Transcriptome variation in human tissues revealed by long-read sequencing.

Regulation of transcript structure generates transcript diversity and plays an important role in human disease1-7. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure8-16. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns.

Transcription factor bHLH121 regulates root cortical aerenchyma formation in maize.

Root anatomical phenotypes present a promising yet underexploited avenue to deliver major improvements in yield and climate resilience of crops by improving water and nutrient uptake. For instance, the formation of root cortical aerenchyma (RCA) significantly increases soil exploration and resource capture by reducing the metabolic costs of root tissue. A key bottleneck in studying such phenotypes has been the lack of robust high-throughput anatomical phenotyping platforms. We exploited a phenotyping approach based on laser ablation tomography, termed Anatomics, to quantify variation in RCA formation of 436 diverse maize lines in the field. Results revealed a significant and heritable variation for RCA formation. Genome-wide association studies identified a single-nucleotide polymorphism mapping to a root cortex-expressed gene-encoding transcription factor bHLH121. Functional studies identified that the bHLH121 Mu transposon mutant line and CRISPR/Cas9 loss-of-function mutant line showed reduced RCA formation, whereas an overexpression line exhibited significantly greater RCA formation when compared to the wild-type line. Characterization of these lines under suboptimal water and nitrogen availability in multiple soil environments revealed that bHLH121 is required for RCA formation developmentally as well as under studied abiotic stress. Overall functional validation of the bHLH121 gene's importance in RCA formation provides a functional marker to select varieties with improved soil exploration and thus yield under suboptimal conditions.

Neurodevelopmental and other phenotypes recurrently associated with heterozygous BAZ2B loss-of-function variants.

The bromodomain adjacent to zinc finger 2B (BAZ2B) gene encodes a chromatin remodeling protein that has been shown to perform a variety of regulatory functions. It has been proposed that loss of BAZ2B function is associated with neurodevelopmental phenotypes, and some recurrent structural birth defects and dysmorphic features have been documented among individuals carrying heterozygous loss-of-function BAZ2B variants. However, additional evidence is needed to confirm that these phenotypes are attributable to BAZ2B deficiency. Here, we report 10 unrelated individuals with heterozygous deletions, stop-gain, frameshift, missense, splice junction, indel, and start-loss variants affecting BAZ2B. These included a paternal intragenic deletion and a maternal frameshift variant that were inherited from mildly affected or asymptomatic parents. The analysis of molecular and clinical data from this cohort, and that of individuals previously reported, suggests that BAZ2B haploinsufficiency causes an autosomal dominant neurodevelopmental syndrome that is incompletely penetrant. The phenotypes most commonly seen in association with loss of BAZ2B function include developmental delay, intellectual disability, autism spectrum disorder, speech delay-with some affected individuals being non-verbal-behavioral abnormalities, seizures, vision-related issues, congenital heart defects, poor fetal growth, and an indistinct pattern of dysmorphic features in which epicanthal folds and small ears are particularly common.

Multiseriate cortical sclerenchyma enhance root penetration in compacted soils.

Mechanical impedance limits soil exploration and resource capture by plant roots. We examine the role of root anatomy in regulating plant adaptation to mechanical impedance and identify a root anatomical phene in maize (Zea mays) and wheat (Triticum aestivum) associated with penetration of hard soil: Multiseriate cortical sclerenchyma (MCS). We characterize this trait and evaluate the utility of MCS for root penetration in compacted soils. Roots with MCS had a greater cell wall-to-lumen ratio and a distinct UV emission spectrum in outer cortical cells. Genome-wide association mapping revealed that MCS is heritable and genetically controlled. We identified a candidate gene associated with MCS. Across all root classes and nodal positions, maize genotypes with MCS had 13% greater root lignin concentration compared to genotypes without MCS. Genotypes without MCS formed MCS upon exogenous ethylene exposure. Genotypes with MCS had greater lignin concentration and bending strength at the root tip. In controlled environments, MCS in maize and wheat was associated improved root tensile strength and increased penetration ability in compacted soils. Maize genotypes with MCS had root systems with 22% greater depth and 49% greater shoot biomass in compacted soils in the field compared to lines without MCS. Of the lines we assessed, MCS was present in 30 to 50% of modern maize, wheat, and barley cultivars but was absent in teosinte and wild and landrace accessions of wheat and barley. MCS merits investigation as a trait for improving plant performance in maize, wheat, and other grasses under edaphic stress.

Prevalence of Low-Acuity Pediatric Emergency Medical Services Transports to a Pediatric Emergency Department in an Urban Area.

OBJECTIVES: Many patients transported by Emergency Medical Services (EMS) do not have emergent resource needs. Estimates for the proportion of pediatric EMS calls for low-acuity complaints, and thus potential candidates for alternative dispositions, vary widely and are often based on physician judgment. A more accurate reference standard should include patient assessments, interventions, and dispositions. The objective of this study was to describe the prevalence and characteristics of low-acuity pediatric EMS calls in an urban area. METHODS: This is a prospective observational study of children transported by EMS to a tertiary care pediatric emergency department. Patient acuity was defined using a novel composite measure that included physiologic assessments, resources used, and disposition. Bivariable and multivariable logistic regression were conducted to assess for factors associated with low-acuity status. RESULTS: A total of 996 patients were enrolled, of whom 32.9% (95% confidence interval, 30.0-36.0) were low acuity. Most of the sample was Black, non-Hispanic with a mean age of 7 years. When compared with adolescents, children younger than 1 year were more likely to be low acuity (adjusted odds ratio, 3.1 [1.9-5.1]). Patients in a motor vehicle crash were also more likely to be low acuity (adjusted odds ratio, 2.4 [1.2-4.6]). All other variables, including race, insurance status, chief complaint, and dispatch time, were not associated with low-acuity status. CONCLUSIONS: One third of pediatric patients transported to the pediatric emergency department by EMS in this urban area are for low-acuity complaints. Further research is needed to determine low-acuity rates in other jurisdictions and whether EMS providers can accurately identify low-acuity patients to develop alternative EMS disposition programs for children.

Genome wide association analysis of root hair traits in rice reveals novel genomic regions controlling epidermal cell differentiation.

BACKGROUND: Genome wide association (GWA) studies demonstrate linkages between genetic variants and traits of interest. Here, we tested associations between single nucleotide polymorphisms (SNPs) in rice (Oryza sativa) and two root hair traits, root hair length (RHL) and root hair density (RHD). Root hairs are outgrowths of single cells on the root epidermis that aid in nutrient and water acquisition and have also served as a model system to study cell differentiation and tip growth. Using lines from the Rice Diversity Panel-1, we explored the diversity of root hair length and density across four subpopulations of rice (aus, indica, temperate japonica, and tropical japonica). GWA analysis was completed using the high-density rice array (HDRA) and the rice reference panel (RICE-RP) SNP sets. RESULTS: We identified 18 genomic regions related to root hair traits, 14 of which related to RHD and four to RHL. No genomic regions were significantly associated with both traits. Two regions overlapped with previously identified quantitative trait loci (QTL) associated with root hair density in rice. We identified candidate genes in these regions and present those with previously published expression data relevant to root hair development. We re-phenotyped a subset of lines with extreme RHD phenotypes and found that the variation in RHD was due to differences in cell differentiation, not cell size, indicating genes in an associated genomic region may influence root hair cell fate. The candidate genes that we identified showed little overlap with previously characterized genes in rice and Arabidopsis. CONCLUSIONS: Root hair length and density are quantitative traits with complex and independent genetic control in rice. The genomic regions described here could be used as the basis for QTL development and further analysis of the genetic control of root hair length and density. We present a list of candidate genes involved in root hair formation and growth in rice, many of which have not been previously identified as having a relation to root hair growth. Since little is known about root hair growth in grasses, these provide a guide for further research and crop improvement.

Genotype-phenotype correlations in RHOBTB2-associated neurodevelopmental disorders.

PURPOSE: Missense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy with early-onset seizures and severe intellectual disability. METHODS: By international collaboration, we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders. By western blotting, we investigated the consequences of missense variants in vitro. RESULTS: In accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe developmental and epileptic encephalopathy in 16 individuals. Now, we also identified de novo missense variants in the GTPase domain in 6 individuals with apparently more variable neurodevelopmental phenotypes with or without epilepsy. In contrast to variants in the BTB domain region, variants in the GTPase domain do not impair proteasomal degradation of RHOBTB2 in vitro, indicating different functional consequences. Furthermore, we observed biallelic splice-site and truncating variants in 9 families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well. CONCLUSION: By identifying genotype-phenotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying biallelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2-related phenotypes, including both autosomal dominant and recessive neurodevelopmental disorders.

Pyuria in Children with Diabetic Ketoacidosis.

Acute kidney injury occurs frequently during pediatric diabetic ketoacidosis (DKA). We reviewed urinalyses from 561 children with DKA; pyuria was detected in 19% overall and in 40% of children with more comprehensive urine testing (≥3 urinalyses) during DKA.

Five siblings expand the spectrum of GPC6-related skeletal dysplasia.

Skeletal dysplasias broadly include disorders of cartilage or bone. Omodysplasia-1 is a type of skeletal dysplasia caused by biallelic loss of function variants in the GPC6 gene. GPC6 codes for the protein glypican 6 (GPC6) (OMIM *604404), which stimulates bone growth. We report a family in which five out of nine children were presented with a skeletal dysplasia characterized phenotypically by mild short stature and rhizomelia. All affected individuals were found to have homozygous missense variants in GPC6: c.511 C>T (p.Arg171Trp). Radiograph findings included rhizomelic foreshortening of all four extremities, coxa breva, and ulna minus deformity. Using a Hedgehog (Hh) reporter assay, we demonstrate that the variant found in this family results in significantly reduced stimulation of Hh activity when compared to the wild-type GPC6 protein, however protein function is still present. Thus, the milder phenotype seen in the family presented is hypothesized due to decreased GPC6 protein activity versus complete loss of function as seen in omodysplasia-1. Given the unique phenotype and molecular mechanism, we propose that this family's findings widen the phenotypic spectrum of GPC6-related skeletal dysplasias.

Intranasal fentanyl and discharge from the emergency department among children with sickle cell disease and vaso-occlusive pain: A multicenter pediatric emergency medicine perspective.

Children with sickle cell disease (SCD) commonly experience vaso-occlusive pain episodes (VOE) due to sickling of erythrocytes, which often requires care in the emergency department. Our objective was to assess the use and impact of intranasal fentanyl for the treatment of children with SCD-VOE on discharge from the emergency department in a multicenter study. We conducted a cross-sectional study at 20 academic pediatric emergency departments in the United States and Canada. We used logistic regression to test bivariable and multivariable associations between the outcome of discharge from the emergency department and candidate variables theoretically associated with discharge. The study included 400 patients; 215 (54%) were female. The median age was 14.6 (interquartile range 9.8, 17.6) years. Nineteen percent (n = 75) received intranasal fentanyl in the emergency department. Children who received intranasal fentanyl had nearly nine-fold greater adjusted odds of discharge from the emergency department compared to those who did not (adjusted odds ratio 8.99, 95% CI 2.81-30.56, p < .001). The rapid onset of action and ease of delivery without intravenous access offered by intranasal fentanyl make it a feasible initial parenteral analgesic in the treatment of children with SCD presenting with VOE in the acute-care setting. Further study is needed to determine potential causality of the association between intranasal fentanyl and discharge from the emergency department observed in this multicenter study.

Clinician and Caregiver Determinations of Acuity for Children Transported by Emergency Medical Services: A Prospective Observational Study.

STUDY OBJECTIVE: Many Emergency Medical Services (EMS) agencies have developed alternative disposition processes for patients with nonemergency problems, but there is a lack of evidence demonstrating EMS clinicians can accurately determine acuity in pediatric patients. Our study objective was to determine EMS and other stakeholders' ability to identify low acuity pediatric EMS patients. METHODS: We conducted a prospective, observational study of children transported to a pediatric emergency department (ED) by EMS. Acuity was defined using a composite measure that included data from the patient's vital signs and examination, resources used (laboratory results, radiographs, etc), and disposition. For each patient, an EMS clinician, patient caregiver, ED nurse, and ED provider completed a survey as soon as possible after the patient's arrival at the ED. The survey asked respondents 2 questions: to state their level of agreement that a patient was low acuity and could the patient have been managed by various alternative dispositions. For each respondent group, we calculated the sensitivity, specificity, and positive and negative predictive values for low acuity versus the composite measure. RESULTS: From August 2020 through September 2021, we approached 1,015 caregivers, of whom 996 (99.8%) agreed to participate and completed the survey. Survey completion varied between 78.7% and 84.1% for EMS and ED nurses and providers. The mean patient age was 7 years, 62.6% were non-Hispanic Black, and 60% were enrolled in public insurance programs. Of the 996 patient encounters, 33% were determined to be low acuity by the composite measure. The positive predictive value for EMS clinicians when identifying low acuity children was 0.60 (95% confidence intervals [CI], 0.58 to 0.67). The positive predictive value for ED nurses and providers was 0.67 (95% CI, 0.61 to 0.72) and 0.68 (95% CI, 0.63 to 0.74) respectively. The negative predictive value for EMS clinicians when identifying not low acuity children was 0.62 (95% CI, 0.58 to 0.67). The negative predictive value for ED nurses and providers was 0.72 (95% CI, 0.68 to 0.76) and 0.73 (95% CI, 0.70 to 0.77) respectively. Caregivers had the lowest positive predictive value 0.34 (95% CI, 0.30 to 0.40) but the highest negative predictive value 0.82 (95% CI, 0.79 to 0.85). The EMS clinicians, ED nurses and providers were more likely than caregivers to think that a child with a low acuity complaint could have been safely managed by alternative disposition. CONCLUSION: All 4 groups studied had a limited ability to identify which children transported by EMS would have no emergency resource needs, and support for alternative disposition was limited. For children to be included in alternative disposition processes, novel triage tools, training, and oversight will be required to prevent undertriage.

Clinical and Laboratory Predictors of Dehydration Severity in Children With Diabetic Ketoacidosis.

STUDY OBJECTIVE: Our primary objective was to characterize the degree of dehydration in children with diabetic ketoacidosis (DKA) and identify physical examination and biochemical factors associated with dehydration severity. Secondary objectives included describing relationships between dehydration severity and other clinical outcomes. METHODS: In this cohort study, we analyzed data from 753 children with 811 episodes of DKA in the Pediatric Emergency Care Applied Research Network Fluid Therapies Under Investigation Study, a randomized clinical trial of fluid resuscitation protocols for children with DKA. We used multivariable regression analyses to identify physical examination and biochemical factors associated with dehydration severity, and we described associations between dehydration severity and DKA outcomes. RESULTS: Mean dehydration was 5.7% (SD 3.6%). Mild (0 to <5%), moderate (5 to <10%), and severe (≥10%) dehydration were observed in 47% (N=379), 42% (N=343), and 11% (N=89) of episodes, respectively. In multivariable analyses, more severe dehydration was associated with new onset of diabetes, higher blood urea nitrogen, lower pH, higher anion gap, and diastolic hypertension. However, there was substantial overlap in these variables between dehydration groups. The mean length of hospital stay was longer for patients with moderate and severe dehydration, both in new onset and established diabetes. CONCLUSION: Most children with DKA have mild-to-moderate dehydration. Although biochemical measures were more closely associated with the severity of dehydration than clinical assessments, neither were sufficiently predictive to inform rehydration practice.

The Management of Children and Youth With Pediatric Mental and Behavioral Health Emergencies.

Mental and behavioral health (MBH) emergencies in children and youth continue to increasingly affect not only the emergency department (ED), but the entire spectrum of emergency medical services for children, from prehospital services to the community. Inadequate community and institutional infrastructure to care for children and youth with MBH conditions makes the ED an essential part of the health care safety net for these patients. As a result, an increasing number of children and youth are referred to the ED for evaluation of a broad spectrum of MBH emergencies, from depression and suicidality to disruptive and aggressive behavior. However, challenges in providing optimal care to these patients include lack of personnel, capacity, and infrastructure, challenges with timely access to a mental health professional, the nature of a busy ED environment, and paucity of outpatient post-ED discharge resources. These factors contribute to prolonged ED stays and boarding, which negatively affects patient care and ED operations. Strategies to improve care for MBH emergencies, including systems level coordination of care, is therefore essential. The goal of this policy statement and its companion technical report is to highlight strategies, resources, and recommendations for improving emergency care delivery for pediatric MBH.

Delineating the genotypic and phenotypic spectrum of HECW2-related neurodevelopmental disorders.

BACKGROUND: Variants in HECW2 have recently been reported to cause a neurodevelopmental disorder with hypotonia, seizures and impaired language; however, only six variants have been reported and the clinical characteristics have only broadly been defined. METHODS: Molecular and clinical data were collected from clinical and research cohorts. Massive parallel sequencing was performed and identified individuals with a HECW2-related neurodevelopmental disorder. RESULTS: We identified 13 novel missense variants in HECW2 in 22 unpublished cases, of which 18 were confirmed to have a de novo variant. In addition, we reviewed the genotypes and phenotypes of previously reported and new cases with HECW2 variants (n=35 cases). All variants identified are missense, and the majority of likely pathogenic and pathogenic variants are located in or near the C-terminal HECT domain (88.2%). We identified several clustered variants and four recurrent variants (p.(Arg1191Gln);p.(Asn1199Lys);p.(Phe1327Ser);p.(Arg1330Trp)). Two variants, (p.(Arg1191Gln);p.(Arg1330Trp)), accounted for 22.9% and 20% of cases, respectively. Clinical characterisation suggests complete penetrance for hypotonia with or without spasticity (100%), developmental delay/intellectual disability (100%) and developmental language disorder (100%). Other common features are behavioural problems (88.9%), vision problems (83.9%), motor coordination/movement (75%) and gastrointestinal issues (70%). Seizures were present in 61.3% of individuals. Genotype-phenotype analysis shows that HECT domain variants are more frequently associated with cortical visual impairment and gastrointestinal issues. Seizures were only observed in individuals with variants in or near the HECT domain. CONCLUSION: We provide a comprehensive review and expansion of the genotypic and phenotypic spectrum of HECW2 disorders, aiding future molecular and clinical diagnosis and management.

The Effect of Documenting Patient Weight in Kilograms on Pediatric Medication Dosing Errors in Emergency Medical Services.

Objectives: Up to 40% of children who receive a medication from emergency medical services (EMS) are subject to a dosing error. One of the reasons for this is difficulties adjusting dosages for weight. Converting weights from pounds to kilograms complicates this further. This is the rationale for the National EMS Quality Alliance measure Pediatrics-03b, which measures the proportion of children with a weight documented in kilograms. However, there is little evidence that this practice is associated with lower rates of dosing errors. Therefore, our objective was to determine whether EMS documentation of weight in kilograms was associated with a lower rate of pediatric medication dosing errors.Methods: We conducted a retrospective cross-sectional study of children 0-14 y/o in the 2016-17 electronic Maryland Emergency Medical Services Data System that received a weight-based medication. Using validated age-based formulas, we assigned a weight to patients without one documented. Doses were classified as errors and severe errors if they deviated from the state protocol by >20% or >50%, respectively. We compared the dosage errors in the two groups and completed secondary analyses for specific medications and age groups.Results: We identified 3,618 cases of medication administration, 53% of which had a documented weight. Patients with a documented weight had a significantly lower overall dose error rate than those without (22 vs. 26%, p<.05). A sensitivity analysis in which we assigned a weight to those patients with a weight recorded did not significantly change this result. Sub-analyses by individual medication showed that only epinephrine (34 vs. 56%, p<.05) and fentanyl (10 vs. 31%, p <.05) had significantly lower dosing error rates for patients with a documented weight. Infants were the only age group where documenting a weight was associated with a lower dosing error rate (33 vs. 53% p<.05).Conclusion: Our findings suggest that documenting a weight in kilograms is associated with a small but significantly lower rate of pediatric dosing errors by EMS. Documenting a weight in kilograms appears particularly important for specific medications and patient age groups. Additional strategies (including age-based standardized dosing) may be needed to further reduce pediatric dosing errors by EMS.

2022 Systematic Review of Evidence-Based Guidelines for Prehospital Care.

INTRODUCTION: Multiple national organizations and federal agencies have promoted the development, implementation, and evaluation of evidence-based guidelines (EBGs) for prehospital care. Previous efforts have identified opportunities to improve the quality of prehospital guidelines and highlighted the value of high-quality EBGs to inform initial certification and continued competency activities for EMS personnel. OBJECTIVES: We aimed to perform a systematic review of prehospital guidelines published from January 2018 to April 2021, evaluate guideline quality, and identify top-scoring guidelines to facilitate dissemination and educational activities for EMS personnel. METHODS: We searched the literature in Ovid Medline and EMBASE from January 2018 to April 2021, excluding guidelines identified in a prior systematic review. Publications were retained if they were relevant to prehospital care, based on organized reviews of the literature, and focused on providing recommendations for clinical care or operations. Included guidelines were appraised to identify if they met the National Academy of Medicine (NAM) criteria for high-quality guidelines and scored across the six domains of the Appraisal of Guidelines for Research and Evaluation (AGREE) II tool. RESULTS: We identified 75 guidelines addressing a variety of clinical and operational aspects of EMS medicine. About half (n = 39, 52%) addressed time/life-critical conditions and 33 (44%) contained recommendations relevant to non-clinical/operational topics. Fewer than half (n = 35, 47%) were based on systematic reviews of the literature. Nearly one-third (n = 24, 32%) met all NAM criteria for clinical practice guidelines. Only 27 (38%) guidelines scored an average of >75% across AGREE II domains, with content relevant to guideline implementation most commonly missing. CONCLUSIONS: This interval systematic review of prehospital EBGs identified many new guidelines relevant to prehospital care; more than all guidelines reported in a prior systematic review. Our review reveals important gaps in the quality of guideline development and the content in their publications, evidenced by the low proportion of guidelines meeting NAM criteria and the scores across AGREE II domains. Efforts to increase guideline dissemination, implementation, and related education may be best focused around the highest quality guidelines identified in this review.

Recommendations for Improving the Quality of Prehospital Evidence-Based Guidelines.

Evidence-based guidelines that provide recommendations for clinical care or operations are increasingly being published to inform the EMS community. The quality of evidence evaluation and methodological rigor undertaken to develop and publish these recommendations vary. This can negatively affect dissemination, education, and implementation efforts. Guideline developers and end users could be better informed by efforts across medical specialties to improve the quality of guidelines, including the use of specific criteria that have been identified within the highest quality guidelines. In this special contribution, we aim to describe the current state of published guidelines available to the EMS community informed by two recent systematic reviews of existing prehospital evidenced based guidelines (EBGs). We further aim to provide a description of key elements of EBGs, methods that can be used to assess their quality, and concrete recommendations for guideline developers to improve the quality of evidence evaluation, guideline development, and reporting. Finally, we outline six key recommendations for improving prehospital EBGs, informed by systematic reviews of prehospital guidelines performed by the Prehospital Guidelines Consortium.

Emergency Medical Services Clinicians' Perspectives on Pediatric Non-Transport.

OBJECTIVES: Emergency medical services clinicians do not transport one-third of all children assessed, even without official pediatric non-transport protocols. Little is known about how EMS clinicians and caregivers decide not to transport a child. Our objectives were to describe how EMS clinicians currently decide whether or not to transport a child and identify barriers to and enablers of successfully implementing an EMS clinician-initiated pediatric non-transport protocol. METHODS: We conducted six virtual focus groups with EMS clinicians from the mid-Atlantic. A PhD trained facilitator moderated all groups using a semi-structured moderator guide. Multiple investigators independently coded a deidentified sample transcript. One team member then completed axial coding of the remaining transcripts. Thematic saturation was achieved. Clusters of similar codes were grouped into themes by consensus. RESULTS: We recruited 50 participants, of whom 70% were paramedics and 28% emergency medical technicians. There was agreement that caregivers often use 9-1-1 for low acuity complaints. Participants stated that non-transport usually occurs after shared decision-making between EMS clinicians and caregivers; EMS clinicians advise whether transport is necessary, but caregivers are responsible for making the final decision and signing refusal documentation. Subthemes for how non-transport decisions were made included the presence of agency protocols, caregiver preferences, absence of a guardian on the scene, EMS clinician variability, and distance to the nearest ED. Participants identified the following features that would enable successful implementation of an EMS clinician-initiated non-transport process: a user-friendly interface, clear protocol endpoints, the inclusion of vital sign parameters, resources to leave with caregivers, and optional direct medical oversight. CONCLUSIONS: EMS clinicians in our study agreed that non-transport is currently a caregiver decision, but noted a collaborative process of shared decision-making where EMS clinicians advise caregivers whether transport is indicated. Further research is needed to understand the safety of this practice. This study suggests there may be a need for EMS-initiated alternative disposition/non-transport protocols.

mTOR Regulation of N-Myc Downstream Regulated 1 (NDRG1) Phosphorylation in Clear Cell Renal Cell Carcinoma.

The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

The Management of Children and Youth With Pediatric Mental and Behavioral Health Emergencies.

Mental and behavioral health (MBH) emergencies in children and youth continue to increasingly affect not only the emergency department (ED), but the entire spectrum of emergency medical services for children, from prehospital services to the community. Inadequate community and institutional infrastructure to care for children and youth with MBH conditions makes the ED an essential part of the health care safety net for these patients. As a result, an increasing number of children and youth are referred to the ED for evaluation of a broad spectrum of MBH emergencies, from depression and suicidality to disruptive and aggressive behavior. However, challenges in providing optimal care to these patients include lack of personnel, capacity, and infrastructure, challenges with timely access to a mental health professional, the nature of a busy ED environment, and paucity of outpatient post-ED discharge resources. These factors contribute to prolonged ED stays and boarding, which negatively affects patient care and ED operations. Strategies to improve care for MBH emergencies, including systems level coordination of care, is therefore essential. The goal of this policy statement and its companion technical report is to highlight strategies, resources, and recommendations for improving emergency care delivery for pediatric MBH.