato J. Giaccia, William J. Greenleaf, Howard Y. Chang
Group: Human Cell Atlas Method Development Community, HYChangLab
Summary: Here we introduce Protein-indexed Assay of Transposase Accessible Chromatin with sequencing (Pi-ATAC) that combines single-cell chromatin and proteomic profiling. In conjunction with DNA transposition, the levels of multiple cell surface or intracellular protein epitopes are recorded by index flow cytometry and positions in arrayed microwells, and then subject to molecular barcoding for subsequent pooled analysis. PiATAC simultaneously identifies the epigenomic and proteomic heterogeneity in individual cells. Pi-ATAC reveals a casual link between transcription factor abundance and DNA motif access, and deconvolute cell types and states in the tumor microenvironment in vivo. We identify a dominant role for hypoxia, marked by HIF1A protein, in the tumor microvenvironment for shaping the regulome in a subset of epithelial tumor cells.

Proper citation: Xingqi Chen, Ulrike Litzenburger, Yuning Wei, Alicia N. Schep, Edward L. LaGory, Hani Choudhry, Amato J. Giaccia, William J. Greenleaf, Howard Y. Chang 2018. Pi-ATAC. protocols.io https://dx.doi.org/10.17504/protocols.io.q6gdzbw Copy   

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