A Large Naturalistic Study on the BRACHA: Confirmation of the Predictive Validity.

Aggression is a major challenge on child/adolescent inpatient psychiatric units. A screening instrument to accurately identify risk is urgently needed. To determine the predictive validity of the Brief Rating of Aggression by Children and Adolescents (BRACHA). Prospective cohort study. BRACHA is administered by clinical staff in the emergency department (ED) prior to inpatient psychiatric admission. A consecutive sample of 10,054 admitted patients from 2010-2021. No patients refused screening nor were excluded. BRACHA administered to patients in the ED prior to admission at Cincinnati Children's Hospital Medical Center (CCHMC). Patient behavioral outcomes measured by Overt Aggression Scale (OAS), categorizing aggression as verbal or physical, then as towards self, others, or objects. Female patients comprised 53.6% (n = 5,386) of the sample. Most patients were white (n = 6,556, 65.2%). Patients ranged in age from 4 to 18 years, with a mean age of 13.6 ± 3.1 years. A single biological parent (n = 5,317, 52.9%) was the predominant living arrangement among patients. The Area Under the Curve (AUC), as an assessment of predictive validity across all possible cut-offs of BRACHA scores ranged from 0.640 (aggression to self) to 0.758 (physical aggression towards others). Our findings support the BRACHA as a useful predictive instrument for aggression in inpatient psychiatric admissions from ED regardless of length of stay. Treating staff are then able to immediately classify risk level and inform care plans for all lengths of hospitalization. Applies to potential risk for aggression, except for self-aggression. Future data analyses will evaluate demographic factors to determine which improve predictive power of the BRACHA and can be used to create a BRACHA calculator. To our knowledge, this naturalistic outcomes study is one of the largest in psychiatry. The BRACHA will continue to be studied to evaluate risk for aggression on inpatient units and aim to assist in keeping unit staff and patients safe.

Systematic Review of Religiosity's Relationship with Suicidality, Suicide Related Stigma, and Formal Mental Health Service Utilization among Black Americans.

Rates of suicide have increased among Black Americans. Suicide is now the 3rd leading cause of death for Black Americans between the ages of 1-19 and the 4th leading cause of death for Black Americans aged 20-44. Due to the increasing need in the community, a marked increase in literature focusing on suicide in the Black community has been published since 2018. To build a better understanding of the current state of the literature on suicidality among Black Americans and to offer suggestions for further areas of research, a systematic review was conducted. Spirituality and religious beliefs are often an important cultural focus in the Black community. Some religious beliefs pose potential unintended regarding the sanctity of life among Black Americans. The focus of this systematic review was religiosity's effect on suicidality among Black Americans. Religiosity was found to have a protective effect against suicidality among Black Americans while discouraging formal mental health services utilization. This systematic review also reveals a dearth of research on the relationship between religiosity and suicide related stigma. Areas for further research are mentioned, and religious institutions as mental health intervention centers are encouraged.

Predicting the time trend of first episodes of aggressive behaviors in pediatric psychiatric inpatient units.

The goal of the current study is to assess whether the scores of Brief Rating of Aggression by Children and Adolescents (BRACHA) at the emergency room (ER) can predict the aggressive incidents at pediatric psychiatric inpatient units. The study aims to identify predictors for two outcome measurements: 1) hazard rates for the first aggressive incident and 2) numbers of days between admission and the first aggressive incident, using the Cox regression model and Poisson regression model, respectively. The clinical records of a total of 5,610 adolescents admitted into the pediatric psychiatric inpatient units of Cincinnati Children's Hospital Medical Center were extracted for the analysis. The aggressive incident was defined as a score >0 from any category of the Overt Aggression Scale (OAS) and the high-aggressive incident was defined as a score ≥ 2 from any category of the OAS. The results indicate that the BRACHA score was not associated with high-aggressive incidents (hazard ratio: 0.98, p = 0.7543). Similarly, the BRACHA scores was only associated with the number of days from admission to the first aggressive incident (Poisson regression coefficient: 0.24, p < 0.0001) but not the number of days from admission to the first high-aggressive incident (Poisson regression coefficient: 0.03, p = 0.3994). Furthermore, the second peak of first aggressive incidents during the hospitalization highlights the importance of interventions at the end of the inpatient treatment course. To summarize, BRACHA scores based on initial assessments at the ER could correlate with first aggressive incidents, but not the first high-aggressive incidents.

Lessons Learned to Building Stakeholder Engagement during the Initial Stages of Pragmatic Research Development and Implementation.

Research does not occur in a vacuum. Effective stakeholder engagement occurs on several levels, including outside influence and cooperation inside the institution. Little guidance around designing and implementing pragmatic mental health research exists. The following paper outlines lessons learned during the initial stages of research design and implementation for a project focused on mental health treatment outcomes.

Finding warning markers: Leveraging natural language processing and machine learning technologies to detect risk of school violence.

INTRODUCTION: School violence has a far-reaching effect, impacting the entire school population including staff, students and their families. Among youth attending the most violent schools, studies have reported higher dropout rates, poor school attendance, and poor scholastic achievement. It was noted that the largest crime-prevention results occurred when youth at elevated risk were given an individualized prevention program. However, much work is needed to establish an effective approach to identify at-risk subjects. OBJECTIVE: In our earlier research, we developed a risk assessment program to interview subjects, identify risk and protective factors, and evaluate risk for school violence. This study focused on developing natural language processing (NLP) and machine learning technologies to automate the risk assessment process. MATERIAL AND METHODS: We prospectively recruited 131 students with or without behavioral concerns from 89 schools between 05/01/2015 and 04/30/2018. The subjects were interviewed with two risk assessment scales and a questionnaire, and their risk of violence were determined by pediatric psychiatrists based on clinical judgment. Using NLP technologies, different types of linguistic features were extracted from the interview content. Machine learning classifiers were then applied to predict risk of school violence for individual subjects. A two-stage feature selection was implemented to identify violence-related predictors. The performance was validated on the psychiatrist-generated reference standard of risk levels, where positive predictive value (PPV), sensitivity (SEN), negative predictive value (NPV), specificity (SPEC) and area under the ROC curve (AUC) were assessed. RESULTS: Compared to subjects' sociodemographic information, use of linguistic features significantly improved classifiers' predictive performance (P < 0.01). The best-performing classifier with n-gram features achieved 86.5 %/86.5 %/85.7 %/85.7 %/94.0 % (PPV/SEN/NPV/SPEC/AUC) on the cross-validation set and 83.3 %/93.8 %/91.7 %/78.6 %/94.6 % (PPV/SEN/NPV/SPEC/AUC) on the test data. The feature selection process identified a set of predictors covering the discussion of subjects' thoughts, perspectives, behaviors, individual characteristics, peers and family dynamics, and protective factors. CONCLUSIONS: By analyzing the content from subject interviews, the NLP and machine learning algorithms showed good capacity for detecting risk of school violence. The feature selection uncovered multiple warning markers that could deliver useful clinical insights to assist personalizing intervention. Consequently, the developed approach offered the promise of an accurate and scalable computerized screening service for preventing school violence.

Linkage of Metabolic Defects to Activated PIK3CA Alleles in Endothelial Cells Derived from Lymphatic Malformation.

BACKGROUND: Lymphatic endothelial cells (LECs) derived from lymphatic malformations (LMs) bear activated PIK3CA alleles yet display an inflammatory gene expression profile. A basis for the inflammatory phenotype was sought by screening for coexisting somatic mutations. METHODS AND RESULTS: Fourteen independent LEC populations bearing activated PIK3CA alleles were isolated from LM. These were characterized by the expression of growth and inflammatory genes (VEGFC, IL-6, COX-2, IL-8, HO-1, E-SEL) by qRT-PCR. Most commonly upregulated gene products were VEGFC, COX2, HO-1, and ANGPTL4. The specific inhibition of PI3K reduced VEGFC expression without resolving inflammation. Whole exome sequencing of six LM-LEC populations identified five novel somatically acquired alleles coexisting with activated PIK3CA alleles. Two affected genes regulate lipid droplet metabolism (FITM2 and ATG2A), two are gene regulators (MTA1 and TAF1L), and the fifth is an isoform of ANK3 (an endosomal/lysosomal protein). Inhibition of AMPK implicated its involvement in regulating COX-2 and HO-1 overexpression. ANGPTL4 expression was independent of AMPK and PI3K activity and reflected lipid stress demonstrated in normal LECs. AMPK activation with AICAR had a selective growth-limiting effect in a subset of LM-LEC isolates. CONCLUSIONS: Inflammatory stress displayed by LM-LECs is consistent with errors in lipid metabolism that may be linked to acquired mutations. The acquisition of PIK3CA alleles may be a permissive event that antagonizes inflammation and metabolic defect.

Laryngotracheal Cleft Repair in Children With Complex Airway Anomalies.

IMPORTANCE: This study provides clinicians with relevant information regarding the surgical outcomes of patients with laryngotracheal cleft in the context of additional airway anomalies. OBJECTIVES: To determine the rates of surgical success in patients who underwent laryngotracheal cleft repair in the context of additional airway anomalies, determine the revision rate for cleft repair, characterize the time to revision among patients who underwent cleft repair, and assess the functional swallowing outcomes after cleft repair. DESIGN, SETTING, AND PARTICIPANTS: A retrospective study was conducted at a quaternary pediatric center of 81 children diagnosed as having laryngotracheal cleft with or without concomitant airway anomalies who underwent laryngotracheal cleft repair between February 1, 2000, and February 28, 2013. Analysis was conducted from October 1, 2012, through March 30, 2013. INTERVENTIONS: Surgical repair of laryngotracheal cleft. MAIN OUTCOMES AND MEASURES: Surgery-specific success, overall surgical success, and revision rate. We defined surgery-specific success as a repair not requiring revision and overall surgical success as absence of a cleft or TEF at the last operative examination, regardless of the number of revisions required. RESULTS: Of 81 patients with laryngotracheal cleft who underwent surgical repair, 48 (59%) had at least 1 additional airway finding; 24 (30%) had tracheomalacia and 21 (26%) had subglottic stenosis. Seventeen patients required a revision of laryngotracheal cleft repair, with a median time to revision of 8.3 months (interquartile range, 4.3-25.1 months). Six patients required a second revision, with a median interval between revisions of 44.2 months (interquartile range, 28.6-53.6 months). The surgery-specific success rate was 77% (37 of 48) in patients with additional airway anomalies and 82% (27 of 33) in those with laryngotracheal clefts alone. The overall surgical success rate was 92% (44 of 48) in patients with additional airway anomalies and 97% (32 of 33) in those with clefts alone. Fourteen (17%) patients demonstrated swallowing dysfunction postoperatively despite closure of the cleft. CONCLUSIONS AND RELEVANCE: Although additional airway findings were common in our cohort of patients with laryngotracheal clefts, these anomalies did not affect surgery-specific or overall surgical success. The overall surgical success rate for those with and without additional airway anomalies was 92% and 97%, respectively. In view of the fact that cleft breakdown after surgical repair is not uncommon and may occur years after the initial repair, we strongly advocate long-term follow-up. Despite successful closure, a significant percentage of children with laryngotracheal cleft will have persistent swallowing dysfunction.

Activating PIK3CA alleles and lymphangiogenic phenotype of lymphatic endothelial cells isolated from lymphatic malformations.

Lymphatic malformations (LMs) are developmental anomalies of the lymphatic system associated with the dysmorphogenesis of vascular channels lined by lymphatic endothelial cells (LECs). Seeking to identify intrinsic defects in affected LECs, cells were isolated from malformation tissue or fluid on the basis of CD31 and podoplanin (PDPN) expression. LECs from five unrelated LM lesions were characterized, including cells derived from one patient previously diagnosed with CLOVES. CLOVES-related LECs carried a known, activating mutation in PIK3CA (p.H1047L), confirmed by direct sequencing. Activating PIK3CA mutations (p.E542K and p.E545A) were identified in lesion-derived cells from the other four patients, also by direct sequencing. The five LM-LEC cultures shared a lymphangiogenic phenotype distinguished by PI3K/AKT activation, enhanced sprouting efficiency, elevated VEGF-C expression and COX2 expression, shorter doubling times and reduced expression of angiopoietin 2 and CXCR4. Nine additional LM-LEC populations and 12 of 15 archived LM tissue samples were shown to bear common PIK3CA variants by allele-specific PCR. The activation of a central growth/survival pathway (PI3K/AKT) represents a feasible target for the non-invasive treatment of LMs bearing in mind that background genetics may individualize lesions and influence treatments.

Swallowing function after laryngeal cleft repair: more than just fixing the cleft.

OBJECTIVES/HYPOTHESIS: To evaluate and describe the swallowing function in children after laryngeal cleft repair. STUDY DESIGN: Ten-year (2002-2012) retrospective chart review. SETTING: Academic tertiary care pediatric otolaryngology practice. METHODS: Records of 60 children who had surgical repair of laryngeal cleft (ages 2 weeks-14 years) and postoperative functional endoscopic evaluation of swallowing or videofluoroscopic swallow studies were examined retrospectively. RESULTS: Twenty-nine children had one postoperative swallow evaluation, 19 children had two, 4 children had three, 5 children had four, and 3 children had five. Median time to the first evaluation was 10.8 weeks (interquartile range [IQR]: 36.5, 231). On the final swallow evaluation, 34 (57%) children demonstrated normal swallowing parameters, 12 (20%) children showed penetration, and 14 (23%) children showed aspiration. Forty-three (72%) children were able to take everything by mouth normally or with minor behavioral modifications, 11 (18%) children required thickened fluids, and six (10%) children were kept nil per os (NPO). Mean improvement on the penetration-aspiration (pen-asp) scale was 2.13. On multivariable analysis, neurodevelopmental issues and gastronomy tube use were associated with the need for NPO status. CONCLUSION: Despite a high rate of surgical success, a substantial minority of children have persistent swallowing dysfunction after laryngeal cleft repair. Swallowing dysfunction after repair is multifactorial and arises from concomitant neurologic, anatomic, or other comorbidities that contribute to oropharyngeal and pharyngeal dysphagia. Based on our results, we recommend a testing schedule for postoperative swallowing evaluations after cleft repair.

Tracheocutaneous fistula closure in the pediatric population: should secondary closure be the standard of care?

OBJECTIVE: Tracheocutaneous fistula (TCF) closure is achieved by excision followed either by primary closure or healing by secondary intention. Although primary closure provides immediate resolution of the fistula, it is associated with more severe potential complications. Healing by secondary intention minimizes these potential complications; however, it is inconvenient for the patient and may be more likely to require revision surgery. We have had 2 life-threatening complications after primary closure, and as a result, we largely changed our practice pattern. We compared complication and success rates of the 2 methods since this change to determine its ramifications. STUDY DESIGN: Historical cohort study. SETTING: Academic tertiary care pediatric otolaryngology practice. Subjects and Methods Two hundred sixteen patients who underwent TCF closure between January 2004 and August 2012. RESULTS: Forty-six (21.3%) fistulae were addressed by primary closure, and 170 (78.7%) were addressed by secondary intention. The complication and revision rates were not significantly different between the 2 methods (8.7% vs 10% and 8.7% vs 14.7%, respectively). CONCLUSION: In our study, we did not see any statistical differences between the 2 methods studied but could not exclude clinically important differences that may have favored one method over the other. Although our comparative results were inconclusive, we have adopted secondary closure as standard practice for management of pediatric TCF. Individual surgeons and patients may use the data presented to help guide decisions concerning which procedure is most appropriate.

Multidisciplinary perinatal management of the compromised airway on placental support: lessons learned.

OBJECTIVE: The aims of this study were to review fetal and maternal outcomes after management of the compromised perinatal airway via operation on placental support or ex utero intrapartum treatment and to discuss implications for future management of these complex and rare cases. METHODS: We have presented a retrospective case series of 12 neonates requiring airway management on placental support at a single tertiary care, academic center. RESULTS: One mother experienced significant blood loss. Operative recovery times were unremarkable. Eight neonates required airway management due to mass obstruction, two for removal of an endotracheal balloon for fetoscopic treatment of congenital diaphragmatic hernia, one for laryngeal atresia, and one for severe retrognathia. One of our series is an unusual case of management on placental support after vaginal delivery. Another child would have ideally been managed on placental support, but an extremely short umbilical cord prevented this. Even though the airway was secured in all 12 cases, five neonates died in the perinatal period. CONCLUSIONS: These procedures have a risk for substantial maternal blood loss. Despite excellent rates of success securing the neonatal airway, children who require management on placental support still have high mortality. A formalized multidisciplinary approach at our institution has enhanced preparedness for these cases.

A simple mechanical device reduces subglottic injury in ventilated animals.

OBJECTIVES/HYPOTHESIS: To test whether a simple inexpensive device that dynamically minimizes endotracheal cuff pressure throughout the respiratory cycle reduces endotracheal cuff pressure-related subglottic injury. STUDY DESIGN: Hypoxic animal model with one control and one experimental group. METHODS: Twelve S. scrofa domesticus piglets (14-16 kg) were intubated with standard endotracheal tubes and maintained in a hypoxic state to accelerate airway injury. Animals in the control group (n = 6) were ventilated with a constant pressure of 20 cm H2O in the endotracheal tube cuff. Animals in the experimental group (n = 6) were ventilated using a custom-designed circuit that altered the pressure in the endotracheal tube cuff in synchrony with the ventilatory cycle. Larynges were harvested at the end of the experiment and examined histologically to determine the degree of airway injury induced by the endotracheal cuff. RESULTS: Animals in the experimental group suffered significantly less airway damage than those in the control group. The differences were seen primarily in the subglottis (aggregate damage score 6.5 vs. 12, P <0.05), where the experimental endotracheal tube cuff exerted the least pressure. There was no difference in damage to the glottic or supraglottic structures. CONCLUSIONS: A simple, reliable, and inexpensive means of modulating endotracheal tube cuff pressure with the ventilatory cycle led to a substantial decrease in airway injury in our animal model. Such reduction in cuff pressure may prove important for humans, particularly those in intensive care units who tend to have underlying conditions predisposing them to tracheal damage from the endotracheal tube cuff.

Clinical indications for canal wall-down mastoidectomy in a pediatric population.

OBJECTIVE: To establish clinically derived indications for performing canal wall-up or canal wall-down surgery when treating children with cholesteatoma. STUDY DESIGN: Case series with chart review. SETTING: Tertiary care academic pediatric otolaryngology practice. SUBJECTS AND METHODS: Retrospective review of 420 children who underwent 700 procedures for cholesteatoma between 1996 and 2010. RESULTS: The canal wall was preserved in 89.5% of cases. Common reasons for removing the canal wall were to provide access to the disease, extensive erosion of key structures, and the desire to avoid further surgery. The mean pure-tone average (PTA) for the canal wall-up group was 30 dB, whereas the canal wall-down group had a mean PTA of 45 dB. A matched-pairs analysis demonstrated that the better performance of the canal wall-up group was independent of preoperative hearing levels. Furthermore, although the presence of the stapes did influence hearing results, the canal wall-up procedure yielded better results even when the condition of the stapes was taken into account. The number needed to treat with canal wall-up to prevent 1 case of hearing loss (ie, mean threshold >30 dB) would be around 6. The need for revision surgery was higher in the canal wall-up group (51%) compared with the canal wall-down group (21%). CONCLUSION: In the setting of adequate follow-up and open access to surgical resources, most children with cholesteatoma can be managed with an intact canal wall technique. The authors believe that the better audiometric outcomes and easier postoperative care outweigh the need for revision surgery in this group.

Decisions regarding intracranial complications from acute mastoiditis in children.

PURPOSE OF REVIEW: To review the clinical findings and treatment algorithms for intracranial complications of acute mastoiditis, such as sigmoid sinus thrombosis, otitic hydrocephalus, intracranial abscess, and otitic meningitis. We also briefly discuss the clinical sequelae of these complications. RECENT FINDINGS: Recent changes in the microbiology and treatment paradigms of otitis media have the potential to influence the rates of intracranial complications of mastoiditis; however, evidence supporting a resultant increase in the rates of these complications is lacking. SUMMARY: Antibiotic therapy and myringotomy with ventilation tube placement, with or without mastoidectomy, are the mainstays of treatment for intracranial complications of acute mastoiditis. Adjunct treatment, such as anticoagulation for sigmoid sinus thrombosis, is often used; however, the rarity of these complications makes establishing appropriate levels of evidence to support their use difficult.

Effect of endoscopic sinus surgery on pulmonary function and microbial pathogens in a pediatric population with cystic fibrosis.

OBJECTIVE: To assess whether improvements in pulmonary function and microbial pathogenic findings can be achieved by endoscopic sinus surgery in a pediatric population with cystic fibrosis. DESIGN: Retrospective medical record review. SETTING: Academic research. PATIENTS: Forty-one patients with cystic fibrosis who had undergone endoscopic sinus surgery at a single tertiary academic pediatric otolaryngology practice. MAIN OUTCOME MEASURES: Changes in pulmonary function test values or respiratory tract microbial pathogens after endoscopic sinus surgery were examined. RESULTS: Endoscopic sinus surgery did not improve pulmonary function test results in this population. Examination of respiratory tract microbial colonization showed that endoscopic sinus surgery did not affect microbial pathogens. The most common organisms isolated were Staphylococcus aureus and Pseudomonas aeruginosa. CONCLUSION: The lack of effect of endoscopic sinus surgery on pulmonary function test results and respiratory tract microbial pathogens in our study highlights the need for prospective assessments of postoperative quality-of-life improvement and of adjunct medical therapy efficacy.

Middle ear volume as an adjunct measure in congenital aural atresia.

OBJECTIVE: To examine middle ear volume in patients with aural atresia and investigate the role of middle ear volume as an adjunct measure in determining surgical candidacy. METHODS: We performed a retrospective review of children with aural atresia in a tertiary academic pediatric otolaryngology practice. High resolution multiplanar CT scans of the temporal bones were analyzed for middle ear volume and staged according to existing clinical grading scales. Atretic ears were compared to the nonatretic ears of the same patient as well as to ears of a control population. RESULTS: The average age of patients at the time CT was performed was 4.7 years (range <0.1-13.8 years). The average middle ear volume of the atretic ears was 0.34 cc compared to an average of 0.51 cc for the nonatretic ears. The mean ratio of the atretic to nonatretic volume was 0.67. In patients who underwent serial scans, no statistically significant difference in rates of growth existed between atretic and nonatretic ears. Finally, measures of middle ear volume correlated well with clinical grading scales. CONCLUSIONS: Both middle ear volume and the ratio of the atretic volume to nonatretic volume serve as useful adjunct measurements in determining surgical candidacy. The practitioner may be better able to assess surgical candidacy by supplementing classic atresia classification systems with middle ear volume measurements.

Deficient forward transduction and enhanced reverse transduction in the alpha tectorin C1509G human hearing loss mutation.

Most forms of hearing loss are associated with loss of cochlear outer hair cells (OHCs). OHCs require the tectorial membrane (TM) for stereociliary bundle stimulation (forward transduction) and active feedback (reverse transduction). Alpha tectorin is a protein constituent of the TM and the C1509G mutation in alpha tectorin in humans results in autosomal dominant hearing loss. We engineered and validated this mutation in mice and found that the TM was shortened in heterozygous Tecta(C1509G/+) mice, reaching only the first row of OHCs. Thus, deficient forward transduction renders OHCs within the second and third rows non-functional, producing partial hearing loss. Surprisingly, both Tecta(C1509G/+) and Tecta(C1509G/C1509G) mice were found to have increased reverse transduction as assessed by sound- and electrically-evoked otoacoustic emissions. We show that an increase in prestin, a protein necessary for electromotility, in all three rows of OHCs underlies this phenomenon. This mouse model demonstrates a human hearing loss mutation in which OHC function is altered through a non-cell-autonomous variation in prestin.

Acute endophthalmitis incidence: intravitreal triamcinolone.

OBJECTIVE: To report the incidence of acute postinjection endophthalmitis following intravitreal injection of triamcinolone acetonide (IVTA) as an office procedure. METHODS: Retrospective, noncomparative, consecutive, interventional case series of all patients who had received IVTA at 2 clinical centers between January 1, 2000, and January 30, 2004. RESULTS: A total of 1006 eyes received IVTA. None of the eyes developed acute, culture-positive, postoperative endophthalmitis in the 6 weeks following the procedure. One patient developed acute, culture-negative, postoperative endophthalmitis 4 days after receiving IVTA, resulting in an incidence of 0.10%. In this case, the presenting symptoms were decreased vision and acute conjunctival erythema. The case was notable for the absence of pain or hypopyon. CONCLUSION: Although acute postoperative endophthalmitis may follow IVTA, our experience suggests that this is a relatively uncommon event.

Spatiotemporal rescue of memory dysfunction in Drosophila.

We have developed a method for temporal and regional gene expression targeting (TARGET) in Drosophila and show the simultaneous spatial and temporal rescue of a memory defect. The transient expression of the rutabaga-encoded adenylyl cyclase in the mushroom bodies of the adult brain was necessary and sufficient to rescue the rutabaga memory deficit, which rules out a developmental brain defect in the etiology of this deficit and demonstrates an acute role for rutabaga in memory formation in these neurons. The TARGET system offers general utility in simultaneously addressing issues of when and where gene products are required.

Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53.

When DNA replication is stalled, a signal transduction pathway is activated that promotes the stability of stalled forks and resumption of DNA synthesis. In budding yeast, this pathway includes the kinases Mec1 and Rad53. Here we report that the Mediator protein Mrc1, which is required for normal DNA replication and for activation of Rad53, is present at replication forks. Mrc1 initially binds early-replicating sequences and moves along chromatin with the replication fork. Blocking initiation of DNA replication blocks Mrc1 loading onto origins, providing an explanation for why so many mutants in DNA replication show checkpoint defects. In the presence of replication blocks, we find that Mec1 is recruited to regions of stalled replication, where it encounters and presumably phosphorylates Mrc1. Mutation of the canonical Mec1 phosphorylation sites on Mrc1 prevents Mrc1 phosphorylation and blocks Rad53 activation, but does not alter Mrc1's role in DNA replication. Our results suggest a model whereby in response to DNA replication interference, the Mec1 kinase is recruited to sites of replication blocks and phosphorylates a component of the DNA replication complex, Mrc1, thereby setting up a solid-state Rad53 activation platform to initiate the checkpoint response.