Impact of Failure of Noninvasive Ventilation on the Safety of Pediatric Tracheal Intubation.

OBJECTIVES: Noninvasive ventilation is widely used to avoid tracheal intubation in critically ill children. The objective of this study was to assess whether noninvasive ventilation failure was associated with severe tracheal intubation-associated events and severe oxygen desaturation during tracheal intubation. DESIGN: Prospective multicenter cohort study of consecutive intubated patients using the National Emergency Airway Registry for Children registry. SETTING: Thirteen PICUs (in 12 institutions) in the United States and Canada. PATIENTS: All patients undergoing tracheal intubation in participating sites were included. Noninvasive ventilation failure group included children with any use of high-flow nasal cannula, continuous positive airway pressure, or bilevel noninvasive ventilation in the 6 hours prior to tracheal intubation. Primary tracheal intubation group included children without exposure to noninvasive ventilation within 6 hours before tracheal intubation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Severe tracheal intubation-associated events (cardiac arrest, esophageal intubation with delayed recognition, emesis with aspiration, hypotension requiring intervention, laryngospasm, pneumothorax, pneumomediastinum) and severe oxygen desaturation (< 70%) were recorded prospectively. The study included 956 tracheal intubation encounters; 424 tracheal intubations (44%) occurred after noninvasive ventilation failure, with a median of 13 hours (interquartile range, 4-38 hr) of noninvasive ventilation. Noninvasive ventilation failure group included more infants (47% vs 33%; p < 0.001) and patients with a respiratory diagnosis (56% vs 30%; p < 0.001). Noninvasive ventilation failure was not associated with severe tracheal intubation-associated events (5% vs 5% without noninvasive ventilation; p = 0.96) but was associated with severe desaturation (15% vs 9% without noninvasive ventilation; p = 0.005). After controlling for baseline differences, noninvasive ventilation failure was not independently associated with severe tracheal intubation-associated events (p = 0.35) or severe desaturation (p = 0.08). In the noninvasive ventilation failure group, higher FIO2 before tracheal intubation (≥ 70%) was associated with severe tracheal intubation-associated events. CONCLUSIONS: Critically ill children are frequently exposed to noninvasive ventilation before intubation. Noninvasive ventilation failure was not independently associated with severe tracheal intubation-associated events or severe oxygen desaturation compared to primary tracheal intubation.

Comment on "RNA-guided DNA insertion with CRISPR-associated transposases".

Strecker et al (Research Articles, 5 July 2019, p. 48) described a system for exploiting a Tn7-type transposon-encoded CRISPR-Cas system to make RNA-guided, programmable insertions. Although this system has great promise, we note that the well-established biochemistry of Tn7 suggests that the particular system used may insert not only the transposon but also the entire donor plasmid.

Apneic Oxygenation As a Quality Improvement Intervention in an Academic PICU.

OBJECTIVES: To evaluate if the use of apneic oxygenation during tracheal intubation in children is feasible and would decrease the occurrence of oxygen desaturation. DESIGN: Prospective pre/post observational study. SETTING: A large single-center noncardiac PICU in North America. PATIENTS: All patients less than 18 years old who underwent primary tracheal intubation from August 1, 2014, to September 30, 2018. INTERVENTIONS: Implementation of apneic oxygenation for all primary tracheal intubation as quality improvement. MEASUREMENTS AND MAIN RESULTS: Total of 1,373 tracheal intubations (661 preimplementation and 712 postimplementation) took place during study period. Within 2 months, apneic oxygenation use reached to predefined adherence threshold (> 80% of primary tracheal intubations) after implementation and sustained at greater than 70% level throughout the postimplementation. Between the preimplementation and postimplementation, no significant differences were observed in patient demographics, difficult airway features, or providers. Respiratory and procedural indications were more common during preintervention. Video laryngoscopy devices were used more often during the postimplementation (pre 5% vs post 75%; p < 0.001). Moderate oxygen desaturation less than 80% were observed in fewer tracheal intubations after apneic oxygenation implementation (pre 15.4% vs post 11.8%; p = 0.049); severe oxygen desaturation less than 70% was also observed in fewer tracheal intubations after implementation (pre 10.4% vs post 7.2%; p = 0.032). Hemodynamic tracheal intubation associated events (i.e., cardiac arrests, hypotension, dysrhythmia) were unchanged (pre 3.2% vs post 2.0%; p = 0.155). Multivariable analyses showed apneic oxygenation implementation was significantly associated with a decrease in moderate desaturation less than 80% (adjusted odds ratio, 0.55; 95% CI, 0.34-0.88) and with severe desaturation less than 70% (adjusted odds ratio, 0.54; 95% CI, 0.31-0.96) while adjusting for tracheal intubation indications and device. CONCLUSIONS: Implementation of apneic oxygenation in PICU was feasible, and was associated with significant reduction in moderate and severe oxygen desaturation. Use of apneic oxygenation should be considered when intubating critically ill children.

Structural insights into the mechanism of double strand break formation by Hermes, a hAT family eukaryotic DNA transposase.

Some DNA transposons relocate from one genomic location to another using a mechanism that involves generating double-strand breaks at their transposon ends by forming hairpins on flanking DNA. The same double-strand break mode is employed by the V(D)J recombinase at signal-end/coding-end junctions during the generation of antibody diversity. How flanking hairpins are formed during DNA transposition has remained elusive. Here, we describe several co-crystal structures of the Hermes transposase bound to DNA that mimics the reaction step immediately prior to hairpin formation. Our results reveal a large DNA conformational change between the initial cleavage step and subsequent hairpin formation that changes which strand is acted upon by a single active site. We observed that two factors affect the conformational change: the complement of divalent metal ions bound by the catalytically essential DDE residues, and the identity of the -2 flanking base pair. Our data also provides a mechanistic link between the efficiency of hairpin formation (an A:T basepair is favored at the -2 position) and Hermes' strong target site preference. Furthermore, we have established that the histidine residue within a conserved C/DxxH motif present in many transposase families interacts directly with the scissile phosphate, suggesting a crucial role in catalysis.

Design and Implementation of a Pediatric ICU Acuity Scoring Tool as Clinical Decision Support.

BACKGROUND AND OBJECTIVE: Pediatric in-hospital cardiac arrest most commonly occurs in the pediatric intensive care unit (PICU) and is frequently preceded by early warning signs of clinical deterioration. In this study, we describe the implementation and evaluation of criteria to identify high-risk patients from a paper-based checklist into a clinical decision support (CDS) tool in the electronic health record (EHR). MATERIALS AND METHODS: The validated paper-based tool was first adapted by PICU clinicians and clinical informaticians and then integrated into clinical workflow following best practices for CDS design. A vendor-based rule engine was utilized. Littenberg's assessment framework helped guide the overall evaluation. Preliminary testing took place in EHR development environments with more rigorous evaluation, testing, and feedback completed in the live production environment. To verify data quality of the CDS rule engine, a retrospective Structured Query Language (SQL) data query was also created. As a process metric, preparedness was measured in pre- and postimplementation surveys. RESULTS: The system was deployed, evaluating approximately 340 unique patients monthly across 4 clinical teams. The verification against retrospective SQL of 15-minute intervals over a 30-day period revealed no missing triggered intervals and demonstrated 99.3% concordance of positive triggers. Preparedness showed improvements across multiple domains to our a priori goal of 90%. CONCLUSION: We describe the successful adaptation and implementation of a real-time CDS tool to identify PICU patients at risk of deterioration. Prospective multicenter evaluation of the tool's effectiveness on clinical outcomes is necessary before broader implementation can be recommended.

An Examination of Peer-Delivered Parenting Skills Programs Across New York State.

Peers are an important adjunct to the public mental health service system, and are being increasingly utilized across the country as a cost-effective solution to workforce shortages. Despite the tremendous growth of peer-delivered support over the past two decades, it has only been within the past few years that peer programs have been the subject of empirical inquiry. The purpose of this study was to examine the prevalence and characteristics of peer-delivered parenting programs across the New York State public mental health service system. We surveyed 46 family peer organizations across New York State regarding their delivery of structured peer-delivered parenting programs. Thirty-four (76%) completed the questionnaire, and of them, 18 (53%) delivered a parenting program. Subsequent interviews with seven of the 18 organizations revealed peer organizations had been delivering eight unique parenting programs for upwards of two decades. Additionally, organizations offered multiple supports to families to participate. Training, supervision, and issues around fidelity are discussed, as well as the implications of this study for states utilizing a peer workforce.

Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase.

The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.

Pulmonary Vasodilator Therapy in Shock-associated Cardiac Arrest.

RATIONALE: Many in-hospital cardiac arrests are precipitated by hypotension, often associated with systemic inflammation. These patients are less likely to be successfully resuscitated, and novel approaches to their treatment are needed. OBJECTIVES: To determine if the addition of inhaled nitric oxide (iNO) to hemodynamic-directed cardiopulmonary resuscitation (HD-CPR) would improve short-term survival from cardiac arrest associated with shock and systemic inflammation. METHODS: In 3-month-old swine (n = 21), LPS was intravenously infused, inducing systemic hypotension. Ventricular fibrillation was induced, and animals were randomized to blinded treatment with either: 1) HD-CPR with iNO, or 2) HD-CPR without iNO. During HD-CPR, chest compression depth was titrated to peak aortic compression pressure of 100 mm Hg, and vasopressor administration was titrated to coronary perfusion pressure greater than or equal to 20 mm Hg. Defibrillation attempts began after 10 minutes of resuscitation. The primary outcome was 45-minute survival. MEASUREMENTS AND MAIN RESULTS: The iNO group had higher rates of 45-minute survival (10 of 10 vs. 3 of 11; P = 0.001). During cardiopulmonary resuscitation, the iNO group had lower pulmonary artery relaxation pressure (mean ± SEM, 10.9 ± 2.4 vs. 18.4 ± 2.4 mm Hg; P = 0.03), higher coronary perfusion pressure (21.1 ± 1.5 vs. 16.9 ± 1.0 mm Hg; P = 0.005), and higher aortic relaxation pressure (36.6 ± 1.6 vs. 30.4 ± 1.1 mm Hg; P < 0.001) despite shallower chest compressions (5.88 ± 0.25 vs. 6.46 ± 0.40 cm; P = 0.02) and fewer vasopressor doses in the first 10 minutes (median, 4 [interquartile range, 3-4] vs. 5 [interquartile range, 5-6], P = 0.03). CONCLUSIONS: The addition of iNO to HD-CPR in LPS-induced shock-associated cardiac arrest improved short-term survival and intraarrest hemodynamics.

A Patient Flow Analysis: Identification of Process Inefficiencies and Workflow Metrics at an Ambulatory Endoscopy Unit.

Background. The increasing demand for endoscopic procedures coincides with the paradigm shift in health care delivery that emphasizes efficient use of existing resources. However, there is limited literature on the range of endoscopy unit efficiencies. Methods. A time and motion analysis of patient flow through the Hotel-Dieu Hospital (Kingston, Ontario) endoscopy unit was followed by qualitative interviews. Procedures were directly observed in three segments: individual endoscopy room use, preprocedure/recovery room, and overall endoscopy unit utilization. Results. Data were collected for 137 procedures in the endoscopy room, 139 procedures in the preprocedure room, and 143 procedures for overall room utilization. The mean duration spent in the endoscopy room was 31.47 min for an esophagogastroduodenoscopy, 52.93 min for a colonoscopy, 30.47 min for a flexible sigmoidoscopy, and 66.88 min for a double procedure. The procedure itself accounted for 8.11 min, 34.24 min, 9.02 min, and 39.13 min for the above procedures, respectively. The focused interviews identified the scheduling template as a major area of operational inefficiency. Conclusions. Despite reasonable procedure times for all except colonoscopies, the endoscopy room durations exceed the allocated times, reflecting the impact of non-procedure-related factors and the need for a revised scheduling template. Endoscopy units have unique operational characteristics and identification of process inefficiencies can lead to targeted quality improvement initiatives.

Diverse Morbidity and Mortality Among Infants Treated with Venoarterial Extracorporeal Membrane Oxygenation.

BACKGROUND: Venoarterial extracorporeal membrane oxygenation (VA-ECMO) is utilized for cardiopulmonary failure. We aimed to qualify and quantify the predictors of morbidity and mortality in infants requiring VA-ECMO. METHODS: Data was collected from 170 centers participating in the extracorporeal life support organization (ELSO) registry. Relationships between in-hospital mortality and risk factors were assessed using logistic regression. Survival was defined as being discharged from the hospital. RESULTS: Six hundred and sixty-two eligible records were reviewed. Mortality occurred in 303 (46%) infants. Congenital diaphragmatic hernia patients (OR=3.83, 95% CI 1.96-7.49, p<0.001), cardiac failure with associated shock (OR= 2.90, 95% CI 1.46-5.77, p=0.002), and pulmonary failure including respiratory distress syndrome (OR=4.06, 95% CI 1.72-9.58, p=0.001) had the highest odds of mortality in this cohort. Birth weight (BW) < 3 kg (OR=1.83, 95% CI 1.21-2.78, p=0.004), E-CPR (OR=3.35, 95% CI 1.57-7.15, p=0.002), hemofiltration (OR=2.04, 95% CI 1.32-3.16, p=0.001), and dialysis (OR=6.13, 95% CI 1.70-22.1, p<0.001) were all independent predictors of mortality. CONCLUSION: Infants requiring VA-ECMO experience diverse sequelae and their mortality are high.

Avoiding the ends: internal epitope tagging of proteins using transposon Tn7.

Peptide tags fused to proteins are used in a variety of applications, including as affinity tags for purification, epitope tags for immunodetection, or fluorescent protein tags for visualization. However, the peptide tags can disrupt the target protein function. When function is disrupted by fusing a peptide to either the N or C terminus of the protein of interest, identifying alternative ways to create functional tagged fusion proteins can be difficult. Here, we describe a method to introduce protein tags internal to the coding sequence of a target protein. The method employs in vitro Tn7-transposon mutagenesis of plasmids for random introduction of the tag, followed by subsequent Gateway cloning steps to isolate alleles with mutations in the coding sequence of the target gene. The Tn7-epitope cassette is designed such that essentially all of the transposon is removed through restriction enzyme digestion, leaving only the protein tag at diverse sites internal to the ORF. We describe the use of this system to generate a panel of internally epitope-tagged versions of the Saccharomyces cerevisiae GPI-linked membrane protein Dcw1 and the Candida glabrata transcriptional regulator Sir3. This internal protein tagging system is, in principle, adaptable to tag proteins in any organism for which Gateway-adapted expression vectors exist.

Structural basis of hAT transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica.

Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA.

The Tn7 transposition regulator TnsC interacts with the transposase subunit TnsB and target selector TnsD.

The excision of transposon Tn7 from a donor site and its insertion into its preferred target site, attachment site attTn7, is mediated by four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC, and TnsD. Transposition requires the assembly of a nucleoprotein complex containing all four Tns proteins and the DNA substrates, the donor site containing Tn7, and the preferred target site attTn7. TnsA and TnsB together form the heteromeric Tn7 transposase, and TnsD is a target-selecting protein that binds specifically to attTn7. TnsC is the key regulator of transposition, interacting with both the TnsAB transposase and TnsD-attTn7. We show here that TnsC interacts directly with TnsB, and identify the specific region of TnsC involved in the TnsB-TnsC interaction during transposition. We also show that a TnsC mutant defective in interaction with TnsB is defective for Tn7 transposition both in vitro and in vivo. Tn7 displays cis-acting target immunity, which blocks Tn7 insertion into a target DNA that already contains Tn7. We provide evidence that the direct TnsB-TnsC interaction that we have identified also mediates cis-acting Tn7 target immunity. We also show that TnsC interacts directly with the target selector protein TnsD.

Developing quality indicators for family support services in community team-based mental health care.

Quality indicators for programs integrating parent-delivered family support services for children's mental health have not been systematically developed. Increasing emphasis on accountability under the Affordable Care Act highlights the importance of quality-benchmarking efforts. Using a modified Delphi approach, quality indicators were developed for both program level and family support specialist level practices. These indicators were pilot tested with 21 community-based mental health programs. Psychometric properties of these indicators are reported; variations in program and family support specialist performance suggest the utility of these indicators as tools to guide policies and practices in organizations that integrate parent-delivered family support service components.

Mobile genetic elements and genome evolution 2014.

The Mobile Genetic Elements and Genome Evolution conference was hosted by Keystone Symposia in Santa Fe, NM USA, 9 March through 14 March 2014. The goal of this conference was to bring together scientists from around the world who study transposable elements in diverse organisms and researchers who study the impact these elements have on genome evolution. The meeting included over 200 scientists who participated through poster presentations, short talks selected from abstracts, and invited speakers. The talks were organized into eight sessions and two workshops. The topics varied from diverse mechanisms of mobilization to the evolution of genomes and their defense strategies against transposable elements.

IPB7 transposase behavior in Drosophila melanogaster and Aedes aegypti.

Transposons are used in insect science as genetic tools that enable the transformation of insects and the identification and isolation of genes though their ability to insert in or near to them. Four transposons, piggyBac, Mos1, Hermes and Minos are commonly used in insects beyond Drosophila melanogaster with piggyBac, due to its wide host range and frequency of transposition, being the most commonly chosen. The utility of these transposons as genetic tools is directly proportional to their activity since higher transposition rates would be expected to lead to higher transformation frequencies and higher frequencies of insertion throughout the genome. As a consequence there is an ongoing need for hyperactive transposases for use in insect genetics, however these have proven difficult to obtain. IPB7 is a hyperactive mutant of the piggyBac transposase that was identified by a genetic screen performed in yeast, a mammalian codon optimized version of which was then found to be highly active in rodent embryonic stem cells with no apparent deleterious effects. Here we report the activity of IPB7 in D. melanogaster and the mosquito, Aedes aegypti. Somatic transposition assays revealed an increase in IPB7's transposition rate from wild-type piggyBac transposase in D. melanogaster but not Ae. aegypti. However the use of IPB7 in D. melanogaster genetic transformations produced a high rate of sterility and a low transformation rate compared to wild-type transposase. This high rate of sterility was accompanied by significant gonadal atrophy that was also observed in the absence of the piggyBac vector transposon. We conclude that IPB7 has increased activity in the D. melanogaster germ-line but that a component of the sterility associated with its activity is independent of the presence of the piggyBac transposon.