This research was funded by the National Institutes of Health-Deafness and Communication Disorders (R01 2DC007428).

1. IPL Video Creation
We design the videos to be interesting and attractive, but also non-aversive to young children with autism, in a number of ways: When animate characters are needed, we use animals rather than humans to make the scenes less socially/emotionally challenging for children with ASD. We use dynamic scenes with brightly colored objects to capture and hold attention. A red blinking light during the intertrial intervals (ITIs) holds the children's attention when the video screens are blank. We produce the voice audio using speech that is exaggerated in both intonation and duration to capture and hold the children's attention.
Figure 1. (Movie) One block of the Noun Bias video. Shows one block of the Noun Bias video, which tests whether children map a novel word onto an object vs. an action. <a href="https://www.jove.com/files/ftp_upload/4331/4331movie1.mov" target="_blank"> Click here to view movie</a>.
Video stimuli are created via commercial nonlinear editing programs such as FinalCut Pro or Avid Media Composer, using 4-8 sec movie clips.
The first 2 clips are arranged sequentially, alternating left and right, paired with the same familiarization or teaching audio.
The baseline trials come next; these present the test stimuli side-by-side, but are paired with nondirecting audios. The baseline trials tell us whether the children have any inherent or visual preference for either stimulus when there is no linguistic or directing audio.
The test trials appear last, paired with the test audios that distinguish the two visuals.
A 1kHz tone goes on the second audio channel, coincident with each trial, providing visual designation to coders of the onset and offset of trials.
2. At the Visit/Child's Home
<ol>
	<li>Perform the consent process with the parent. Describe the study format in more detail and ask parent to sign the consent forms. Emphasize that the child should watch with minimal interaction with the parent, and introduce the mp3 player for the parent to use, if the child is to sit on the parent's lap. The parent listens to music on the mp3 player while watching the IPL video, so that s/he does not hear the IPL test audio and so potentially influence which way the child should look.</li>
	<li>Set up the IPL components in the designated room, and connect to power sources using extension cords. It is helpful to bring a research assistant along who can play with the child during setup. Set up the big screen with its back to the major light source. Place the speaker centered behind the screen; adjust both volume knobs to halfway levels. Place the camera on the tripod atop the tone adaptor box, centered in front of screen. Load the tape into the camera and place the DTE on the floor next to the camera. Set up the projector stand 7-10 feet away from the screen; place the projector on top. Place the laptop next to the projector stand.</li>
</ol>
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/4331/4331fig2highres.jpg" src="/files/ftp_upload/4331/4331fig2.jpg" /> 
Figure 2. The portable IPL components and the arrangement of their setup in the home. Shows the portable IPL components and the spatial arrangement of their setup in the home. <a href="https://www.jove.com/files/ftp_upload/4331/4331fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<ol start="3">
	<li>Connect the IPL components: Connect the laptop so that it sends video signal to the projector. The laptop also sends out two audio signals (via a splitter), with the voice audio going to the speaker and the tone audio going to the camera. Connect the camera so that it sends video and audio signal to the DTE. Once connected, power up all components. The DTE screen should show "AVI Type 1" on left and "Counter" underneath.</li>
	<li>Play the video(s) for the child: Load the first video on the laptop into Quicktime.
	<ol>
		<li>Place a chair 2.5 to 3 feet from camera, in front of screen. Invite the child to sit on the chair, or on the parent's lap. If on parent's lap, give the parent the mp3 player and earbuds, tell her/him to start it. If child sits alone, parent may sit alongside. Children who get fidgety during the video may be encouraged in general terms to "watch the movie."</li>
		<li>Ensure that the child's face is visible in the camcorder. Re-check whenever the child moves around. Adjust lighting in room as needed.</li>
		<li>Write child's information on a dry erase whiteboard (pseudonym, gender, visit, video, date, birthdate, age).Turn on camcorder and press record. Press record on DTE. Film the whiteboard for 10 sec.</li>
		<li>On the laptop, play the first video by clicking "View" followed by "Enter Full Screen."</li>
		<li>After the video is completed, press the stop button on the DTE. If the child is willing, another video may be presented. Alternatively, this may be a good time for a break. Some children with ASD may need frequent breaks, but others may prefer to view all of the videos in a row.</li>
	</ol>
	</li>
	<li>Reward children with a gift at the end of the visit.</li>
</ol>
3. Coding Back in the Lab
<ol>
	<li>Check that the child's eye movements are already digitized on the DTE.
	<ol>
		<li>Import this film into a nonlinear editing program, convert to the format (.avi) used by our custom coding program, and then export to a secure storage device.</li>
	</ol>
	</li>
	<li>Code each video following its specific layout (i.e,. arrangement of familiarization/teaching, baseline, and test trials).
	<ol>
		<li>Tab through the child's film and find the frames where the waveform of the 1kHz tone is visible, indicating that a visual stimulus is being presented to the child.</li>
		<li>Begin coding during the intertrial interval (ITI) before each target trial (i.e., there's no tone and the centering light is presented; the child should be centering or looking away). Tab ahead frame by frame, recording each change of gaze as L (to the left), R (to the right), C (to the center) or A (away: up, down, far left, far right, back).</li>
	</ol>
	</li>
</ol>
The coding program outputs columns of numbers indicating the timing and type of each code entered. So, TSC 33499 means that at the onset of the trial, 3.3499 sec from the start of the video, the child looked to the center.
The coding arrays are analyzed by a custom matlab analysis program, which accesses the specific layout-which side is the match for each trial-of each video.
The analysis programs then calculate the child's duration and direction of looking during each coded trial, the child's latency to first look at the matching stimulus, and the number of times the child switches attention during each coded trial.
We then compare children's looking during the baseline vs. test trials on (a) duration of gaze to the matching image, (b) latency of first look to the matching image, (c) timecourse of looking to both images during the entire trial, and (d) number of switches of attention.
The rationale is that children who understand the linguistic stimulus will look longer at the matching image during the test trial than they had during the baseline trial, that they will look more quickly (once the trial begins) at the matching image than the nonmatching image, and that they will switch attention less during the test trials than during the baseline trials, because the linguistic stimulus is guiding their looking.
The only difference between baseline and test trials is the linguistic audio; the visual stimuli are the same but the baseline trials have a nondirecting audio (e.g., "look here!") while the test trials have a directing audio (e.g., "Where's toopen?") Therefore, comparing test trials to baseline trials for the duration and switches of attention measures tells us how much the directing audio is guiding the children's looking patterns. Also, if the children understand the directing audio, they should look more quickly at the matching image than at the nonmatching image.

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No conflicts of interest declared.

Specific attention is needed to ensure that coders be 'deaf' to/unaware of the specific stimuli that the child is experiencing on a given trial, so that they don't know which side is the matching one and inadvertently bias their coding. We ensure this via the inclusion of the 1kHz tone on the 2nd audio channel of the video, which is then copied onto the film of the child's eye movements. The waveform of this tone provides the visual indication of the onset and offset of each trial, thereby delimiting the trials without the coder knowing their video or audio content.
It is very important to ensure that the analysis layout takes account of the left-right switch that occurs when children are coded (i.e., if the matching scene is on the left side while the child is watching, it will appear to be on the right side to the eye-movement coder, and vice versa).
Intercoder reliability assessments yield correlations of about 0.98 for pairs of experienced coders on random selections of 10% of the data;3 with less experienced coders or if there is high turn-over in the coders used across the participants of the study, we recommend having every child's eye-movements coded by at least 2 people and requiring that their codes (usually duration to match) be within 0.3 sec of each other for each trial. If they are not, then a third, fourth or fifth person should code the child until reliability is achieved.17
With participants of toddler age and/or children with developmental delays, it is inevitable that some will not look at either scene for some proportion of each trial, and for some trials, never. The following conventions are generally applied for these lapses of attention: (a) Children need to look to at least one scene for a minimum of 0.3 sec for that trial to be counted. Otherwise, it is a missing trial. (b) For a given video, children need to provide data for more than half of the test trials in order to be included in the final dataset. (c) Missing trials are replaced with the mean across children in that age group/condition for that item.
IPL is a method that taps children's earliest mappings of linguistic forms (i.e., words and sentences) onto referential (i.e., objects and actions) or propositional (i.e., relations and events) meanings. It can be used to assess the processes by which children of different ages learn new words, as well as the ages at which they understand different types of grammatical forms. By requiring only eye movements as overt indicators of understanding (or not), IPL can be used with toddlers whose behavioral compliance is generally low, as well as with some special populations such as children with ASD. Moreover, because this technology can be brought to children's homes, it may also be used with populations which are hard to reach in conventional laboratories, such as children from low social economic groups, children in rural communities, and bilingual children of monolingual non-native speaking parents. Newer ways of analyzing these eye movements are revealing interesting and important effects of learning different types of languages, and of learning words at different ages. Future plans include using IPL concurrently with neuroimaging methodologies, so that children's brain activity during language comprehension can be compared with their behavioral indicators.

<table border="1">
 <tbody>
 <tr>
 <td>Name of component</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Screen</td>
 <td>Draper</td>
 <td>RoadWarrior Portable Projection Screen</td>
 <td></td>
 </tr>
 <tr>
 <td>Laptop</td>
 <td>Apple</td>
 <td>15 inch MacBook Pro</td>
 <td></td>
 </tr>
 <tr>
 <td>Projector</td>
 <td>NEC</td>
 <td>NP40 Projector </td>
 <td>Digital Lens Projector</td>
 </tr>
 <tr>
 <td>Projector stand </td>
 <td>DA-LITE </td>
 <td>Project-O-Stand </td>
 <td></td>
 </tr>
 <tr>
 <td>Speaker </td>
 <td>YAMAHA </td>
 <td>Monitor Speaker Model MS101II </td>
 <td></td>
 </tr>
 <tr>
 <td>Tripod </td>
 <td>Velbon </td>
 <td>QB-4W DF Mini</td>
 <td></td>
 </tr>
 <tr>
 <td>Camcorder </td>
 <td>Canon </td>
 <td>VIXIA HV30 HDV </td>
 <td>High Definition Video </td>
 </tr>
 <tr>
 <td>DTE </td>
 <td>DTE Technology </td>
 <td>FS-H200 </td>
 <td></td>
 </tr>
 <tr>
 <td>Noise cancelling headphones </td>
 <td>Sony</td>
 <td>MDR-NC60 </td>
 <td></td>
 </tr>
 </tbody>
</table>

portable intermodal preferential looking (ipl): investigating language comprehension in typically developing toddlers and young children with autism

One of the defining characteristics of autism spectrum disorder (ASD) is difficulty with language and communication.1 Children with ASD's onset of speaking is usually delayed, and many children with ASD consistently produce language less frequently and of lower lexical and grammatical complexity than their typically developing (TD) peers.6,8,12,23 However, children with ASD also exhibit a significant social deficit, and researchers and clinicians continue to debate the extent to which the deficits in social interaction account for or contribute to the deficits in language production.5,14,19,25
Standardized assessments of language in children with ASD usually do include a comprehension component; however, many such comprehension tasks assess just one aspect of language (e.g., vocabulary),5 or include a significant motor component (e.g., pointing, act-out), and/or require children to deliberately choose between a number of alternatives. These last two behaviors are known to also be challenging to children with ASD.7,12,13,16
We present a method which can assess the language comprehension of young typically developing children (9-36 months) and children with autism.2,4,9,11,22 This method, Portable Intermodal Preferential Looking (P-IPL), projects side-by-side video images from a laptop onto a portable screen. The video images are paired first with a 'baseline' (nondirecting) audio, and then presented again paired with a 'test' linguistic audio that matches only one of the video images. Children's eye movements while watching the video are filmed and later coded. Children who understand the linguistic audio will look more quickly to, and longer at, the video that matches the linguistic audio.2,4,11,18,22,26
This paradigm includes a number of components that have recently been miniaturized (projector, camcorder, digitizer) to enable portability and easy setup in children's homes. This is a crucial point for assessing young children with ASD, who are frequently uncomfortable in new (e.g., laboratory) settings. Videos can be created to assess a wide range of specific components of linguistic knowledge, such as Subject-Verb-Object word order, wh-questions, and tense/aspect suffixes on verbs; videos can also assess principles of word learning such as a noun bias, a shape bias, and syntactic bootstrapping.10,14,17,21,24 Videos include characters and speech that are visually and acoustically salient and well tolerated by children with ASD.

An example of looking patterns from children tested using P-IPL is presented in Figure 3. Viewing the NounBias video, TD children 21 months of age looked longer at the matching image during test compared with baseline trials, indicating they had mapped the novel words onto objects rather than actions.21 Children with ASD averaging 33 months of age behaved similarly, with similar effect sizes.21,24 Therefore, children with ASD show a similar word learning bias as TD children. Viewing the SVO word order video, children in both groups also showed longer looking to matching images during test over baseline (i.e., significant comprehension), demonstrating understanding of English word order (e.g., that "The girl pushes the boy" is a different relationship from "The boy pushes the girl").3,14,21 Developmental differences are seen with the wh-question video, as TD children showed significant comprehension at 28 months of age, whereas significant comprehension was first found in children with ASD at 54 months of age.10 Therefore, children with ASD are delayed in learning how to correctly interpret questions such as "What did the apple hit?"
<img alt="Figure 3" src="/files/ftp_upload/4331/4331fig3.jpg" /> 
 Figure 3. Children's percent of looking time to the match (i.e., object rather than action), while viewing the Noun Bias video. Shows children's percent of looking time to the match (i.e., object rather than action), while viewing the Noun Bias video.
Figure 4 presents the more detailed timecourse analyses of TD children viewing the ShapeBias video at four successive visits, spanning 20-32 months. Blue lines indicate looking to the match (i.e., same shape object); notice that the height of the blue lines during the test trials (right side of each graph) increases with age, showing increasing shape preferences. Moreover, as the children get older, the blue lines rise to the match earlier in the trial, indicating that they are understanding the directing audio more quickly with age. Red lines indicate looking away; notice that the breadth of the red lines diminishes with age as children spend less time looking away. Pink lines indicate looking to the Center during the ITI; we use these to make sure that children aren't biased before the trial starts. These IPL data confirm those from other methods that the Shape Bias is an increasingly strong word learning principle for TD children. However, three replications have not yet succeeded in demonstrating a significant word-based shape bias in the children with ASD.17,24
<img alt="Figure 4" src="/files/ftp_upload/4331/4331fig4.jpg" /> 
 Figure 4. TD children's timecourse of looking while viewing the Shape Bias video. Shows TD children's timecourse of looking to the match, nonmatch, center, and away, while viewing the Shape Bias video. <a href="https://www.jove.com/files/ftp_upload/4331/4331fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
Figure 5 presents the timecourse data for the same children with ASD, when they averaged 41 months of age, while viewing the syntactic bootstrapping video, when they are asked to learn novel verbs using surrounding sentence frames (e.g., to determine that gorping in "The duck is gorping the bunny" refers to a causative action rather than to a noncausative action). Syntactic Bootstrapping is another word learning principle replicated in many studies of TD children. Notice that the children with ASD look longer at the nonmatching image (green lines) during the baseline trials, but then their looking is directed more at the matching images (blue lines) during the second half of the test trials, when they are asked to find the referent of the novel verb (e.g., "Find gorping!"). As a group, these children with ASD were thus able to correctly map the verb onto the causative action. Cross-visit regressions reveal that the children with ASD who looked longer at the match during the Syntactic Bootstrapping video had shorter latencies to the match when they viewed the Word Order video, 8 months earlier.14 
<img alt="Figure 5" src="/files/ftp_upload/4331/4331fig5.jpg" /> 
 Figure 5. Children with ASD's timecourse of looking while viewing the Syntactic Bootstrapping video. Shows children with ASD's timecourse of looking to the match, nonmatch, center, and away while viewing the Syntactic Bootstrapping video.

Watch this Scientific Journal Video about Portable Intermodal Preferential Looking IPL: Investigating Language Comprehension in Typically Developing Toddlers and Young Children with Autism at JoVE.com

Portable Intermodal Preferential Looking IPL: Investigating Language Comprehension in Typically Developing Toddlers and Young Children with Autism

A reliable home-based way to assess the language comprehension of very young typically developing children, as well as those with autism, is described. The method analyzes children's eye gaze while viewing side-by-side images but hearing an audio that matches only one image. Stimuli are designed with young participants in mind.

Portable Intermodal Preferential Looking (IPL): Investigating Language Comprehension in Typically Developing Toddlers and Young Children with Autism

One  of the defining characteristics of autism spectrum disorder (ASD) is difficulty  with language and communication.1  Children with ASD's onset of ...

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Research

JoVE Journal

Medicine

18.0K 次观看.  University of Connecticut. 一个可靠的以家庭为基础的方法来评估非常年轻的典型发展儿童的语言理解，以及那些患有自闭症，描述。该方法分析孩子的眼睛凝视着观看的图像，但听到的声音只匹配了一个图像。刺激的设计考虑到青少年参加。

视频: Portable Intermodal Preferential Looking IPL: Investigating Language Comprehension in Typically Developing Toddlers and Young Children with Autism

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Probing the Brain in Autism Using fMRI and Diffusion Tensor Imaging

Newly emerging theories suggest that the brain does not function as a cohesive unit in autism, and this discordance is reflected in the behavioral symptoms displayed by individuals with autism. While structural neuroimaging findings have provided some insights into brain abnormalities in autism, the consistency of such findings is questionable. Functional neuroimaging, on the other hand, has been more fruitful in this regard because autism is a disorder of dynamic processing and allows examination of communication between cortical networks, which appears to be where the underlying problem occurs in autism. Functional connectivity is defined as the temporal correlation of spatially separate neurological events1. Findings from a number of recent fMRI studies have supported the idea that there is weaker coordination between different parts of the brain that should be working together to accomplish complex social or language problems2,3,4,5,6. One of the mysteries of autism is the coexistence of deficits in several domains along with relatively intact, sometimes enhanced, abilities. Such complex manifestation of autism calls for a global and comprehensive examination of the disorder at the neural level. A compelling recent account of the brain functioning in autism, the cortical underconnectivity theory,2,7 provides an integrating framework for the neurobiological bases of autism. The cortical underconnectivity theory of autism suggests that any language, social, or psychological function that is dependent on the integration of multiple brain regions is susceptible to disruption as the processing demand increases. In autism, the underfunctioning of integrative circuitry in the brain may cause widespread underconnectivity. In other words, people with autism may interpret information in a piecemeal fashion at the expense of the whole. Since cortical underconnectivity among brain regions, especially the frontal cortex and more posterior areas 3,6, has now been relatively well established, we can begin to further understand brain connectivity as a critical component of autism symptomatology.
A logical next step in this direction is to examine the anatomical connections that may mediate the functional connections mentioned above. Diffusion Tensor Imaging (DTI) is a relatively novel neuroimaging technique that helps probe the diffusion of water in the brain to infer the integrity of white matter fibers. In this technique, water diffusion in the brain is examined in several directions using diffusion gradients. While functional connectivity provides information about the synchronization of brain activation across different brain areas during a task or during rest, DTI helps in understanding the underlying axonal organization which may facilitate the cross-talk among brain areas. This paper will describe these techniques as valuable tools in understanding the brain in autism and the challenges involved in this line of research.

Neuroimaging techniques, such as functional MRI and Diffusion Tensor Imaging have become increasingly useful in characterizing the cognitive and neural deficits in autism. An examination of brain connectivity in autism at a network level along with adaptations for scanning children with developmental disabilities is presented.

Newly emerging theories suggest that the brain does not function as a cohesive unit in autism, and this discordance is reflected in the behavioral ...

Eye Tracking Young Children with Autism

The rise of accessible commercial eye-tracking systems has fueled a rapid increase in their use in psychological and psychiatric research. By providing a direct, detailed and objective measure of gaze behavior, eye-tracking has become a valuable tool for examining abnormal perceptual strategies in clinical populations and has been used to identify disorder-specific characteristics1, promote early identification2, and inform treatment3. In particular, investigators of autism spectrum disorders (ASD) have benefited from integrating eye-tracking into their research paradigms4-7. Eye-tracking has largely been used in these studies to reveal mechanisms underlying impaired task performance8 and abnormal brain functioning9, particularly during the processing of social information1,10-11. While older children and adults with ASD comprise the preponderance of research in this area, eye-tracking may be especially useful for studying young children with the disorder as it offers a non-invasive tool for assessing and quantifying early-emerging developmental abnormalities2,12-13. Implementing eye-tracking with young children with ASD, however, is associated with a number of unique challenges, including issues with compliant behavior resulting from specific task demands and disorder-related psychosocial considerations. In this protocol, we detail methodological considerations for optimizing research design, data acquisition and psychometric analysis while eye-tracking young children with ASD. The provided recommendations are also designed to be more broadly applicable for eye-tracking children with other developmental disabilities. By offering guidelines for best practices in these areas based upon lessons derived from our own work, we hope to help other investigators make sound research design and analysis choices while avoiding common pitfalls that can compromise data acquisition while eye-tracking young children with ASD or other developmental difficulties.

Eye tracking has long been used to study gaze patterns in typically-developing individuals, but recent technological advancements have made its use with clinical populations, including autism, more feasible. While eye-tracking young children with autism can offer insight into early symptom manifestations, it involves methodological challenges. Suggestions for best practices are provided.

The rise of accessible commercial eye-tracking systems has fueled a rapid increase in their use in psychological and psychiatric research. By providing a ...

Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this experiment, we demonstrate the administration of low-frequency repetitive TMS (rTMS) to an optimal stimulation site in the right hemisphere in patients with chronic non-fluent aphasia. A battery of standardized language measures is administered in order to assess baseline performance. Patients are subsequently randomized to either receive real rTMS or initial sham stimulation. Patients in the real stimulation undergo a site-finding phase, comprised of a series of six rTMS sessions administered over five days; stimulation is delivered to a different site in the right frontal lobe during each of these sessions. Each site-finding session consists of 600 pulses of 1 Hz rTMS, preceded and followed by a picture-naming task. By comparing the degree of transient change in naming ability elicited by stimulation of candidate sites, we are able to locate the area of optimal response for each individual patient. We then administer rTMS to this site during the treatment phase. During treatment, patients undergo a total of ten days of stimulation over the span of two weeks; each session is comprised of 20 min of 1 Hz rTMS delivered at 90% resting motor threshold. Stimulation is paired with an fMRI-naming task on the first and last days of treatment. After the treatment phase is complete, the language battery obtained at baseline is repeated two and six months following stimulation in order to identify rTMS-induced changes in performance. The fMRI-naming task is also repeated two and six months following treatment. Patients who are randomized to the sham arm of the study undergo sham site-finding, sham treatment, fMRI-naming studies, and repeat language testing two months after completing sham treatment. Sham patients then cross over into the real stimulation arm, completing real site-finding, real treatment, fMRI, and two- and six-month post-stimulation language testing.

We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Toddlers - Resting and Developmental Challenges

The autonomic nervous system (ANS) consists of two branches, the parasympathetic and sympathetic nervous systems, and controls the function of internal organs (e.g., heart rate, respiration, digestion) and responds to everyday and adverse experiences 1. ANS measures in children have been found to be related to behavior problems, emotion regulation, and health 2-7. Therefore, understanding the factors that affect ANS development during early childhood is important. Both branches of the ANS affect young children&#8217;s cardiovascular responses to stimuli and have been measured noninvasively, via external monitoring equipment, using valid and reliable measures of physiological change 8-11. However, there are few studies of very young children with simultaneous measures of the parasympathetic and sympathetic nervous systems, which limits understanding of the integrated functioning of the two systems. In addition, the majority of existing studies of young children report on infants&#8217; resting ANS measures or their reactivity to commonly used mother-child interaction paradigms, and less is known about ANS reactivity to other challenging conditions. We present a study design and standardized protocol for a non-invasive and rapid assessment of cardiac autonomic control in 18 month old children. We describe methods for continuous monitoring of the parasympathetic and sympathetic branches of the ANS under resting and challenge conditions during a home or laboratory visit and provide descriptive findings from our sample of 140 ethnically diverse toddlers using validated equipment and scoring software. Results revealed that this protocol can produce a range of&#160;physiological responses to both resting and developmentally challenging conditions, as indicated by changes in heart rate and indices of parasympathetic and sympathetic activity. Individuals demonstrated&#160;variability in resting levels, responses to challenges, and challenge reactivity, which provides additional evidence that this protocol is useful for the examination of ANS individual differences for toddlers.

Measuring Cardiac Autonomic Nervous System ANS Activity in Toddlers - Resting and Developmental Challenges

We describe the methods for continuous monitoring of the autonomic nervous system under resting and challenge conditions with 18 month old children. Results revealed that this protocol can produce meaningful physiological responses in both branches of the autonomic nervous system and elicit significant individual variability in patterns of responses.

The autonomic nervous system (ANS) consists of two branches, the parasympathetic and sympathetic nervous systems, and controls the function of internal ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

Oral Biofilm Sampling for Microbiome Analysis in Healthy Children

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition leads to a better understanding of cariogenic and periopathogenic mechanisms. Microbial changes taking place in the oral cavity during childhood are of interest for several reasons. The evolution of the child oral microbiota and shifts in its composition need to be analyzed further to understand and possibly prevent the onset of disease. At the same time, advanced knowledge of the natural composition of oral biofilm is needed. Early stages of caries-free permanent dentition with healthy gums provide a widely unaffected subgingival habitat that can serve as an in situ baseline for studying features of oral health and disease. Analysis of children's oral biofilm during different stages in life is thus an important theme in the field. Modern molecular analysis methods can provide comprehensive information about the bacterial diversity of such biofilms. To enable microbiota data comparison, it is important to standardize each step in the procedure for molecular data generation. This procedure spans from clinical sampling, Next Generation Sequencing (NGS), bioinformatic data processing, to taxonomic interpretation. One of the most critical factors here is biofilm sampling. Sampling in children is even more challenging in particular due to limited space in subgingival areas. We thus focus on the use of paper points for subgingival sampling. This article provides a detailed protocol for oral biofilm sampling of the subgingival sulcus, the mucosa, and saliva in children.

Changes in the oral microbiome throughout childhood are of growing interest. Comparison of different microbiome studies reveals a lack of standardized sampling protocols. Limited space makes sampling the sound subgingival sulcus of children challenging. Paper point sampling is presented here in detail as the method of choice for this area.

Oral biofilm and its molecular analysis provide a basis for investigating various dental research and clinical questions. Knowledge of biofilm composition ...

Measurements of Motor Function and Other Clinical Outcome Parameters in Ambulant Children with Duchenne Muscular Dystrophy

While the number of new treatment options tested in patients with Duchenne muscular dystrophy (DMD) is increasing, there is still no defining of the most reliable assessments regarding therapeutic efficacy. We present clinical and radiological outcome measures used in ambulatory patients participating in our trial &#34;Treatment with L-citrulline and metformin in Duchenne muscular dystrophy&#34;. The motor function measure is a validated test in patients with neuromuscular disorders that consists of 32 items and assesses all three dimensions of motor performance including standing and transfer (D1 subscore), axial and proximal motor function (D2 subscore), and distal motor function (D3 subscore). The test shows high intra- and inter-rater variability but only when strictly following guidelines of the materials, examination steps, and calculation of scores. The 6-minute walk test, timed 10-meter walk/run test, and supine-up time are commonly used timed functional tests that also sufficiently monitor changes in muscle function; however, they strongly depend on patient collaboration. Quantitative MRI is an objective and sensitive biomarker to detect subclinical changes, though the examination costs may be a reason for its limited use. In this study, a high correlation between all clinical assessments and quantitative MRI scans was found. The combinational use of these methods provides a better understanding about disease progression; however, longitudinal studies are needed to validate their reliability.

The aim of this study is to present the most reliable clinical outcome measures and their correlations with quantitative muscle MRI in ambulant patients with Duchenne muscular dystrophy.

While the number of new treatment options tested in patients with Duchenne muscular dystrophy (DMD) is increasing, there is still no defining of the most ...