Stepwise Instructions Research Articles

Single-cell mechanogenetics using monovalent magnetoplasmonic nanoparticles.

Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to answer a host of important biological questions. This protocol describes a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. The key components of this tool are a magnetoplasmonic nanoparticle (MPN) actuator that delivers defined spatial and mechanical cues to receptors through target-specific one-to-one engagement and a micromagnetic tweezers (μMT) that remotely controls the magnitude of force exerted on a single MPN. In our approach, a SNAP-tagged cell-surface receptor of interest is conjugated with a single-stranded DNA oligonucleotide, which hybridizes to its complementary oligonucleotide on the MPN. This protocol consists of four major stages: (i) chemical synthesis of MPNs, (ii) conjugation with DNA and purification of monovalent MPNs, (iii) modular targeting of MPNs to cell-surface receptors, and (iv) control of spatial and mechanical properties of targeted mechanosensitive receptors in live cells by adjusting the μMT-to-MPN distance. Using benzylguanine (BG)-functionalized MPNs and model cell lines expressing either SNAP-tagged Notch or vascular endothelial cadherin (VE-cadherin), we provide stepwise instructions for mechanogenetic control of receptor clustering and for mechanical receptor activation. The ability of this method to differentially control spatial and mechanical inputs to targeted receptors makes it particularly useful for interrogating the differential contributions of each individual cue to cell signaling. The entire procedure takes up to 1 week.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project.

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Undergraduate Laboratory Exercises Specific to Food Spoilage Microbiology

AbstractFood spoilage has an enormous economic impact, and microbial food spoilage plays a significant role in food waste and loss; subsequently, an equally significant portion of undergraduate food microbiology instruction should be dedicated to spoilage microbiology. Here, we describe a set of undergraduate microbiology laboratory exercises that focus specifically on food spoilage which were taught in 2 lab periods as part of the undergraduate food microbiology lab curriculum at Cornell University. The lab was broken down into 3 exercises. Two exercises lead students to determine the likely source of contamination in a canned salsa through (exercise 1a) plating and observation of colony morphology and (exercise 1b) determination of the thermal resistance for those isolates. The final exercise (2) involved detection of the spoilage bacterium Alicyclobacillus in apple juice. Spoiled juice demonstrations were also prepared in this exercise for students to observe sensorial changes resulting from spoilage, emphasizing that spoilage is not always visually detectable. Students were able to successfully determine the source of contamination based on the results of their laboratory findings, which they used to make recommendations for production to reduce microbial food spoilage in the canned salsa product. Based on student answers to discussion questions provided following lab exercises, participants were able to (a) identify the significance of microbial spoilage and how spoilage is principally different from food safety, (b) describe varying sensorial changes associated with microbial spoilage, and (c) employ methods and analysis to evaluate sources and type of contamination. Downloadable handouts and stepwise instructions are available as supporting information.

Global Curve Fitting Approach to Receptor Allosterism

G‐protein coupled receptors (GPCRs) are known to be modulated by both orthosteric agonists and positive/negative allosteric modulators (PAMs/NAMs). Proper experimental design and data analysis for dual dose dependence between agonist and PAM or NAM are critical to elucidate key parameters for understanding molecular mechanism and structure‐activity relationship (SAR) in drug discovery. Toward that end, we have developed a global curve fitting approach to analyze simultaneously all curves in dual dose dependence data of receptor allosterism to report all key parameters. A general equation based on operational model of receptor allosterism is reformulated in GraphPad Prism to allow global curve fitting. Specific stepwise instructions with graphic illustrations will be outlined.

Development of instructions and tape measure to improve accuracy of parental report of preschool height (1024.5)

Accurate height measurements are essential to calculating BMI. An iterative process was used to develop materials to improve the accuracy with which parents in an obesity‐prevention program measured and reported child height. Phase A used brief, written, stepwise instructions that were cognitively tested, operationalized by parents using equipment provided by researchers, and refined using parent feedback and researcher observations after each of 3 testing rounds (n=6,13,5). Researcher and parent child height measurements did not differ significantly in Round 1 & 2 (mean difference within +0.18”+0.58SD). Round 3 revealed instructions were too difficult for parents with less formal education. Phase B used a brief narrated video that was tested and refined over a series of 4 rounds (n=9,13,5,5; 56% had high school or less; 44% had some college). Common errors were not verifying TM straightness, hanging TM over a baseboard, not removing child shoes, not having child breath in‐out before taking measurements, and misreading fractions. The TM was revised to label each ¼” mark. The video was refined and retested until differences between researcher and parent measurements were within +0.38”+0.48SD in the last testing round. Compared to written instructions, parents rated the video as clearer (4.88±0.34 vs 4.5±0.75SD) and felt the video would improve their child height measurements (4.98±0.09SD vs 4.09±1.16SD).Grant Funding Source: Supported by USDA, National Institute of Food and Agriculture, Grant Number 2011‐68001‐30170

V12 Novel tissue simulator for robotic assisted radical prostatectomy: Face, content, and construct validation and stepwise instructions for creation

V12 Novel tissue simulator for robotic assisted radical prostatectomy: Face, content, and construct validation and stepwise instructions for creation

A New Predoctoral Endodontic Module: Evaluating Learning and Effectiveness

The teaching of advanced endodontic courses at the predoctoral level is common, but it can be difficult to assess teaching effectiveness. Advanced modules placed later in the dental curriculum provide the opportunity to introduce a new topic, revisit and reinforce concepts learned previously, and instill the notion of lifelong learning. At any level, the introduction of new techniques to novices must be based on recognition of their prior knowledge and experience and their need for explicit direction, stepwise instruction, and comprehensive feedback. Assessment of students' performance should not only provide insights into what they know and can do, but also steer them towards desired outcomes. In addition, assessment can provide valuable feedback on teaching effectiveness. In this article, we describe a module piloted for inclusion in the University of Otago (New Zealand) fourth-year dental curriculum. This involved the use of tapered hand and rotary nickel-titanium files for root canal preparation and was taught through a didactic program (lectures and problem-based learning seminars) and a series of preclinical hands-on sessions. Findings from formative and summative assessments as well as student, peer, and self-evaluation indicated that the objectives of the module were met and that it was effective in both providing students with the basic skills for using this type of instrumentation and increasing their understanding and enthusiasm for endodontics. We conclude by discussing curriculum changes resulting from our module evaluation, directions for future research, and suggestions for teaching advanced endodontic techniques.

Reliable Assessment of Sedation Level in Routine Clinical Practice by Adding an Instruction to the Ramsay Scale

The level of sedation in mechanically ventilated patients is most often assessed with the Ramsay Scale. Its reliability, however, has never been evaluated in a large group of professionals using the Ramsay Scale in daily clinical practice, while differences in interpretations among professionals have been indicated. We developed a written stepwise instruction to optimize the inter-observer reliability of the Ramsay Scale within a large group of Intensive Care (IC) nurses. Reliability study. The Intensive Care Cardiology (ICC) and the Intensive Care Thoracic surgery (ICT) units of a university hospital. The study population comprises randomly selected mechanically ventilated patients and IC nurses with a bachelor's degree in Nursing and an IC certification. In total 2x105 Ramsay measures were performed in 45 patients by 24 nurses. Analysis of 105 paired Ramsay scores showed an almost perfect agreement between observers (weighted K (Kw)=0.90). In both ICC patients and ICT patients, agreement between Ramsay scores was high (Kw=0.95 and Kw=0.86, respectively). By using a written stepwise instruction with the Ramsay Scale, the inter-observer reliability of the level of sedation measurements, performed in daily clinical practice within a large team of IC nurses, proved to be almost perfect.

How do People Use Instructions in Procedural Tasks?

Instructions for procedural tasks are often designed to be read before starting the task, but evidence suggests that people often do not use instructions that way. Participants ( N = 20) completed knot and assembly tasks using stepwise instructions and the time viewing the instructions in the course of completing the tasks was measured. Half the participants reported that they read the instructions first and half say they usually attempt the task first, but no difference was found between those groups in how they used the instructions. Rather, it was found to depend on the type of task; instructions were used more in the course of completing the knot tasks than the assembly tasks. This indicates that the nature of the task determines how instructions are used and that people might not consistently adhere to a preferred strategy for using instructions.

Anatomic Analysis of the Genial Bone Advancement Trephine System’s Effectiveness at Capturing the Genial Tubercle and its Muscular Attachments

The Genial Bone Advancement Trephine (GBAT) System (Stryker Leibinger Inc, Kalamazoo, MI) is a 1-step system that advances the genial tubercle for treatment of retrolingual obstruction in obstructive sleep apnea. The purpose of this project was to anatomically analyze the effectiveness of the GBAT System to capture the genial tubercle and its muscular attachments in the circular trephine. Eight cadaveric heads (ages 54-89 years; 3 female and 5 male) were examined, and Panorex radiographs (Stryker Leibinger Inc, Kalamazoo, MI) were obtained. Seven specimens were selected on the basis of mandibular height and dentition to undergo the procedure. The procedure was performed using the GBAT system with the 14-mm trephine according to the stepwise instructions. The mandibles were dissected, and analysis of the location of the osteotomy with respect to the genial tubercle, genioglossus muscles, geniohyoid muscles, and mandibular dentition was performed. All 7 of the specimens had complete capture of the genial tubercle. The mean percentage of the genioglossus muscle captured in the circular trephine was 85% (50%-100%). All specimens had preservation of the posterior muscle fibers. The mean percentage of the geniohyoid muscle captured was 78% (15%-100%). No tooth roots were transected or contained in the bone plug. The GBAT System is an effective 1-step system for capturing and advancing the genial tubercle and its muscular attachments in the treatment of OSA.

Anatomic analysis of the GBAT system’s effectiveness capturing genial tubercle and attachments

Objectives: The Genial Bone Advancement Trephine (GBAT) System is a one-step system that advances the genial tubercle for the treatment of retroligual obstruction in obstructive sleep apnea syndrome. The purpose of this project was to anatomically analyze the effectiveness of the GBAT System to capture the genial tubercle and its muscular attachments in the circular trephine. Methods: Eight cadaveric heads were examined and panorex x-rays were obtained. Seven specimens were selected based on mandibular height and dentition to undergo the procedure. Using the GBAT System with the 14 mm trephine, the procedure was performed following the stepwise instructions. The mandibles were dissected and analysis of the location of the osteotomy with respect to the genial tubercle, genioglossus muscles, geniohyoid muscles, and mandibular dentition was performed. Results: All 7 of the specimens had complete capture of the genial tubercle. The mean percentage of the genioglossus muscle captured in the circular osteotomy was 85% (50%-100%). All specimens had preservation of the posterior genioglossus muscle fibers. The mean percentage of the geniohyoid muscle captured was 78% (15%-100%). No tooth roots were transected or contained in the bone plug. Conclusions: The GBAT System is an effective, one-step system for capturing and advancing the genial tubercle and its muscular attachments in the treatment of obstuctive sleep apnea syndrome.

Several Applications of Advanced Scientific Calculators: Nonlinear Least-Squares Analysis and Titration of a Weak Acid

Several advanced applications of scientific graphing calculators are demonstrated. These include (a) nonlinear least squares analysis and (b) construction of titration curves for weak acids with a strong base. Stepwise instructions for obtaining the titration curves with TI-80 Series Calculators are presented. Immediate display of graphical results of a solution from a quantitative chemical problem in a classroom setting is one of the advantages of the advanced scientific calculator.

’n Plastiek Cartesiese duiker as respirometer

The working principles of the Cartesian diver as a respirometer for terrestrial micro-athropods are discussed. The advan­tages and disadvantages of Perspex as thermoplastic diver material are compared with those of Pyrex or Jena glass. With the aid of diagrammes on which physical dimensions are indicated, the functional parts of a Perspex diver with a diver cons­tant of about 80 microliters are described. Stepwise instructions for the operation of the diver system (flotation vessel, manometer, glassmanifold) within the enclosed waterbath are presented to enable one to measure the equilibrium pressure (ΔP) of each individual animal with a mass less than 1 mg. The calculations used to determine the diver constant for each diver are fully described. Practical examples are used to enable one to calculate the oxygen consumption rate (VO₂) for a mite species from the equilibrium pressure measurement.

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: Study design

The most commonly used genotoxicity assays for cultured mammalian cells are mammalian cell mutagenesis, chromosome aberrations/SCE, hepatocyte UDS, and cell transformation. Since their inception, protocols for these assays have been modified in various laboratories. It has been observed that minor but potentially significant method modifications frequently remain unpublished (Swierenga et al., 1983) but should be considered in the development of recommended protocols. The present study was undertaken to determine the current ‘state of the art’ for these tests. Detailed questionnaires on culture conditions and testing protocols for both stock and test cell populations were designed with the assistance of an international advisory committee and sent to all research and contract laboratories that could be readily identified in Canada, U.S.A. and Europe. Responses from 425 completed questionnaires were analyzed to determine the most commonly used approach and modifications for each procedural step. As expected, the results show a large degree of interlaboratory variation. Detailed protocols for conducting each assay have been prepared and include:stepwise instructions, precautionary measures and practical solutions to common problems associated with each assay; recipes for media and solutions; formulas for quantifying genotoxic responses; reference lists of related assays; guidelines for interpretation; and discussion of the applications, advantages and disadvantages of each test.