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Principal Component Analysis: Mathematical procedure that transforms a number of possibly correlated variables into a smaller number of uncorrelated variables called principal components.
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An Overview of Genetic Analysis

JoVE 5540

An organism’s physical traits, or phenotype, are a product of its genotype, which is the combination of alleles (gene variants) inherited from its parents. To varying degrees, genes interact with each other and environmental factors to generate traits. The distribution of alleles and traits within a population is influenced by a number of factors, including natural selection, migration, and random genetic drift.In this video, JoVE introduces some of the foundational discoveries in genetics, from Gregor Mendel’s elucidation of the genetic basis of inheritance, to how natural processes affect allele distributions within populations, to the modern synthesis of biology that brought together Mendelian genetics and Darwinian evolution. We then review the questions asked by geneticists today regarding how genes influence traits, and some of the main tools used to answer these questions. Finally, several applications of techniques such as genetic crosses, screens and evolution experiments will be presented.


 Genetics

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Expression Profiling with Microarrays

JoVE 5547

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and nucleic acids derived from samples. Using these arrays, scientists can simultaneously evaluate the expression of thousands of genes. In addition, the expression profiles of different cells or tissue types can be compared, allowing researchers to deduce how the expression of different genes change during biological processes, and thus gain insight into how the genes may function in pathways or networks.Here, JoVE explains the principles behind microarrays. This is followed by a general protocol for performing a microarray experiment, and a brief introduction to analyzing microarray data. We end on a discussion of how scientists are currently using microarrays, for example to compare gene expression between different cell types derived from cancerous and non-cancerous tissues, to study important biological problems.


 Genetics

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Recombineering and Gene Targeting

JoVE 5553

One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that allow them to be joined together. However, this technique has certain restrictions that limit its applicability for large or complex DNA construct generation. A newer technique that addresses some of these shortcomings is recombineering, which modifies DNA using homologous recombination (HR), the exchange between different DNA molecules based on stretches of similar or identical sequences. Together with gene targeting, which takes advantage of endogenous HR to alter an organism’s genome at a specific loci, HR-based cloning techniques have greatly improved the speed and efficacy of high-throughput genetic engineering.In this video, we introduce the principles of HR, as well as the basic components required to perform a recombineering experiment, including recombination-competent organisms and genomic libraries such as bacterial artificial chromosomes (BAC). We then walk through a protocol that uses recombineering to generate a gene-targeting vector that can ultimately be transfected into embryonic stem cells to generate a transgenic animal. Finally, several applications that highlight the utility and variety of recombineering techniques wi


 Genetics

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Ethics in Psychology Research

JoVE 10045

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco—Monmouth University

When a researcher finds an interesting topic to study such as aggression, the goal is often to study it in a way that is as true to life as possible. However, researchers must act in an ethical manner.  To do this, they must balance their research goals with the best interests of the participants. Ethics often enter into the planning process when researchers identify all of the ways they can manipulate or measure a variable, but then make their final decision based on how they should manipulate or measure a variable. After receiving a poor grade on a test or paper, a college student may appear to take it out on (i.e., act in an aggressive manner toward) their roommates by being mean or nasty, screaming, throwing things, or even becoming physically violent. Aggression is an important human behavior to study and understand due to the implications it has for interpersonal violence. However, for safety reasons, a study cannot expose participants to the risk that serious types of violence presents. As a result, researchers must identify similar but benign behaviors that can help us understand more aggressive behaviors without harming participants.


 Experimental Psychology

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Nuclear Magnetic Resonance (NMR) Spectroscopy

JoVE 5680

Source: Laboratory of Dr. Henrik Sundén – Chalmers University of Technology

Nuclear magnetic resonance (NMR) spectroscopy is a vital analysis technique for organic chemists. With the help of NMR, the work in the organic lab has been facilitated tremendously. Not only can it provide information about the structure of a molecule but also determine the content and purity of a sample. Compared with other commonly encountered techniques for organic chemists — such as thermal analysis and mass spectrometry (MS) — NMR is a non-destructive method that is valuable when recovery of the sample is important. One of the most frequently used NMR techniques for an organic chemist is proton (1H) NMR. The protons present in a molecule will behave differently depending on its surrounding chemical environment, making it possible to elucidate its structure. Moreover, it is possible to monitor the completion of a reaction by comparing NMR spectra of the starting material to that of the final product. This video exemplifies how NMR spectroscopy can be used in the everyday work of an organic chemist. The following will be shown: i) preparation of an NMR sample. ii) Using 1H NMR to monitor a reaction. iii) Identifying the product obtained from


 Organic Chemistry

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Pelvic Exam II: Speculum Exam

JoVE 10141

Source:

Alexandra Duncan, GTA, Praxis Clinical, New Haven, CT

Tiffany Cook, GTA, Praxis Clinical, New Haven, CT

Jaideep S. Talwalkar, MD, Internal Medicine and Pediatrics, Yale School of Medicine, New Haven, CT

Providing comfortable speculum placement is an important skill for providers to develop, since the speculum is a necessary tool in many gynecological procedures. Patients and providers are often anxious about the speculum exam, but it is entirely possible to place a speculum without patient discomfort. It's important for the clinician to be aware of the role language plays in creating a comfortable environment; for instance, a provider should refer to the speculum "bills" rather than "blades" to avoid upsetting the patient. There are two types of speculums: metal and plastic (Figure 1). This demonstration utilizes plastic, as plastic speculums are most commonly used in clinics for routine testing. When using a metal speculum, it's recommended to use a Graves speculum if the patient has given birth vaginally, and a Pederson speculum if the patient has not. Pederson and Graves speculums are different shapes, and both come in many different sizes (me


 Physical Examinations II

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Observational Research

JoVE 10048

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco—Monmouth University

If you want to know how someone thinks or feels, you can ask that person questions.  Another approach is to observe how the person is acting or look for indicators of how they acted in the past. While observations may seem revealing, it isn’t always easy to know if they are truly accurate. For instance, you may see a person smiling and assume they are happy, when in reality they are annoyed and merely being polite. The purpose of science is to move beyond an individual’s own views of the self because they are inherently skewed by that individual’s expectations, previous experience, personal biases, motivations, emotions, etc. While a person may have unique insight into one’s self, these insight may not accurately represent reality. Put more simply, what a person says, does not always match up well with what they actually do. For this reason, researchers should incorporate a variety of measures (e.g., asking participants to report how they feel, but also observing actual behavior) in order to more accurately capture how the person truly feels. This video demonstrates a correlational des


 Experimental Psychology

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An Introduction to Cell Division

JoVE 5640

Cell division is the process by which a parent cell divides and gives rise to two or more daughter cells. It is a means of reproduction for single-cell organisms. In multicellular organisms, cell division contributes to growth, development, repair, and the generation of reproductive cells (sperms and eggs). Cell division is a tightly regulated process, and aberrant cell division can cause diseases, notably cancer. JoVE's Introduction to Cell Division will cover a brief history of the landmark discoveries in the field. We then discuss several key questions and methods, such as cell cycle analysis and live cell imaging. Finally, we showcase some current applications of these techniques in cell division research.


 Cell Biology

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The Transwell Migration Assay

JoVE 5644

Cells migration in response to chemical cues is crucial to development, immunity and disease states such as cancer. To quantify cell migration, a simple assay was developed in 1961 by Dr. Stephen Boyden, which is now known as the transwell migration assay or Boyden chamber assay. This set-up consists an insert which separates the wells of a multiwell plate into top and bottom compartments. Cells whose migration is to be studied are seeded into the top compartment and the chemoattractant solution is placed in the bottom compartment. After incubation, counting the cells in the bottom compartment allows quantification of migration induced by chemoattractants. This video will review the commonly used experimental set-up for cell migration studies. Then we'll highlight a few key considerations, and outline a generalized protocol for running an experiment involving adherent cells. Lastly, we'll review various adaptations of this set-up currently being used to study different factors that affect migration.


 Cell Biology

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Scanning Electron Microscopy (SEM)

JoVE 5656

Source: Laboratory of Dr. Andrew J. Steckl — University of Cincinnati

A scanning electron microscope, or SEM, is a powerful microscope that uses electrons to form an image. It allows for imaging of conductive samples at magnifications that cannot be achieved using traditional microscopes. Modern light microscopes can achieve a magnification of ~1,000X, while typical SEM can reach magnifications of more than 30,000X. Because the SEM doesn’t use light to create images, the resulting pictures it forms are in black and white.  Conductive samples are loaded onto the SEM’s sample stage. Once the sample chamber reaches vacuum, the user will proceed to align the electron gun in the system to the proper location. The electron gun shoots out a beam of high-energy electrons, which travel through a combination of lenses and apertures and eventually hit the sample. As the electron gun continues to shoot electrons at a precise position on the sample, secondary electrons will bounce off of the sample. These secondary electrons are identified by the detector. The signal found from the secondary electrons is amplified and sent to the monitor, creating a 3D image. This video will demonstrate SEM sample preparation, operation, and imaging capabilities.


 Analytical Chemistry

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Dialysis: Diffusion Based Separation

JoVE 5684

Dialysis is a common technique used in biochemistry for separating molecules based on diffusion. In this procedure, a semipermeable membrane allows the movement of certain molecules based on size. This method can be applied to the removal of buffer, known as desalting, or exchanging buffer molecules or ions from a protein solution.

This video covers the principles of dialysis along with a general procedure.  Several applications of dialysis are reviewed, including the removal of gradient reagents following ultracentrifugation, removing detergent after a membrane protein extraction, and the reconstitution of proteins by changing the solution environment. Biochemical samples typically have high buffer concentrations that can disrupt downstream processing and analysis. Dialysis is a common, inexpensive technique used to separate molecules based on diffusion. The method utilizes a semi-permeable membrane that allows the movement of certain components, based on size. This video will show the concepts of dialysis, a general procedure, and some of its uses in biochemistry. The most important aspect of dialysis is a semi-permeable membrane, which has pores that impose a molecular weight cut-off, allowing molecules below a certain size to pass through. For example, a 10k


 Biochemistry

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Within-subjects Repeated-measures Design

JoVE 10034

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco—Monmouth University

A within-subjects, or repeated-measures, design is an experimental design where all the participants receive every level of the treatment, i.e., every independent variable. For example, in a candy taste test, the researcher would want every participant to taste and rate each type of candy. This video demonstrates a within-subjects experiment (i.e., one where there is an independent variable with several variations or levels) that examines how different motivational messages (e.g., hard work, self-affirmation, outcomes, and positive affect) influence willingness to exert physical effort. As a within-subjects design, the participant will read each of the four types of motivational messages and then lift weights to measure physical effort. By providing an overview of how a researcher conducts a repeated-measures experiment, this video allows viewers to see how to address order effects through counterbalancing, which involves a systematic approach to making sure all possible orders of the conditions occur in the study. Psychological studies often use higher


 Experimental Psychology

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Realism in Experimentation

JoVE 10033

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco—Monmouth University

In an ideal world researchers would conduct their studies in real world settings where behaviors naturally happen. For example, if you want to see what influences individuals’ voting behavior, it would be best to watch them vote. However, research in these settings is not always ethical or even practical. Further, a researcher may want more control over the setting to better pinpoint the exact variables that are influencing an outcome.  When researchers need to conduct studies in a lab, they try to optimize mundane realism, which means that they do everything they can to make the lab feel like a real-life experience. This video demonstrates a two-group design that examines how researchers use mundane realism in a lab to determine whether positive restaurant reviews are connected to diners’ level of tipping. Psychological studies often use higher sample sizes than studies in other sciences. A large number of participants helps to ensure that the population under study is better represented and the margin of error accompanied by studying human behavior is sufficiently accounted for. In this vid


 Experimental Psychology

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Analysis of Earthworm Populations in Soil

JoVE 10002

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

Using mustard, Lumbricus terrestris earthworm populations can be sampled directly from soil depths without landscape disturbance or toxicity. Earthworms can then be counted for data and statistical analysis using a bar graph and student’s t-test. Monitoring earthworm populations is a vital technique for environmental scientists, as multiple species of earthworms (most notably those from the suborder Lumbricina) have been invasively spreading throughout North America and South America. Exotic earthworms can be found on nearly every land mass and in nearly every ecosystem on the planet, and where and when these species become invasive has been a focus of international environmental research.1 Ecological invasion typically lowers biodiversity of an ecosystem by directly outcompeting, endangering, or otherwise contributing to the extirpation of native species. As ecosystem engineers, invasive earthworm species alter the cycling of nutrients through decomposition rates of organic matter on the upper horizons of soil, where plant roots mine for nutrients. Invasive Lumbricus species have both extirpated native earthworm species and have been shown to


 Environmental Science

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Pilot Testing

JoVE 10031

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco—Monmouth University

In any experiment researchers have the challenge of creating experiences for participants that are consistent (i.e., reliable) and authentic (i.e., valid). Yet there are many ways to manipulate any one variable. For example, if you want participants to feel sad, you can have them think of their own sad memory, watch a sad video, or read a sad story. Researchers must find the best way to operationalize a psychological construct in order to produce the most effective manipulation possible. Often, before running the main study, researchers will pilot test (i.e., try out) their manipulations to check their effectiveness. This video demonstrates how to operationalize the same independent variable (acute stress) in three different ways. Specifically, this study seeks to identify the best sound (static, ticking clock, or crying baby) to play during a difficult task (solving complex math problems) to optimally manipulate stress. Psychological studies often use higher sample sizes than studies in other sciences. A large number of participants helps to ensure that the population under study is


 Experimental Psychology

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Testing For Genetically Modified Foods

JoVE 10044

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

Genetic modification of foods has been a controversial issue due to debated concerns over health and environmental safety. This experiment demonstrates technical understanding of how food DNA is genetically identified, allowing for educated decision making about the safety and potential dangers of using genetically modified organisms (GMOs) in food supplies. Polymerase Chain Reaction (PCR) is used to amplify food DNA to test for the presence of genetically modified DNA in food products. Presence of specific DNA bands is detected by using gel electrophoresis to pull extracted food DNA through a 3% agarose gel, a concentration dense enough to separate the bands of DNA containing the genetically modified DNA. Several controls are used in the electrophoresis procedure to ensure DNA is successfully extracted from test foods (plant primer), and to provide known examples of both genetically modified DNA (purchased genetically modified DNA) and non-genetically modified DNA (purchased certified non-GMO food control).


 Environmental Science

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Filamentous Fungi

JoVE 10030

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz

Fungi are heterotrophic eukaryotic organisms, and with the exception of yeasts, are aerobic. They are abundant in surface soils and are important for their role in nutrient cycling and the decomposition of organic matter and organic contaminants. White rot fungi (phanerochaete chryosporium) for example, (Figure 1) are known to degrade aromatics. Figure 1. White rot on birch.


 Environmental Microbiology

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Force and Acceleration

JoVE 10346

Source: Nicholas Timmons, Asantha Cooray, PhD, Department of Physics & Astronomy, School of Physical Sciences, University of California, Irvine, CA

The goal of this experiment is to understand the components of force and their relation to motion through the use of Newton's second law by measuring the acceleration of a glider being acted upon by a force. Nearly every aspect of motion in everyday life can be described using Isaac Newton's three laws of motion. They describe how objects in motion will tend to stay in motion (the first law), objects will accelerate when acted upon by a net force (the second law), and every force exerted by an object will have an equal and opposite force exerted back onto that object (the third law). Almost all of high school and undergraduate mechanics is based on these simple concepts.


 Physics I

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Water Quality Analysis via Indicator Organisms

JoVE 10025

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Luisa Ikner

Water quality analysis monitors anthropogenic influences such as pollutants, nutrients, pathogens, and any other constituent that can impact the water’s integrity as a resource. Fecal contamination contributes microbial pathogens that threaten plant, animal, and human health with disease or illness. Increasing water demands and strict quality standards require that water being supplied for human or environmental resources be monitored for low pathogen levels. However, monitoring each pathogen associated with fecal pollution is not feasible, as laboratory techniques involve extensive labor, time, and costs. Therefore, detection for indicator organisms provides a simple, rapid, and cost effective technique to monitor pathogens associated with unsanitary conditions.


 Environmental Microbiology

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Dissolved Oxygen in Surface Water

JoVE 10016

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

Dissolved oxygen (DO) measurements calculate the amount of gaseous oxygen dissolved in surface water, which is important to all oxygen-breathing life in river ecosystems, including fish species preferred for human consumption (e.g. bluegill and bass), as well as decomposer species critical to the recycling of biogeochemical materials in the system. The oxygen dissolved in lakes, rivers, and oceans is crucial for the organisms and creatures living in it. As the amount of dissolved oxygen drops below normal levels in water bodies, the water quality is harmed and creatures begin to die. In a process called eutrophication, a body of water can become hypoxic and will no longer be able to support living organisms, essentially becoming a “dead zone.” Eutrophication occurs when excess nutrients cause algae populations to grow rapidly in an algal bloom. The algal bloom forms dense mats at the surface of the water blocking out two essential inputs of oxygen for water: gas exchange from the atmosphere and photosynthesis in the water due to the lack of light below the mats. As dissolved oxygen levels decline below the surface, oxygen-breathing organisms die-off in large amounts, creati


 Environmental Science

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Turbidity and Total Solids in Surface Water

JoVE 10015

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

Turbidity and total solids are related measurements addressing clarity of surface waters. Turbidity is an indirect measure of water clarity that determines the amount of light that can pass through the water. Total solids is a direct measurement of solid particles suspended in water determined by weight. High levels of turbidity and total solids are caused by soil erosion, waste discharge, runoff, or changes in ecological communities including algal growth or abundance of benthic organisms that can disrupt sediments up into the water. Higher levels of turbidity and suspended solids can lower water quality by absorbing heat causing an increase in water temperature and a decrease in oxygen levels (warm water holds less oxygen). These conditions can also cause a decrease in photosynthesis as less sunlight penetrates the water, making the water unable to support some aquatic life. Suspended solids can also clog gills, smother eggs, reduce growth rates, and disrupt microhabitats of many aquatic organisms. One method of measuring turbidity includes using a Secchi disk. A Secchi disk is a metal disk with alternate black and white quarters (Figure 1). It is attached to a rope that


 Environmental Science

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Reliability in Psychology Experiments

JoVE 10046

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco—Monmouth University

In order to study something scientifically, a researcher needs to determine a way to quantify it. However, psychological constructs can be challenging to measure and quantify. This video examines reliability in the context of content analysis.  A recent study in the journal Pediatrics reported that 4-year-olds who watched a fast-paced cartoon had worse performance on cognitive tasks, such as following rules in a game, listening to direction from an adult, and delaying gratification, compared to other children who watched a slower paced cartoon.1 In addition to the pace of the cartoon, the content of the cartoon may also have deleterious effects on its young viewers. This video uses a simple two-group design, to exemplify the issue of reliability, in examining the question of whether the cartoon SpongeBob SquarePants has more inappropriate content than does the cartoon Caillou.


 Experimental Psychology

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The Factorial Experiment

JoVE 10058

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco—Monmouth University

A factorial design is a common type of experiment where there are two or more independent variables. This video demonstrates a 2 x 2 factorial design used to explore how self-awareness and self-esteem may influence the ability to decipher nonverbal signals. This video leads students through the basics of a factorial design including, the nature of a factorial design and what distinguishes it from other designs, the benefits of factorial design, the importance and nature of interactions, main effect and interaction hypotheses, and how to conduct a factorial experiment.


 Experimental Psychology

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The Simple Experiment: Two-group Design

JoVE 10056

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco—Monmouth University

A two-group design is the simplest way to establish a cause-effect relationship between two variables. This video demonstrates a simple experiment (two-group design).  In providing an overview of how a researcher conducts a simple experiment (two-group design), this video shows viewers the process of turning ideas into testable ideas and forming hypothesis, the identification and effect of experiment variables, the formation of experimental conditions and controls, the process of conducting the study, the collection of results, and the consideration their implications. This research technique is demonstration in the context of answering the research question: “How does physiological arousal/excitement influence perceived attraction?”


 Experimental Psychology

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The Multi-group Experiment

JoVE 10057

Source: Laboratories of Gary Lewandowski, Dave Strohmetz, and Natalie Ciarocco—Monmouth University

A multi-group design is an experimental design that has 3 or more conditions/groups of the same independent variable. This video demonstrates a multi-group experiment that examines how different interethnic ideologies (multiculturalism and color-blind) influence feelings about diversity and actions toward and out-group member. In providing an overview of how a researcher conducts a multi-group experiment, this video shows viewers how to distinguish levels in variables, common types of conditions/groups to use (including placebo and empty-control conditions/groups), the process of conducting the study, the collection of results, and the consideration of their implications.   


 Experimental Psychology

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Visual Search for Features and Conjunctions

JoVE 10062

Source: Laboratory of Jonathan Flombaum—Johns Hopkins University

How do people find objects in cluttered visual scenes? Think, for example, of looking for keys on a messy desk, finding the ripest-looking fruit at the grocery store, locating your car when you can’t quite remember where you parked it, or finding an old friend at an airport exit gate. Clearly, an understanding of visual perception is going to play a role in any answers, and more specifically, an understanding of visual attention will be crucial. Visual attention refers to the ability to focus in on just part of an image, mustering one’s processing resources selectively to determine whether the thing being looked for—the target, in the standard experimental jargon—is present. To study search and attention, experimental psychologists have developed a widely used paradigm known (unsurprisingly) as visual search. Psychologists have also motivated a great deal of research by the intuition that any good theory of search is going to have, to explain why some things are easy to find and others are hard to find. So in the context of the visual search paradigm, perceptual psychologists have often focused on contrasting easy searches with more difficult one


 Cognitive Psychology

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Two-Dimensional Gel Electrophoresis

JoVE 5686

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights. The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM). Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially a


 Biochemistry

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Metabolic Labeling

JoVE 5687

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR. This video introduces the concepts of metabolic labeling and show two general procedures.  The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos. Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications. Metabolic lab


 Biochemistry

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Photometric Protein Determination

JoVE 5688

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more a sample contains light-absorbing substances, the less the light will transmit through it. Since the relationship between concentration and absorption is linear, this phenomenon can be used to measure the concentration in samples where it is unknown. This video describes the basics of photometric protein determination and introduces the Bradford Assay and the Lowry Method. The procedure in the video will cover a typical Bradford assay. Applications covered include direct measurement of very small volumes of nucleic acids to characterize concentration and purity, determination of coupling efficiency of a biomimetic material, and another variation of photometric protein determination using Remazol dye. Determining the concentration of a protein in samples is a fundamental step in many biochemical assays. Photometric determination can be done with small sample sizes. The more a sample contains light-absorbing substances, the less the light will transmit through it. This provides a quantitative measurement of the absorbing substances. These concepts are so fundamental to science that the articles that introduced two of the techniques are in the three most cited papers of


 Biochemistry

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Protein Crystallization

JoVE 5689

Protein crystallization, obtaining a solid lattice of biomolecules, elucidates protein structure and enables the study of protein function. Crystallization involves drying purified protein under a combination of many factors, including pH, temperature, ionic strength, and protein concentration. Once crystals are obtained, the protein structure can be elucidated by x-ray diffraction and computation of an electron density model. This video introduces protein crystallization and shows a general procedure. Protein expression and purification, crystallization, and x-ray diffraction are covered in the procedure. Applications of protein crystallization include in silico drug design, binding site determination, and membrane protein structure analysis. Protein crystallization is the process of obtaining a latticed solid form of a protein. These crystals are especially valuable to structural biologists, assisting in the study of protein function. Other techniques, such as mass spec or SDS-PAGE, can only provide information on the one-dimensional structure of proteins. Protein crystallization is complemented by the techniques of recombinant protein expression and x-ray diffraction. This video will show the principles of protein crystallization, a general laboratory procedure, and several of its applications in the


 Biochemistry

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MALDI-TOF Mass Spectrometry

JoVE 5691

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry ion source ideal for the analysis of biomolecules. Instead of ionizing compounds in the gaseous state, samples are embedded in a matrix, which is struck by a laser. The matrix absorbs the majority of the energy; some of this energy is then transferred to the sample, which ionizes as a result. Sample ions can then be identified using a time-of-flight analyzer (TOF). This video covers principles of MALDI-TOF, including matrix selection and how TOF is used to elucidate mass-to-charge ratios. This procedure shows the preparation of a MALDI plate, the loading of samples onto the plate, and the operation of the TOF-mass spectrometer. In the final section, applications and variations are shown, including whole-cell analysis, characterization of complex biological samples, and electron spray ionization. Matrix-assisted laser desorption ionization, or MALDI, is a mass spectrometry ion source ideal for the analysis of biomolecules. Most ion sources remove structural information from large, fragile biomolecules. MALDI maintains structural integrity, and therefore information, while accelerating the molecules into the mass analyzer, which separates the compounds based on size and charge. The most commonly coupled with MALDI is the time of flight, or TOF,


 Biochemistry

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Enzyme Assays and Kinetics

JoVE 5692

Enzyme kinetics describes the catalytic effects of enzymes, which are biomolecules that facilitate chemical reactions necessary for living organisms. Enzymes act on molecules, referred to as substrates, to form products. Enzyme kinetic parameters are determined via assays that directly or indirectly measure changes in substrate or product concentration over time. 

This video will cover the basic principles of enzyme kinetics (including rate equations) and kinetic models. The concepts governing enzyme assays are also discussed, followed by a typical colorimetric assay. The applications section discusses an enzyme assay via Förster resonance energy transfer (FRET) analysis, characterizing extracellular enzyme activity in the environment, and investigating DNA repair kinetics using molecular probes. Enzymes are biochemical catalysts that are essential for life. Enzyme assays are used to study the kinetic properties of enzymatic reactions, elucidating the catalytic effects of enzymes. This video will cover enzyme kinetics and assays, go over a general procedure, and show some applications. Enzymes are proteins or protein-like molecules that act on a reactant molecule, referred to as the substrate. Enzymes reduce the activation energy of bioc


 Biochemistry

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Electrophoretic Mobility Shift Assay (EMSA)

JoVE 5694

The electrophoretic mobility shift assay (EMSA) is a biochemical procedure used to elucidate binding between proteins and nucleic acids. In this assay a radiolabeled nucleic acid and test protein are mixed. Binding is determined via gel electrophoresis which separates components based on mass, charge, and conformation.

This video shows the concepts of EMSA and a general procedure, including gel and protein preparation, binding, electrophoresis, and detection. Applications covered in this video include the analysis of chromatin-remodeling enzymes, a modified EMSA that incorporates biontinylation, and the study of binding sites of bacterial response regulators. EMSA, the electrophoretic mobility shift assay, also known as the gel shift assay, is a versatile and sensitive biochemical procedure. EMSA elucidates binding between proteins and nucleic acids by detecting a shift in bands in gel electrophoresis. This video describes the principles of EMSA, provides a general procedure, and discusses some applications. DNA replication, transcription, and repair, as well as RNA processing are all critical biochemical processes. They all involve binding between proteins and nucleic acids. Many serious diseases and disorders are associated with modifications in this


 Biochemistry

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Förster Resonance Energy Transfer (FRET)

JoVE 5696

Förster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent molecule can non-radiatively transfer energy to an acceptor molecule if their respective emission and absorbance spectra overlap. The amount of energy transferred—and consequently the overall emission of sample—depends on the proximity of an acceptor-donor pair of photoluminescent molecules. FRET analysis is combined with other biochemistry techniques to obtain detailed information of biomolecular structures and interactions from this “spectroscopic ruler.” This video covers the principles and concepts of FRET analysis. The procedure focuses on preparing samples for FRET and ways to present and interpret data. Finally, the applications include monitoring conformational and cellular processes by labeling parts of a cell or protein, monitoring enzyme reactions that alter protein structures, and using FRET to monitor aggregation of monomers expressed by cells. Förster Resonance Energy Transfer, or FRET, is a non-radiative transfer of energy between light-emitting molecules, and is often used to investigate close-range biochemical interactions. FRET only occurs when fluorescent molecules are spaced within 10 nm of each other. FRET analysis


 Biochemistry

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Surface Plasmon Resonance (SPR)

JoVE 5697

Surface plasmon resonance (SPR) is the underlying optical phenomenon behind label-free biosensors to evaluate the molecular affinity, kinetics, specificity, and concentration of biomolecules. In SPR, biomolecular interactions occur on a biosensor made of a thin layer of metal on a prism. Real-time interactions of biomolecules can be monitored by measuring the changes of light reflected off the underside of the metal. This video describes the basic concepts of SPR and how it is used to analyze and visualize biomolecular interactions. This is followed by a sample preparation and experimental protocol for investigating binding rates using SPR. In the applications section, SPR imaging, localized SPR, and quantum dot enhanced SPR are explored. Surface plasmon resonance, or SPR, is the underlying phenomenon behind certain label-free biosensors for evaluating binding and adsorption interactions of biomolecules. Binding assays that require labeling, such as ELISA, can be a time-consuming process, and may alter the functionality of the analyte. In SPR, biomolecular interactions occur on a special sensor made of a thin layer of metal on one face of a prism. By monitoring the changes in light reflected off of the underside of the metal, SPR instruments visualize these interactions in real-time without the use of labels. This video


 Biochemistry

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Electrochemical Measurements of Supported Catalysts Using a Potentiostat/Galvanostat

JoVE 5698

Source: Laboratory of Dr. Yuriy Román — Massachusetts Institute of Technology

A potentiostat/galvanostat (often referred to as simply a potentiostat) is an instrument that measures current at an applied potential (potentiostatic operation) or measures potential at an applied current (galvanostatic operation) (Figure 1). It is the most commonly used instrument in the electrochemical characterization of anode and cathode materials for fuel cells, electrolyzers, batteries, and supercapacitors. Conventionally, these anode and cathode materials are interfaced with a potentiostat via a three-electrode electrochemical cell. The electrode leads from the potentiostat are connected to the reference electrode, the counter electrode (often called the auxiliary electrode), and the working electrode (which contains the test material of interest). The electrochemical cell is then filled with a high ionic strength electrolyte solution, such as an acidic, alkaline, or salt solution. The media for this high ionic strength solution is typically aqueous; however, for applications necessitating higher operating cell potential windows, such as batteries and supercapacitors, non-aqueous media is often used. The cell media is degassed with an inert gas (to prevent unwanted side react


 Analytical Chemistry

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Nutrients in Aquatic Ecosystems

JoVE 10023

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

Nitrogen and phosphorus are essential plant nutrients found in aquatic ecosystems and both are monitored as a part of water quality testing because in excess amounts they can cause significant water quality problems. 

Nitrogen in water is measured as the common form nitrate (NO3-) that is dissolved in water and readily absorbed by photosynthesizers such as algae. The common form of phosphorus measured is phosphate (PO43-), which is strongly attracted to sediment particles as well as dissolved in water. In excess amounts, both nutrients can cause an increase in aquatic plant growth (algal bloom, Figure 1) that can disrupt the light, temperature, and oxygen levels in the water below and lead to eutrophication and hypoxia (low dissolved oxygen in water) forming a “dead zone” of no biological activity. Sources of nitrates and phosphorus include wastewater treatment plants, runoff from fertilized lawns and agricultural lands, faulty septic systems, animal manure runoff, and industrial waste discharge. Figu


 Environmental Science

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Measuring Tropospheric Ozone

JoVE 10024

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

Ozone is a form of elemental oxygen (O3), a molecule of three oxygen atoms bonded in a structure that is highly reactive as an oxidizing agent. Ozone occurs in both the stratosphere and the troposphere levels of the atmosphere. When in the stratosphere (located approximately 10-50 km from the earth’s surface), ozone molecules form to the ozone layer and help prevent harmful UV rays from reaching Earth’s surface. In lower altitudes of the troposphere (surface - approximately 17 km), ozone is harmful to human health and is considered an air pollutant contributing to photochemical smog (Figure 1). Ozone molecules can cause damage directly by harming respiratory tissue when inhaled or indirectly by harming plant tissues (Figure 2) and softer materials including tires on automobiles. Outdoor tropospheric ozone is formed at ground level when nitrogen oxides (NOx) and volatile organic compounds (VOCs) from automobile emissions are exposed to sunlight. Consequently, health concerns over ozone concentrations escalate in sunny conditions or when and where automobile use is increased. Reaction: NO2 + VOC + sunlight &


 Environmental Science

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Sonication Extraction of Lipid Biomarkers from Sediment

JoVE 10055

Source: Laboratory of Jeff Salacup - University of Massachusetts Amherst

The material comprising the living "organic" share of any ecosystem (leaves, fungi, bark, tissue; Figure 1) differs fundamentally from the material of the non-living "inorganic" share (rocks and their constituent minerals, oxygen, water, metals). Organic material contains carbon linked to a series of other carbon and hydrogen molecules (Figure 2), which distinguishes it from inorganic material. Carbon's wide valency range (-4 to +4) allows it to form up to four separate covalent bonds with neighboring atoms, usually C, H, O, N, S, and P. It can also share up to three covalent bonds with a single other atom, such as the triple bond in the often-poisonous cyanide, or nitrile, group. Over the past 4.6 billion years, this flexibility has led to an amazing array of chemical structures, which vary in size, complexity, polarity, shape, and function. The scientific field of organic geochemistry is concerned with the identification and characterization of the whole range of detectable organic compounds, called biomarkers, produced by life on this planet, as we


 Earth Science

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Soil Nutrient Analysis: Nitrogen, Phosphorus, and Potassium

JoVE 10077

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

In this experiment, three soil macronutrients are chemically extracted, combined with color-based reagents, then analyzed using color to determine the nutrient concentration present in the soil sample.

Nitrogen, phosphorus, and potassium are the main components of soil fertilizer. These methods isolate each nutrient from the soil into a solution that can be analyzed using turbidity and color to determine the concentration of nutrients present in the soil sample. Knowing present concentration informs environmental scientists of a nutrient deficiency or surplus in soils used to support plant production, and also provides general insight into basic biogeochemical cycles of an ecosystem.


 Environmental Science

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Habituation: Studying Infants Before They Can Talk

JoVE 10068

Source: Laboratories of Nicholaus Noles, Judith Danovitch, and Cara Cashon—University of Louisville

Infants are one of the purest sources of information about human thinking and learning, because they’ve had very few life experiences. Thus, researchers are interested in gathering data from infants, but as participants in experimental research, they are a challenging group to study. Unlike older children and adults, young infants are unable to reliably speak, understand speech, or even move and control their own bodies. Eating, sleeping, and looking around are the only activities babies can perform reliably. Given these limitations, researchers have developed clever techniques for exploring infants’ thoughts. One of the most popular methods makes use of a characteristic of attention called habituation. Like adults, infants prefer to pay attention to new and interesting things. If they are left in the same environment, over time they become accustomed to their surroundings and pay less attention to them. This process is called habituation. However, the moment something new happens, infants are waiting and ready to pay attention again. This reengagement of attention following habituation is referred to as dishabituation. Scientists can use these characteristic changes in attention as


 Developmental Psychology

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Using Your Head: Measuring Infants' Rational Imitation of Actions

JoVE 10069

Source: Laboratories of Judith Danovitch and Nicholaus Noles—University of Louisville

One of the main challenges of infancy is to learn how to achieve one’s goals in the world, whether they are to pick up a toy or to express desires to another person, and one of the most powerful tools in this learning process is imitation. However, imitation is not always as simple as copying other people’s actions; it is also necessary to realize that goals and intentions guide behavior. The world is a complicated place, and the actions that babies imitate are not perfectly presented. For example, consider a baby watching their father drinking from a plastic water bottle. As he picks up the bottle, he accidentally drops it on the floor. He picks it up and dusts it off, before turning the cap and taking a drink. If the baby wants to drink from the bottle on their own, they have to decode this complex set of events and determine which actions are related to their goal. Do they have to drop it and dust it off, or can they simply turn the cap and take a drink? One way to solve this problem is to view many examples of the same behavior, but some behaviors are rare or different each time they are performed. Thus, it is beneficial for infants to think more about the person they are imitating and less


 Developmental Psychology

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Tree Survey: Point-Centered Quarter Sampling Method

JoVE 10060

A number of methods are available for sampling forest communities. Point-centered quarter is one such method. It is used to gather information on the density, frequency, and coverage of tree species found in a forest. This information provides the ability to estimate the number of individual trees encountered, how often a certain tree occurs, how common the tree is compared to other trees, and the size of the tree. Compared to the standard plot analysis, the point-centered quarter method is more efficient, which is a major advantage. In a fixed-area plot sampling, a small portion of the total area of the forest is examined. In this small subsample, the density is determined directly by counting and identifying each tree. The ratio between size of the subplot and the overall forest size is used to determine the density for the entire forest.


 Environmental Science

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Carbon and Nitrogen Analysis of Environmental Samples

JoVE 10012

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

Elemental Analysis is a method used to determine elemental composition of a material. In environmental samples such as soils, scientists are particularly interested in the amounts of two ecologically important elements, nitrogen and carbon. Elemental analysis by the flash combustion technique works by oxidizing the sample with a catalyst through combustion in a high-temperature chamber. The products of combustion are then reduced to N2 and CO2 and detected with a thermal conductivity detector. Unlike other methods for total nitrogen determination (Kjeldahl method) and total carbon determination (Walkley-Black, Heanes or Leco methods), the flash combustion technique does not use toxic chemicals and is therefore much safer to use. This video will demonstrate combustion-based elemental analysis using the Flash EA 1112 instrument from Thermo Fisher Scientific.


 Environmental Science

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Multiple Object Tracking

JoVE 10019

Source: Laboratory of Jonathan Flombaum—Johns Hopkins University

In a staggeringly complex and engaging world, it is crucial to selectively process some stimuli at the expense of others. Experimental psychologists call this ability attention. Specifically, visual attention refers to the ability to selectively process aspects of a visual scene. Many paradigms used to study visual attention involve brief, punctuated, and repeated trials. However, everyday situations often place sustained demands on attention, as opposed to requiring only brief focus. For example, compare driving through busy city streets, which demands sustained attention, with crossing a busy street, which demands just a few moments of caution. To investigate sustained visual attention, experimental psychologists typically rely on a paradigm called multiple object tracking. This video demonstrates standard procedures for investigating sustained visual attention through multiple object tracking.


 Cognitive Psychology

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Are You Smart or Hardworking? How Praise Influences Children's Motivation

JoVE 10112

Source: Laboratories of Judith Danovitch and Nicholaus Noles—University of Louisville

Imagine teaching two children how to skate. It is a hard task for both of them, and they fall down frequently. After falling down for the first time, one child says that skating is too hard and wants to go home. The other child seems to enjoy the challenge and eagerly gets back up after falling down each time. Why do the children have such different attitudes about the same task? One reason may be that they have different mindsets or beliefs about the nature of their ability. According to psychologist Carol Dweck, some people have a fixed mindset, and some people have a growth mindset. People with a fixed mindset believe that intelligence or abilities are fixed and cannot change. When these people face a challenge, like learning how to skate, they tend to believe that if a new skill does not come easily, then they are simply no good at it. They do not see their skills as capable of changing, and thus they decide that it’s useless to continue trying. People with a growth mindset have the opposite attitude. They believe that abilities can be developed through hard work, and they continue trying to improve even if they do not initially succeed. How do these different m


 Developmental Psychology

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The Rouge Test: Searching for a Sense of Self

JoVE 10111

Source: Laboratories of Nicholaus Noles and Judith Danovitch—University of Louisville

Humans are different from other animals in many ways, but one of the abilities that sets humans apart is their advanced ability to understand other people and simulate their thoughts and feelings, even when the thoughts and feelings do not align with their own. In scientific terms, these abilities are referred to as theory of mind, and this understanding is necessary for activities like giving compliments, working in groups, asking for favors, and telling white lies. Humans are not born with a fully developed theory of mind. An individual’s understanding that they are separate from other people and that they have different desires and knowledge requires an established sense of self. Thus, developing self-recognition and self-awareness are some of the initial steps on the path to developing a mature theory of mind. Studying a child’s emerging sense of self is complicated, because children’s conceptual development exceeds their mastery of language. To solve this problem, researchers borrowed methods used to detect self-recognition in animals and applied them to young children. Thus, with a mirror and a bit of make-up, the rouge task was born. This


 Developmental Psychology

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Numerical Cognition: More or Less

JoVE 10093

Source: Laboratories of Nicholaus Noles and Judith Danovitch—University of Louisville

One of the goals of the modern education system is to teach children mathematical literacy. They are taught to add, subtract, multiply, and divide, and this base knowledge is used to support learning about geometry, algebra, calculus, physics, and statistics. School-aged children usually acquire these skills in formal educational settings, but the foundation of mathematical understanding is developed much earlier in life. As infants, humans begin to form the rough representations that allow them to make judgments about number, and perhaps the first numerical concept that humans develop is the idea of less versus more. However, probing these concepts can be challenging, because even if babies have some understanding of number, they have very few ways of showing off what they know. What they can do is crawl, eat, cry, and sleep. Thus, researchers developed a task using this limited set of responses to investigate whether babies can mentally represent number. This experiment demonstrates how researchers can creatively use food to study concepts of numerical cognition in infants using the method by Feigenson, Carey, and Hauser.1


 Developmental Psychology

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Executive Function and the Dimensional Change Card Sort Task

JoVE 10085

Source: Laboratories of Nicholaus Noles and Judith Danovitch—University of Louisville

Infants are born with amazing cognitive resources at their disposal, but they don’t know how to use them effectively. In order to harness the power of their brains, humans must develop high-level cognitive processes that manage basic brain functions. These processes make up what psychologists refer to as executive function. Executive function is a key factor in many self-regulatory behaviors, including forming plans to solve problems, negotiating between desires and actions, and directing attention. For example, a child must use several executive processes to stop playing with toys and start cleaning their room. These processes include inhibition (to stop what they’re doing), planning (to determine what actions need to be performed to clean the room), and attentional control (to stay on task until the cleaning is done). A breakdown of executive function during any of these steps would lead to the room remaining dirty. Developing executive function is one of the key challenges faced by children as they mature. Some elements of executive function can only be mastered with practice, and brain areas linked to executive function, specifically the prefrontal cortex, develop slowly throughout


 Developmental Psychology

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Children's Reliance on Artist Intentions When Identifying Pictures

JoVE 10117

Source: Laboratories of Judith Danovitch and Nicholaus Noles—University of Louisville

Children are not the best artists. Sometimes it’s easy to pick out the characteristic triangular head, whiskers, and tail of a cat, but children often describe elaborate scenarios that they depict as a beautifully unrecognizable mess. Thus, given children’s questionable artistic talent, how do they know what their drawings, and the drawings of others, represent? One way children identify pictures is by relying on resemblance. If it looks like a cat, then it’s a cat. However, some pictures do not clearly resemble any real object. In this situation, children must use other means to figure out what the picture represents, including their understanding of what the person who created the picture intended it to represent. By their first birthday, children are sensitive to the intentions of other people. They know that people’s actions are driven by their goals, and they can infer a person’s intentions even if the goal-directed action is not successful (e.g., they understand a person struggling to turn a lid intends to open a jar, even if they never see them succeed in opening it). By about age 3, children can use this understanding of intention to guide their interpretation


 Developmental Psychology

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