The authors would like to thank the parents and adolescents who participated in the CHAMACOS study. We also wish to thank the CHAMACOS staff who worked on the study, especially Katherine Kogut, M.S., for all the work she did as project manager for this study. The authors would also like to acknowledge the National Institute of Drug Abuse grant 1R01DA035300.

This study was approved by the Institutional Review Board from the University of California, Berkeley and the University of California, San Francisco. Participants were scheduled to arrive at the lab between 4:30-5:00 PM.
1. Rinse Mouth
<ol>
	<li>Ask the participant to go to the restroom and rinse out their mouth with water only. Have the participant swish with cold water for 5-10 s and spit in the sink. Do this only once.</li>
	<li>Say to participant, &#34;This is to make sure there&#39;s no food or crumbs in your teeth for saliva collection.&#34;</li>
</ol>
2. Resting Period
<ol>
	<li>Usher participant into a solitary room and allow them to sit for at least 10 min. Have the participant complete non-invasive or non-provoking questionnaires such as a contact information sheet and daily living questionnaires, as well as watch a 3 min peaceful video of sea life.</li>
</ol>
3. Saliva collection #1 (Pre-stress)
<ol>
	<li>Place a line to indicate 1 mL of saliva on a 2 mL cryovial, and tell the participant to fill the cryovial to this line. Collect participant&#39;s saliva via passive drool through a two-inch straw into the 2 mL cryovial36 and freeze immediately (-80 &#176;C).</li>
	<li>Say to participant, &#34;I will ask you to fill this vial with saliva right up to this line. The way to do it is to let saliva collect in your mouth and then let it run through the straw into the vial. It&#39;s best if you don&#39;t spit because that gets bubbles in your sample - just let the saliva run through the straw. It will help if you don&#39;t talk while you&#39;re doing this. Please try not to cough up phlegm, we just want normal saliva. Keep an eye on your saliva vial and how full it is. If there are bubbles at the top of your saliva, they should go over the line, so there is just liquid saliva right up to the line. Tell me when it fills up to the line. Here are some pictures of food posted on the wall (POINT OUT PICTURES), since thinking of favorite foods makes peoples&#39; mouths water.&#34;</li>
	<li>Offer water after the participant has completed the saliva sample. Do not allow the participant to keep the water; take it back when he/she is finished.</li>
</ol>
4. Preparation Period
<ol>
	<li>Say to participant: &#34;In a few min, we would like you to give a five-minute speech about how you are a good friend. There is a committee of two trained reviewers who are in the room next door who are going to be listening to your speech. Your job is to make the reviewers believe that you are a good friend. They want you to explain what things make you a good friend to others. You are competing against a lot of other people for how good you are at giving your speech and convincing people that you are a good friend. The reviewers are experts in judging facial expression and body movement, and they are going to compare your speech to other people in the study. You may notice that the reviewers are taking notes. This is so we can review their notes at a later time. You will also be videotaped so that we can rate your performance later. 
	 
	You&#39;ll have 3 min now to prepare for your speech. Remember, your speech should explain what it is about you that makes you a good friend, and you are competing against other people for how believable you are. After that, I will take you to the next room, where you will introduce yourself to the reviewers and give your 5-minute speech. There will be another task that comes after the speech, but the reviewers will let you know what the task is after the speech. Do you have any questions? You may write notes if you want, but you won&#39;t be able to take them into the room with you. Ok, please start preparing. I&#39;ll return in a few minutes.&#34;</li>
	<li>Hand participant paper, pen, and clipboard to allow them to take notes.</li>
	<li>Leave the participant alone in the room.</li>
	<li>Wait 3 min and then come back in the room.</li>
</ol>
5. TSST Set Up
<ol>
	<li>Say to participant, &#34;Okay, it&#39;s time to go next door and get started.&#34; If the participant has written notes, take them from the participant. Say, &#34;I can take those from you now. Thanks.&#34;</li>
	<li>Walk the participant to TSST room and indicate that they should face the confederates and the video camera.</li>
	<li>Start the video recording before the participant enters the room. Record the entire speech and math tasks (approximately 10 min total).</li>
	<li>Have the Youth Interviewer introduce the committee. &#34;This is_(participant&#39;s name)_. _(Participant)_, do you know either of these people?&#34;
	<ol>
		<li>If the participant answers no, have the Youth Interviewer says &#34;Okay, you can get started.&#34;</li>
		<li>If the participant answers yes, have the Participant indicate whom s/he knows, and have the Youth Interviewer say &#34;Okay, _(confederate)__, please excuse yourself. The judges need to be people who don&#39;t know you.&#34;</li>
	</ol>
	</li>
</ol>
6. TSST Speech Task
<ol>
	<li>Have Confederate 1 say, &#34;Please tell us why you are a good friend. Begin speaking now.&#34;</li>
	<li>Set the timer for 5 min. Start timer.</li>
	<li>After 30 s of silence, use one of the following prompts to keep the participant talking. Use prompts in order each time there is a 30 s pause.
	<ol>
		<li>Say &#34;You still have time. Please continue.&#34;</li>
		<li>Say &#34;Why else are you a good friend?&#34;</li>
		<li>Say &#34;Please give an example of when you were a good friend.&#34;</li>
		<li>Say &#34;Be as creative as you like. Why else are you a good friend?&#34;</li>
		<li>After all prompts above have been used, continue prompting thereafter with, &#34;You still have time. Please continue.&#34;</li>
	</ol>
	</li>
	<li>After 5 min, have Confederate 1 say: &#34;Please stop. Your time is up.&#34; (Proceed to math task.)</li>
</ol>
7. TSST Math Task
<ol>
	<li>Have Confederate 1 say, &#34;We are now going to ask you to count out loud backward. Start at 423, and count backward by 7&#39;s. Keep counting backward by 7&#39;s out loud until we tell you to stop. If you make a mistake, we will let you know and ask you to start over. Please be as quick and accurate as possible. Now we would like you to begin counting backward - start at 423, and subtract 7. Continue subtracting 7&#39;s out loud until we tell you to stop.&#34;</li>
	<li>Set the timer for 5 min. Start timer.</li>
	<li>If participant looks confused, have Confederate 1 say, &#34;Would you like me to repeat that?&#34;</li>
	<li>If the participant makes a mistake, have Confederate 1 say, &#34;That&#39;s incorrect. Please start again. 423 minus 7.&#34;</li>
	<li>If the participant does not start counting out loud after 30 s, move to the next easier math problem, using same instructions as above with same prompts.</li>
	<li>Use the following as the easier math problems include (in this order): 
	423 - 5 = ? 
	95 - 5 = ? 
	66 - 3 = ? 
	6 + 3 = ?</li>
	<li>If the participant stops after each correct answer to wait for confirmation, have Confederate 1 say, &#34;Continue counting backward by 7. We will only correct you if you make a mistake - otherwise, keep going.&#34;</li>
	<li>After 5 min, have Confederate 1 say, &#34;Time is up. Please go next door to give your next saliva sample.&#34;</li>
	<li>Escort participant back to solitary room.</li>
</ol>
8. Stop Protocol
<ol>
	<li>Empower both confederates to stop the protocol early. Both confederates are not required to consult with each other before doing so.</li>
	<li>If participant cries or appears teary-eyed, becomes obviously angry or aggressive, or otherwise shows distress or if participant begins to posture in a way that appears aggressive or odd, stop protocol immediately but as normally as possible.</li>
	<li>Say, &#34;Time&#39;s up. Please go next door to give your next saliva sample.&#34;</li>
	<li>Direct participant to the next room.</li>
</ol>
9. Saliva Sample # 2 (15 min Post-stressor Onset)
<ol>
	<li>When youth re-enters next room, set the timer for 15 min.</li>
	<li>Say to the participant, &#34;That&#39;s the toughest part of your visit; thanks for doing that and we&#39;re going to talk about it in a second. It&#39;s time to collect another saliva.&#34;</li>
	<li>If participant mentions TSST, say to the participant, &#34;We can talk about that in a minute, but please do the saliva sample first. </li>
	<li>Place a line to indicate 1 mL of saliva on a 2 mL cryovial, and tell participant to fill the cryovial to this line. Collect participant&#39;s saliva via passive drool through a two-inch straw into the 2 mL cryovial36 and freeze immediately (-80 &#176;C).</li>
	<li>Offer water to drink after the participant has completed the saliva sample. Do not allow the participant to keep the water; take it back when he/she is finished.</li>
</ol>
10. Debrief
<ol>
	<li>Say to participant, &#34;Okay, let&#39;s talk about the speech and math activities. You may have guessed this already, but the whole purpose of those activities was to create stress. You may have noticed that the &#34;reviewers&#34; weren&#39;t exactly the friendliest in the world, right?&#34; 
	 
	&#34;Well, first of all, I want to tell you that the &#39;reviewers&#39; are just research assistants. Their job is to make teens do the activities but not give any positive feedback no matter how great you do. So their behavior in there had nothing to do with your performance on those tasks - they act the same way with every single participant they meet. &#34; 
	 
	&#34;You&#39;re probably wondering why we did that. We had a scientific reason for doing it. We are studying whether the amount of stress you experience in your daily life affects how strongly you respond to a stressful situation like this one. One point of the study is to understand why teens react differently to stress. We were measuring something called cortisol in your saliva samples as a way to see your stress response.&#34; 
	 
	&#34;If you found the speech and math stressful, you&#39;re not alone! We chose making speeches and doing math because we know that even though they&#39;re safe to do, they make a lot of people nervous. Let me tell you, we needed to do those activities ourselves, and we all found it really hard! Even the scientists from Berkeley messed up the math. And we all said &#39;those were the longest 10 minutes of my life!&#39; Did you feel that way?&#34; 
	 
	&#34;A few more things I want to mention&#8230;&#34; 
	 
	[IF VIDEOTAPED] &#34;We videotaped this for 2 reasons. The main reason is so our bosses can make sure that WE are doing the activity correctly. In the future, we may also look at the videos to get more information about people&#39;s facial expressions. It&#39;s just another way of looking at how teens respond differently to stress.&#34; 
	 
	[IF NOT VIDEOTAPED] &#34;The truth is we weren&#39;t actually videotaping you. The camera wasn&#39;t really turned on. This was another part of the acting to make it more stressful.&#34; 
	 
	&#34;The people in there weren&#39;t really writing notes about your performance. They were just writing nonsense notes as part of their acting. 
	 
	&#34;We would like to ask that you please not mention this activity to anyone else who might be in the study, even if they&#39;re your best friend, brother, sister, cousin, or whatever. If other people learn about this activity before they come in for the visit, they may practice their speech at home or get nervous before they even get here, and they will have a totally different experience than you just did. We don&#39;t want that to happen. Does this make sense? For the same reason, if you talk with your parents about it, please ask them not to tell friends or family in the study.&#34; 
	 
	&#34;Do you have any questions?&#34;</li>
</ol>
11. Saliva Sample #3 (30 min Post-stressor Onset)
<ol>
	<li>Place a line to indicate 1 mL of saliva on a 2 mL cryovial, and tell participant to fill the cryovial to this line. Collect participant&#39;s saliva via passive drool through a two-inch straw into the 2 mL cryovial36 and freeze immediately (-80 &#176;C).</li>
	<li>Set timer for 15 min.</li>
	<li>Offer water to drink after the participant has completed the saliva sample. Do not allow the participant to keep the water; take it back when he/she is finished.</li>
</ol>
12. Saliva Sample #4 (45 min Post-stressor Onset)
<ol>
	<li>Place a line to indicate 1 mL of saliva on a 2 mL cryovial, and tell participant to fill the cryovial to this line. Collect participant&#39;s saliva via passive drool through a two-inch straw into the 2 mL cryovial36 and freeze immediately (-80 &#176;C).</li>
	<li>Offer water to drink after the participant completed the saliva sample. Do not allow the participant to keep the water; take it back when he/she is finished.</li>
</ol>

There are no competing financial interests.

The current study sought to confirm the efficacy of a modified version of the TSST with vulnerable youth. The CHAMACOS project presented the optimal sample to test the effectiveness of a modified contextually appropriate TSST, as it is both unique (Mexican American samples are often excluded from research) and represents a sample of youth who have experienced significant early life adversity37. Results confirm the success of the modified TSST in provoking a significant increase in cortisol from pre-stress to post-stress samples, as well as a normal relative recovery from the TSST by the time the last saliva sample was collected. This study provides a valuable tool to the field through both the contextually appropriate protocol of the TSST as well as an exploration of the factors that influenced the modifications of the original TSST.
There are several critical steps for the set up and administration of the TSST that must be attended to. First, both confederates need to wear white lab coats, sit behind a table, and have a video camera recording and pointed towards where the participant will be standing before the participant is ushered into the room. The participant stands throughout the entire TSST, while the committee sits. Second, there needs to be 1 confederate of each sex, and the confederates need to be of similar race/ethnicity to the participant, and appearing of similar age as the participant (this study used slightly older youth who looked similar in age to the participants). Third, confederates must demonstrate flat affect, with slight boredom throughout the protocol. Confederates are to look at the participant as much as possible without showing interest during the TSST. Confederate 2 writes notes, whereas Confederate 1 is responsible for administering prompts and instructions.
It should be noted that conducting the TSST in an emotionally &#34;neutral&#34; state takes skill and practice. Some individuals may not be appropriate for the confederate role if they are not able to maintain a neutral face. Confederates in this study were hand-selected for being able to maintain the neutral stance during the entire TSST. However, should a participant become tearful, sad, or aggressive, these personnel need to know how and when to intervene to stop the protocol. The integrity of the TSST also needs to be assessed periodically throughout the study to ensure there is no drift in the emotional demeanor or script. In this study, weekly check-ins were provided to confederates when they were able to troubleshoot any unusual occurrences with experts in TSST administration and check in with questions. TSST &#34;refreshers&#34; were also provided every few months to retrain confederates on the TSST. Videos were recorded during the TSST for participants whose mothers provided consent, and these videos were reviewed to ensure veracity to the TSST script. Finally, some researchers may think that participants who expressed betrayal or anger in response to the TSST will have high cortisol responses. However, this study ruled out this notion in the data, as it turned out that these youths were most likely to be non-responders (i.e., individuals whose cortisol either decreased or remained stable across the task) as assessed via their cortisol reactions. Thus, strictly adhering to a sensitive protocol did not undermine the overall reactivity rates, but rather ensured that the stressor&#39;s challenge was titrated to the appropriate level for these youth. The standard TSST protocol would thus have been inappropriate both ethically but also scientifically as attrition, nonresponse, and &#34;giving up&#34; was a predominant response.
After running a few participants through the original TSST, the original TSST was modified to better match the contextual factors of this population due to non-optimal responses (i.e., crying, disengaging from the task, aggressive posturing). Study personnel consulted with numerous researchers who have used the TSST with vulnerable populations (e.g., orphanages, juvenile justice settings, abused populations) but none of the modifications fit this study&#39;s needs perfectly - some veered too far from the protocol and we were concerned that these changes would not elicit a cortisol response12,27. Thus, three primary modifications were applied to the TSST in our study, and the modifications and motivations for these modifications are illuminated below.
First, it is important not to alienate participants. Administration of a stress task was important to this study, yet it was imperative that participants were not lost to attrition, as the youth had been followed longitudinally since their mothers became pregnant with them. In this study, participants were debriefed immediately after the TSST in order to ensure that the stress ceased for participants after the conclusion of the TSST. For this sample, the staff administering the debriefing needed to be someone as unfamiliar as possible but still empathic, without an emotional connection to the youth. While that may seem counterintuitive, when staff who were familiar and emotionally connected to the youth debriefed them on the TSST, the youth tended to have more proclivity towards rumination over the TSST and talking more about it, which sometimes ended with crying or feelings of resentment towards being &#34;tricked.&#34; Unfamiliar staff administering the debriefing helped ensure that we did not sever the longitudinal connections that the cohort had made with the familiar staff by implementing the TSST. This is noted as important within the Latino community but is something that many longitudinal studies that develop a strong rapport with participants should consider. Using unfamiliar staff also reduced the likelihood that the debrief served to further stress the youth, given the more optimal behavioral responses we observed using unfamiliar staff. While debriefing before the collection of two more saliva samples may concern researchers using this modification, it should be noted that this study observed the prototypical cortisol response pattern after the conclusion of the TSST (with the highest concentration coming 15 min after the conclusion of the stressor, and recovery 30 min after the conclusion of the stressor). This modification was made to be more culturally sensitive to the youth whom may have continued to have a stress response after the TSST due to their vulnerable state.
Second, the TSST protocol needs to be flexible to the individual factors of the participant. The Mexican American youth in this study reside in low-resourced and impoverished agricultural communities in the Salinas Valley. CHAMACOS families labor under difficult conditions, specifically low-wage, intermittent seasonal employment and long working hours that circumvent opportunities for positive familial interactions during a time of rapid and salient adolescent development. Stressors associated to low SES/poverty in the cohort contribute to food insecurity, overcrowded housing, substandard living conditions, and inadequate medical services to address cognitive disabilities and mental health needs. Compounding this is that youth in the Salinas Valley are disproportionately exposed to family turmoil and youth violence, which spill into local schools and neighborhoods alike. These risk factors become increasingly accentuated as youth either learn to adapt to rapidly changing social and economic challenges or gradually become sensitized (or in some cases desensitized) to the unpredictable nature of their daily lives. One example of making the TSST more flexible for this population was to vary the difficulty of the math section. Given that this study was privy to previous cognitive testing and had knowledge of environmental pathogen exposure, which may impact IQ38, it was clear that the &#34;one size fits all&#34; math task typically associated with the TSST was not going to accommodate this population. Following other stressors39,40, math difficulty was therefore titrated to be challenging but not impossible for the youth.
Third, the TSST protocol needs to recognize and build upon the high sense of community and social connection within certain cultural settings. It is important to use confederates that youth participants connect with, so this study recruited confederates that were slightly older than the participants so as to appear of similar age and ethnicity and came from the same community so SET came from &#34;their peers&#34;. Conversely, too much social connection was problematic. It is important to ensure that confederates are appropriately neutral and that confederates were unknown to the participants. This was critical for the current study because although the Salinas Valley is a relatively large region, the youth community can be very small and interconnected. Thus, it can be useful to ensure before the TSST commences that the participants and confederates do not know each other. If the participant or confederate recognized the other in this study, the TSST was run with just one confederate. The fact that this study&#39;s confederates came from the same community is important to note in this context (Salinas), which is far from a university setting and difficult when finding staff who can work an hour or two at a time. This study&#39;s field office researchers were very savvy and highly trained regarding research, which should not be taken for granted in terms of maintaining protocol.
While these results are promising for validating the use of a modified TSST with vulnerable adolescents, more research is needed to confirm the replicability of these effects across different samples. First, while this project was fortunate to be able to access such a unique and vulnerable sample of Mexican American adolescents, these results need to be replicated in other vulnerable ethnicities in order to confirm that this modified TSST is potent enough to elicit a cortisol response in other populations. Second, this sample was limited to 14-year-olds. While the narrow sampling controlled for the effect of age on TSST response in this sample, future research could attempt to replicate these effects in different adolescent and child ages, and perhaps across pubertal development. Finally, it should be noted that data was not collected from a comparison group utilizing a non-modified TSST, as it would be unethical with this sample given the high rate of poor behavioral and emotional responses we were experiencing in the beginning with a less modified version of the TSST.
There are also limitations of the TSST technique in general. As mentioned earlier, it takes significant practice for confederates to maintain neutral stance throughout the entire TSST. At least 3 people are needed to run the TSST appropriately (2 confederates and 1 experimenter), and the TSST runs better if these people are unknown to the participant. Thus, this protocol is easier to run in college campus settings where undergraduates can work in the lab 1-2 h at a time, but less easy in remote settings or in settings where confederates are being paid as a job (as they are only paid for ~1 h of work per TSST). This technique could also be a hurdle for longitudinal studies with personnel that work on the project over time &#8211; these well-known personnel should not be the ones administering the TSST. Also, there is significant overhead in terms of maintaining the integrity of the TSST over time &#8211; experts are needed to review the video tapes of the TSST, maintain weekly check ins with the confederates, and provide periodical &#34;refresher&#34; sessions to ensure there is no drift in the technique. Further, the TSST is difficult to do repeatedly for a given participant, as it relies on minor deception and participants may habituate to the technique. The speech and math portions are not counterbalanced, which would be more optimal for other methodologies such as autonomic nervous system functioning. There are no parts to the TSST that are non-stressful, so it requires another session (i.e., friendly TSST or a placebo TSST) or something else (i.e., a basal day) to have a non-stress comparison. Finally, a non-lab-based stressor might be more effective (i.e., karaoke, roller coasters, or haunted houses) or more ecological41.
This study provides the foundation for future research to utilize a modified TSST with vulnerable youth. Future longitudinal studies seeking to retain participants across multiple waves of data collection may also need to modify their approach utilizing some of these modifications so as to limit attrition. Further, this study shows that it is indeed possible to create a modified version of the TSST that is culturally sensitive while still titrating stress reactivity to an appropriate level to capture individual differences in cortisol responsivity.

The Trier Social Stress Test (TSST)1 has become the gold standard for inducing stress in laboratory settings2. It is a well-validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis1,3. A meta-analysis concluded that two key aspects are integral to inducing the most robust HPA reactivity across the lifespan: unpredictability/uncontrollability and social evaluative threat (SET), both of which characterize the TSST3.
Adolescence may be a key period of time when SET is especially stressful, marked by a sensitive period of development, whereby social interactions facilitate learning4. For example, during early puberty and adolescence, integration with peer groups becomes motivationally salient5. Rapid increases in hormone levels at the start of puberty influence development of limbic circuits, which leads to increases in the processing of status-relevant social stimuli (i.e., admiration) as having increased motivational salience6. This increase in social affective engagement not only influences motivations and behaviors in the moment but also influences patterns of behavior and motivational learning over long intervals. Over time, this change in prioritizing social incentives can lead to health exploration and risk taking, which can promote social and affective learning and skills that underpin adult social functioning. Thus, it is no surprise that aspects of social functioning involved in the TSST speech phase are a potent form of stress for adolescents, i.e., the &#34;good friend&#34; topic7,8,9. However, individuals sensitized by a history of trauma or stressful life events may be primed to react poorly to socially evaluative or unpredictable situations due to poor emotional control or augmented sensitivity to stress, and this pattern of negative response could contribute to negative healthy outcomes, such as drug use or dangerous thrill seeking. Thus, understanding biological responses to SET is of utmost importance in vulnerable youth.
A handful of studies have explored how variations of SET and uncontrollability influence stress responsivity. SET can be increased or decreased based on the number of confederate judges. The TSST calls for 2 confederates, but it is more effective with four10, and less effective with 1 - 2 confederates11,12,13 or with a virtual or distant audience14,15,16. Adding more negative commentary increases SET and responder rates11,17, whereas positive commentary reduces SET and responder rates18. Rates of responsivity are influenced by changing uncontrollability by (a) modifying the length of time participants have to prepare the speech14 (b) increasing participants&#39; psychosocial resources19,20,21, or (c) increasing participants&#39; self-confidence prior to the TSST22. Modifications that enhance emotion suppression23, vigilance or attention to the stressor24,25, or thoughts about the self26 all increase TSST responsivity. Finally, the sex and age of confederate can influence stress reactivity to SET27,28. These studies illustrate that responsivity rates can be experimentally increased or decreased. In the current study, the TSST was modified by including timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief.
A recent review of stressor paradigms demonstrated particularly robust effects of social evaluative threat on cortisol elevation (HPA response) during middle adolescence29. However, there are valid concerns about using the TSST with vulnerable youth. Specifically, human subjects&#39; protection concerns may be raised to ensure that the benefits outweigh the risks, as there is the potential trauma associated with enduring the TSST when youth are already vulnerable (e.g., coming from environments of poverty and early life adversity). Thus, there is the need to ensure that the youth do not leave in a state of elevated anxiety or agitation. However, modifications to the TSST to alleviate/mitigate some of these concerns must not reduce the overall efficacy or validity of the stressor. For example, prior studies have documented the effects of modified versions of the TSST for vulnerable youth but have had limited success in terms of provoking a rise in cortisol30,31. Further, modifications may need to be made to make the TSST more culturally sensitive and appropriate. For example, there is literature that suggests interacting with individuals of different races can induce stress and threat reactions32. Differing the racial makeup of the participant and TSST committee has been shown not to impact cortisol reactivity to the TSST33, but rather to impact cortisol recovery from the TSST34. Thus, using same-race/ethnic confederates for the TSST may eliminate any unnecessary confound of the confederates&#39; race on the perceived stereotype threat and cortisol recovery from the TSST.
The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). This sample is characterized as highly vulnerable due to high rates of poverty (76% of the families were at or below the Federal Poverty level) and high numbers of adverse early life events (mean number of lifetime events = 4.5 for the sample, SD = 3.3). In comparison, a large retrospective study conducted by a partnership between the U.S. Centers for Disease Control and Prevention and Kaiser Permanente based on thousands or respondents found only 13% to have reported 4 or more adverse childhood events35.
The TSST was administered to the youth in this study as part of a large NIH grant aimed at researching the link between adverse early life events, biological reactivity to social evaluative threat at age 14 (when the TSST was administered), and risk-taking behaviors (at age 16; data collection is still ongoing; grant1R01DA035300). During the early stages of piloting the TSST, we observed that the approach needed to be shifted to become contextually sensitive to the unique needs of this population and to mitigate poor responses (i.e., crying, becoming angry, disengaging from the TSST). To be precise, we had a 20% rate of poor behavioral responses to the TSST prior to modifications (i.e., 4 of 20 TSST participants either cried or responded with anger), whereas only 5% responded with poor responses after the modifications were implemented. Study personnel were also keenly aware of the importance of retaining youth in this longitudinal study. It should be noted that during the pilot phase, individuals who responded to the TSST with extreme stress reactions behaviorally during the debrief were, at the biological level, &#34;non-responders,&#34; i.e., they did not demonstrate a cortisol rise. This emotional/biological juxtaposition from these initial results also highlighted the need to modify the TSST to create the optimal conditions to induce an appropriate biological response to the TSST. Thus, this study provides preliminary evidence from a subsample of youth of the efficacy of the modified TSST to induce cortisol reactivity.

<table border="0" cellpadding="0" cellspacing="0" width="651">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">TSST</td>
			<td style="width:128px;"></td>
			<td style="width:128px;"></td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 white lab coats</td>
			<td>Dickies</td>
			<td>83403</td>
			<td>For confederates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 clip boards</td>
			<td>Staples</td>
			<td>21423</td>
			<td>For confederates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 pens</td>
			<td>BIC</td>
			<td>GSM609-Blk</td>
			<td>For confederates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table</td>
			<td>Ikea</td>
			<td>B00EYU3E7I</td>
			<td>For confederates to sit behind</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Video Camera &#38; Tripod</td>
			<td>Sony</td>
			<td>K-86674-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paper&#160;</td>
			<td>Generic</td>
			<td></td>
			<td>For participant&#39;s preparation period</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saliva samples</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Latex gloves</td>
			<td>Fisher Scientific</td>
			<td>19041171C</td>
			<td>To collect saliva samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 mL cryovials</td>
			<td>Wheaton</td>
			<td>W985872-100</td>
			<td>4 per participant</td>
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		<tr height="21">
			<td height="21" style="height:21px;">80 C freezer</td>
			<td>So-Low</td>
			<td>U85-13</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">2&#39;&#39; straws&#160;</td>
			<td>Genuine Joe</td>
			<td>GJO58925</td>
			<td></td>
		</tr>
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a modified trier social stress test for vulnerable mexican american adolescents

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis in humans. While studies have explored how modifications to the TSST influence stress responsivity, few studies have created a modified TSST appropriate for vulnerable youth that elicits a significant cortisol stress response. Thus, the current study sought to modify or adjust the TSST in a culturally sensitive way for a vulnerable sample of 14 year-old adolescents. The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). The CHAMACOS sample was optimal to test the effectiveness of a modified culturally appropriate TSST, as it is comprised of Mexican American youth, who are often excluded from research. These youths also have experienced significant early life adversity. Example modifications included timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief. Saliva samples were collected at baseline (after a resting phase), and then again at 15, 30, and 45 min post-TSST onset to assess cortisol concentration. A pilot study of 50 participants (50% female) have been analyzed for cortisol reaction to the TSST. Results confirmed that this modified version of the TSST was successful at eliciting a significant cortisol reaction, with a wide range of variability likely due to individual differences. Goals for modifications and ethnicity considerations are discussed. This study provides the foundation for future research to utilize a modified TSST with vulnerable youth.

The sample consisted of 14 year-olds who have been a part of a richly characterized, established cohort study, the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). This study consists of the first cohort of 325 Mexican American families living in a farmworker community in California and followed prospectively since they were in utero. The TSST was not administered to the second CHAMACOS cohort of 300 children followed since age 9 years-old. Adolescents were screened from participating in the TSST if their full scale IQ was &#60;70 from a prior visit (age 12), if the adolescent was ever diagnosed with autism per maternal report, if extreme atypical behaviors shown in clinic at past visits (i.e. pronounced distrust/paranoia about study, seeing things or people who are not there - per maternal report), if their depression screener score was &#8805;3 SDs above the mean for age and sex, and if the adolescent reported involvement in a gang fight in the previous year. These exclusion criteria were added based on over-responding initially to the TSST, but it is unclear whether these exclusion criteria made a difference in the final results. Other than these exclusion criteria, participants were enrolled no matter their psychological and physical health. In the end, 264 adolescents completed the TSST in full with either no reported problems or no problems until debrief. Table 1 lists the number of participants who were excluded and why they were excluded. There were no demographic differences between those who completed versus did not complete the TSST.
Saliva samples were stored at -80 &#176;C until they were shipped on dry ice to colleagues at Iowa State University (i.e., biosafety level II lab). Upon arrival, frozen saliva samples were cataloged and placed again in a -80 &#176;C freezer. On the day of assay, each saliva sample was thawed and assayed within 24 h in duplicate using well-established highly sensitive enzyme immunoassay kits. Mean intra-assay coefficients of variation (CVs) were less than 2.25% and mean inter-assay CV was 5.13% for cortisol. The sample was reanalyzed if the CV for the duplicate measurements were greater than 7%.
Descriptive statistics for the cortisol measurements along with standard deviations across each sample are provided in Table 2 and depicted in Figure 1. A 2 x 4 repeated measures ANOVA was run to test the effects of sex on the four saliva samples. There was a main effect for cortisol response to the TSST (F = 9.09, p &#60;0.001), but sex was not a significant predictor of cortisol response to stress (F = 1.03, p = .39). Average body mass index was 23.53, and was also not a significant predictor of the repeated measures cortisol ANOVA (F = 1.03, p = 0.38). As would be expected, paired samples t-tests (provided in Table 3) revealed significant changes in cortisol from pre-stress to 15 min post-stressor onset, from 15 min post-stressor onset to 30 min post-stressor onset, and from 30 min post-stressor to 45 min post-stressor. The greatest change in cortisol occurred when comparing the pre-stress cortisol concentration to the 30 min post-stressor concentration, consistent with prior research (Cohen&#39;s d = .80)1,3. Individuals who were marked as having a difficult time with the TSST tended to report heightened stress reactivity to the stressor but reported coming back to a normal state prior to the end of the study. While the 30 min post-stressor concentration was the highest concentration from these samples, the 45 min post-stressor cortisol concentration suggests that individuals began to recover from the stressor, as the 45 min post-stressor concentration was significantly lower than the 30 min concentration (see Table 3).
<img alt="Figure 1" src="/files/ftp_upload/55393/55393fig1.jpg" /> 
Figure 1: Cortisol Reactivity to the TSST. Cortisol reactivity means plotted with standard deviations. <a href="http://cloudflare.jove.com/files/ftp_upload/55393/55393fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/55393/55393table1.jpg" /> 
Table 1: Reasons for Exclusion. Participants were excluded from the study if they started the TSST but stopped early, refused to participate, were screened out of the project, or were skipped due to project related issues.
<img alt="Table 2" src="/files/ftp_upload/55393/55393table2.jpg" /> 
Table 2: Mean Cortisol Concentrations and Standard Deviations. Cortisol increased from samples 1-3, and then declined by sample 4.
<img alt="Table 3" src="/files/ftp_upload/55393/55393table3.jpg" /> 
Table 3: Paired Samples t-tests. Compares samples to one another in cortisol concentration (&#181;g/dL) and standard deviations (SD) for cortisol change. Significant increases in cortisol were observed from the baseline sample to post-stress samples.

Watch this Scientific Journal Video about A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents at JoVE.com

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

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12.7K Views.  University of California, Berkeley. Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

Video: A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

Automated Quantification of Hematopoietic Cell &amp;#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.