Ubiquitin Modification by the E3 Ligase/ADP-Ribosyltransferase Dtx3L/Parp9.

ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases either conjugate a single ADP-ribose to a target or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+-dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly(ADP-ribose) binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly(ADP-ribose) regulation of E3 activity.

Optimizing Outcomes After Cleft Palate Repair: Design and Implementation of a Perioperative Clinical Care Pathway.

OBJECTIVE: To evaluate the development process and clinical impact of implementing a standardized perioperative clinical care pathway for cleft palate repair. DESIGN: Medical records of patients undergoing primary cleft palate repair prior to pathway implementation were retrospectively reviewed as a historical control group (N = 40). The historical cohort was compared to a prospectively collected group of patients who were treated according to the pathway (N = 40). PATIENTS: Healthy, nonsyndromic infants undergoing primary cleft palate repair at a tertiary care pediatric hospital. INTERVENTIONS: A novel, standardized pathway was created through an iterative process, combining literature review with expert opinion and discussions with institutional stakeholders. The pathway integrated multimodal analgesia throughout the perioperative course and included intraoperative bilateral maxillary nerve blocks. Perioperative protocols for preoperative fasting, case timing, antiemetics, intravenous fluid management, and postoperative diet advancement were standardized. MAIN OUTCOME MEASURES: Primary outcomes include: (1) length of hospital stay, (2) cumulative opioid consumption, (3) oral intake postoperatively. RESULTS: Patients treated according to the pathway had shorter mean length of stay (31 vs 57 hours, P < .001), decreased cumulative morphine consumption (77 vs 727 µg/kg, P < .001), shorter time to initiate oral intake (9.3 vs 22 hours, P = .01), and greater volume of oral intake in first 24 hours postoperatively (379 vs 171 mL, P < .001). There were no differences in total anesthesia time, total surgical time, or complication rates between the control and treatment groups. CONCLUSIONS: Implementation of a standardized perioperative clinical care pathway for primary cleft palate repair is safe, feasible, and associated with reduced length of stay, reduced opioid consumption, and improved oral intake postoperatively.

Children's Pain During IV Induction: A Randomized-Controlled Trial With the MEDi® Robot.

OBJECTIVE: This study examined the impact of a humanoid robot (MEDi®) programmed to teach deep breathing as a coping strategy, on children's pain and fear as primary and secondary outcomes, respectively, during intravenous (IV) line placement. The completion of IV induction was also examined as an exploratory outcome. METHODS: In this randomized controlled, two-armed trial, 137 children (4-12 years) were recruited in Short Stay Surgery at a tertiary pediatric hospital. Patients were randomly assigned to standard care (SC) with Ametop© only (N = 60) or SC and robot-facilitated intervention (N = 59) before induction. Pain and fear before, during, and after IV insertion were rated by patients and observers. RESULTS: No significant differences were found between groups and there were no changes over time for pain or fear (ps > .05). Exploratory analyses show that patients in the MEDi® group were 5.04 times more likely to complete IV induction, compared to SC, Fisher's exact test: X2 (1) = 4.85, p = .04, φc = 0.22, odds ratio = 5.04, 95% CI [1.06, 24.00]. CONCLUSION: This study was the first to examine children's IV induction experience when provided MEDi® support. Reasons for nonsignificance, limitations, and research suggestions were made.

Validation of sonographic assistance for placement of a nasogastric tube in pediatric patients.

PURPOSE: Enteral access via nasogastric tube (NGT) placement can be essential in the provision of care in pediatric patients. Methods exist to confirm correct placement with success rates between 80% and 85%. Radiographic confirmation remains the "gold-standard," however; it exposes patients to ionizing radiation and fails to provide "real-time" information. In this study, we determined the feasibility of using sonography to assist in the placement of NGT insertions in pediatric patients that have difficulty cooperating. METHODS: Thirty patients requiring NGT placement were stratified into three age groups. Upon NGT insertion, transverse and longitudinal ultrasound images were acquired to visualize tube progression through the esophagus. Subsequently, a focused ultrasonographic exam of the gastric antrum and body were performed. If amenable, an air bolus (1 mL/kg) was injected in the stomach if the NGT was not directly visualized. Following intubation, standard guidelines for NGT position confirmation were performed. RESULTS: The NGT was visualized in all esophageal views and 52% of gastric views. Subgroup analysis showed that successful visualization of tube placement in the stomach ranged from 40% (7-18 years) to 70% (3-6 years). Eighty percentage of air boluses injected were visualized. CONCLUSION: The use of ultrasonography may assist NGT placement in pediatric patients and reduce the incidence of suboptimal placement during insertion. We demonstrated successful NGT visualization through the esophagus regardless of age. NGT visualization in the stomach was challenging; however, injection of an air bolus may improve visualization. Further studies are required to improve the success rate of obtaining gastric views of the NGT.

Suprazygomatic maxillary nerve block: an ultrasound and cadaveric study to identify correct sonoanatomical landmarks.

​PURPOSE: Suprazygomatic maxillary nerve blocks (SMB) are used in adult and pediatric patients to provide analgesia for midface surgery and chronic maxillofacial pain syndromes. The ultrasound-guided SMB technique ensures visualisation of the needle tip, avoidance of the maxillary artery and confirmation of local anesthetic spread. The goal of this study was to correctly identify SMB sonoanatomical landmarks to ensure the nerve block is performed safely and effectively. METHODS: Following an ultrasound-guided SMB with dye injection on 2 embalmed cadavers, pre-tragal face-lift style incision with a full thickness flap dissection was performed allowing accurate visualization of the bony landmarks being used for sonography and identification of the location of the injected dye. RESULTS: This study identifies the correct sonoanatomic landmarks as the maxilla and the coronoid process of the mandible which suggests that the block needle tip and local anesthetic injection are within the infratemporal fossa as opposed to the previously reported pterygopalatine fossa. CONCLUSION: An improved understanding of the sonoanatomy will aid clinicians who are learning, performing and teaching the ultrasound-guided suprazygomatic approach to the maxillary nerve block.

Twin Peaks: Covid-19 and the labor market.

This paper develops a choice-theoretic equilibrium model of the labor market in the presence of a pandemic. It includes heterogeneity in productivity, age and the ability to work from home. Worker and firm behavior changes in the presence of the virus, which itself has equilibrium consequences for the infection rate. The model is calibrated to the UK and counterfactual lockdown measures are evaluated. We find a different response in both the evolution of the virus and the labor market with different lockdown policies. A laissez-faire approach results in lives lost and acts as negative shock to the economy. A lockdown policy, absent any other intervention, will reduce the lives lost but increase the economic burden. Consistent with recent evidence, we find that the economic costs from lockdown are most felt by those earning the least. Finally, we introduce a job retention scheme as implemented by the UK Government and find that it spreads the economic hardship more equitably.

De novo mutations in SIK1 cause a spectrum of developmental epilepsies.

Developmental epilepsies are age-dependent seizure disorders for which genetic causes have been increasingly identified. Here we report six unrelated individuals with mutations in salt-inducible kinase 1 (SIK1) in a series of 101 persons with early myoclonic encephalopathy, Ohtahara syndrome, and infantile spasms. Individuals with SIK1 mutations had short survival in cases with neonatal epilepsy onset, and an autism plus developmental syndrome after infantile spasms in others. All six mutations occurred outside the kinase domain of SIK1 and each of the mutants displayed autophosphorylation and kinase activity toward HDAC5. Three mutations generated truncated forms of SIK1 that were resistant to degradation and also showed changes in sub-cellular localization compared to wild-type SIK1. We also report the human neuropathologic examination of SIK1-related developmental epilepsy, with normal neuronal morphology and lamination but abnormal SIK1 protein cellular localization. Therefore, these results expand the genetic etiologies of developmental epilepsies by demonstrating SIK1 mutations as a cause of severe developmental epilepsy.

The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

BACKGROUND: Phosphoinositide-3 (PI-3) kinase signaling has a pervasive role in cancer. One of the key effectors of PI-3 kinase signaling is AKT, a kinase that promotes growth and survival in a variety of cancers. Genetically engineered mouse models of prostate cancer have shown that AKT signaling is sufficient to induce prostatic epithelial neoplasia (PIN), but insufficient for progression to adenocarcinoma. This contrasts with the phenotype of mice with prostate-specific deletion of Pten, where excessive PI-3 kinase signaling induces both PIN and locally invasive carcinoma. We reasoned that additional PI-3 kinase effector kinases promote prostate cancer progression via activities that provide biological complementarity to AKT. We focused on the PKN kinase family members, which undergo activation in response to PI-3 kinase signaling, show expression changes in prostate cancer, and contribute to cell motility pathways in cancer cells. METHODS: PKN kinase activity was measured by incorporation of 32 P into protein substrates. Phosphorylation of the turn-motif (TM) in PKN proteins by mTOR was analyzed using the TORC2-specific inhibitor torin and a PKN1 phospho-TM-specific antibody. Amino acid substitutions in the TM of PKN were engineered and assayed for effects on kinase activity. Cell motility-related functions and PKN localization was analyzed by depletion approaches and immunofluorescence microscopy, respectively. The contribution of PKN proteins to prostate tumorigenesis was characterized in several mouse models that express PKN transgenes. The requirement for PKN activity in prostate cancer initiated by loss of phosphatase and tensin homolog deleted on chromosome 10 (Pten), and the potential redundancy between PKN isoforms, was analyzed by prostate-specific deletion of Pkn1, Pkn2, and Pten. RESULTS AND CONCLUSIONS: PKN1 and PKN2 contribute to motility pathways in human prostate cancer cells. PKN1 and PKN2 kinase activity is regulated by TORC2-dependent phosphorylation of the TM, which together with published data indicates that PKN proteins receive multiple PI-3 kinase-dependent inputs. Transgenic expression of active AKT and PKN1 is not sufficient for progression beyond PIN. Moreover, Pkn1 is not required for tumorigenesis initiated by loss of Pten. Triple knockout of Pten, Pkn1, and Pkn2 in mouse prostate results in squamous cell carcinoma, an uncommon but therapy-resistant form of prostate cancer.

Failed caudal block due to physiologic changes associated with a cerebrospinal fluid leak: a case report.

PURPOSE: The sum of the volumes of brain tissue, cerebrospinal fluid (CSF), and intracranial blood remain constant. This tenet of the Monroe-Kellie hypothesis is most often considered in the setting of intracranial hypertension, but it can also be applied in the setting of CSF volume depletion. We used this hypothesis to explain a case of failed caudal block in a patient with an iatrogenic CSF leak. CLINICAL FEATURES: A one-month-old baby (3 kg) born at 35 weeks' gestation presented for right inguinal hernia repair. His past medical history was significant for arthrogryposis (congenital joint contractures in two or more areas of the body) as well as ongoing apneic episodes that required continuous positive airway pressure therapy and neonatal intensive care. An ultrasound confirmed caudal block was completed and within five minutes of the procedure, the patient's heart rate increased, with an accompanying slight increase in T-wave amplitude. Pinch tests revealed anesthesia to the feet bilaterally but insufficient anesthesia to the abdomen. The surgery was delayed but successfully completed under general anesthesia the following week. Magnetic resonance imaging of the brain and spine following the surgery showed a significant CSF leak with engorgement of the epidural venous plexus along the entire spine. These findings were consistent with a CSF leak likely secondary to a prior lumbar puncture (at age 13 days) that was part of the investigation of his respiratory issues. CONCLUSIONS: The possible mechanism of this failed caudal block was high systemic absorption of anesthetic given the epidural venous plexus engorgement thus leaving less anesthetic acting within the CSF and on the exiting spinal nerves. Decreased CSF flow in the thecal sac might also have contributed, as might dilution of the remaining local anesthetic caused by large amounts of leaking CSF within the epidural space.

Ultrasound assessment of gastric volume in the fasted pediatric patient undergoing upper gastrointestinal endoscopy: development of a predictive model using endoscopically suctioned volumes.

BACKGROUND: Aspiration of gastric contents can be a serious anesthetic-related complication. Gastric antral sonography prior to anesthesia may have a role in identifying pediatric patients at risk of aspiration. We examined the relationship between sonographic antral area and endoscopically suctioned gastric volumes, and whether a 3-point qualitative grading system is applicable in pediatric patients. METHODS: Fasted patients presenting to a pediatric hospital for upper gastrointestinal endoscopy were included in the study. Sonographic measurement of the antral cross-sectional area (CSA) in supine (supine CSA) and right lateral decubitus (RLD CSA) position was completed, and the antrum was designated as empty or nonempty. Gastric contents were endoscopically suctioned and measured. Multiple regression analysis was used to fit a mathematical model to estimate gastric volume. RESULTS: One hundred patients (aged 11-216 months) were included. The gastric antrum was measured in 94% and 99% of patients in the supine and RLD positions, respectively. Gastric antral CSA correlated with total gastric volume in both supine (ρ = 0.63) and RLD (ρ = 0.67) positions. A mathematical model incorporating RLD CSA and age (R(2)  = 0.60) was determined as the best-fit model to predict gastric volumes. Increasing gastric antral grade (0-2) was associated with increasing gastric fluid volume. CONCLUSION: The results suggest that sonographic assessment of the gastric antrum provides useful information regarding gastric content (empty versus nonempty) and volume (ml·kg(-1) ) in pediatric patients. Results suggest that the three-point grading system may be a valuable tool to assess gastric 'fullness' based on a qualitative exam of the antrum.

The Androgen Receptor Does Not Directly Regulate the Transcription of DNA Damage Response Genes.

The clinical success of combined androgen deprivation therapy (ADT) and radiotherapy (RT) in prostate cancer created interest in understanding the mechanistic links between androgen receptor (AR) signaling and the DNA damage response (DDR). Convergent data have led to a model where AR both regulates, and is regulated by, the DDR. Integral to this model is that the AR regulates the transcription of DDR genes both at a steady state and in response to ionizing radiation (IR). In this study, we sought to determine which immediate transcriptional changes are induced by IR in an AR-dependent manner. Using PRO-seq to quantify changes in nascent RNA transcription in response to IR, the AR antagonist enzalutamide, or the combination of the two, we find that enzalutamide treatment significantly decreased expression of canonical AR target genes but had no effect on DDR gene sets in prostate cancer cells. Surprisingly, we also found that the AR is not a primary regulator of DDR genes either in response to IR or at a steady state in asynchronously growing prostate cancer cells. IMPLICATIONS: Our data indicate that the clinical benefit of combining ADT with RT is not due to direct AR regulation of DDR gene transcription, and that the field needs to consider alternative mechanisms for this clinical benefit.

Enabling genomic island prediction and comparison in multiple genomes to investigate bacterial evolution and outbreaks.

Outbreaks of virulent and/or drug-resistant bacteria have a significant impact on human health and major economic consequences. Genomic islands (GIs; defined as clusters of genes of probable horizontal origin) are of high interest because they disproportionately encode virulence factors, some antimicrobial-resistance (AMR) genes, and other adaptations of medical or environmental interest. While microbial genome sequencing has become rapid and inexpensive, current computational methods for GI analysis are not amenable for rapid, accurate, user-friendly and scalable comparative analysis of sets of related genomes. To help fill this gap, we have developed IslandCompare, an open-source computational pipeline for GI prediction and comparison across several to hundreds of bacterial genomes. A dynamic and interactive visualization strategy displays a bacterial core-genome phylogeny, with bacterial genomes linearly displayed at the phylogenetic tree leaves. Genomes are overlaid with GI predictions and AMR determinants from the Comprehensive Antibiotic Resistance Database (CARD), and regions of similarity between the genomes are also displayed. GI predictions are performed using Sigi-HMM and IslandPath-DIMOB, the two most precise GI prediction tools based on nucleotide composition biases, as well as a novel blast-based consistency step to improve cross-genome prediction consistency. GIs across genomes sharing sequence similarity are grouped into clusters, further aiding comparative analysis and visualization of acquisition and loss of mobile GIs in specific sub-clades. IslandCompare is an open-source software that is containerized for local use, plus available via a user-friendly, web-based interface to allow direct use by bioinformaticians, biologists and clinicians (at https://islandcompare.ca).

Canadian surveillance study of complex regional pain syndrome in children.

ABSTRACT: This study describes the minimum incidence of pediatric complex regional pain syndrome (CRPS), clinical features, and treatments recommended by pediatricians and pain clinics in Canada. Participants in the Canadian Paediatric Surveillance Program reported new cases of CRPS aged 2 to 18 years monthly and completed a detailed case reporting questionnaire from September 2017 to August 2019. Descriptive analysis was completed, and the annual incidence of CRPS by sex and age groupings was estimated. A total of 198 cases were reported to the Canadian Paediatric Surveillance Program, and 168 (84.8%) met the case definition. The minimum Canadian incidence of CRPS is estimated at 1.14/100,000 (95% confidence interval 0.93-1.35/100,000) children per year. Incidence was highest among girls 12 years and older (3.10, 95% confidence interval 2.76-3.44/100,000). The mean age of CRPS diagnosis was 12.2 years (SD = 2.4), with the mean time from symptom onset to diagnosis of 5.6 months (SD = 9.9) and no known inciting event for 19.6% of cases. Most cases had lower limb involvement (79.8%). Nonsteroidal anti-inflammatory drugs (82.7%) and acetaminophen (66.0%) were prescribed more commonly than antiepileptic drugs (52.3%) and antidepressants (32.0%). Referrals most commonly included physical therapy (83.3%) and multidisciplinary pain clinics (72.6%); a small number of patients withdrew from treatment because of pain exacerbation (5.3%). Pain education was recommended for only 65.6% of cases. Treatment variability highlights the need for empiric data to support treatment of pediatric CRPS and development of treatment consensus guidelines.

How does the rate of dentoalveolar distraction affect the bone regenerate produced?

INTRODUCTION: This experimental study was designed to evaluate how the rate of dentoalveolar distraction into a bony defect affects bone quality and quantity. METHODS: Using 6 adult foxhound dogs and a randomized split-mouth design, we evaluated the differences between regenerate bone produced by distracting segments of bone containing the second premolars at either 1 or 2 mm per day for 5 days, followed by a 6-week consolidation period. Microcomputed tomography was used to evaluate bone density, percent bone volume, trabecular number, trabecular separation, and trabecular thickness. RESULTS: The lingual aspect of the regenerate exhibited more bone than did the buccal aspect, and all but one of the 12 specimens showed less than 1 mm of vertical bone deficiency in the regenerate area. No differences were found between the 1-mm per day and the 2-mm per day rates for bone density, percent bone volume, trabecular number, trabecular separation, and trabecular thickness. With the exception of trabecular separation on the 2-mm per day side (P = 0.030), there were no statistically significant differences between the mesial, middle, and distal segments of the regenerate. Compared with control bone, the regenerate bone was less dense, and had less bone volume, a higher trabecular number, and approximately half the trabecular thickness. CONCLUSIONS: Bone regenerate produced by rates of 1 and 2 mm per day of dentoalveolar distraction was similar in quality and quantity. Although less mature, the size and shape of the regenerate bone produced by rapid dentoalveolar distraction was comparable with the control bone.