Potentiation of neuronal activity by tonic GluD1 current in brain slices.

Ion channel function of native delta glutamate receptors (GluDR ) is incompletely understood. Previously, we and others have shown that activation of Gαq protein-coupled receptors (GqPCR) produces a slow inward current carried by GluD1R . GluD1R also carries a tonic cation current of unknown cause. Here, using voltage-clamp electrophysiological recordings from adult mouse brain slices containing the dorsal raphe nucleus, we find no role of ongoing G-protein-coupled receptor activity in generating or sustaining tonic GluD1R currents. Neither augmentation nor disruption of G protein activity affects tonic GluD1R currents, suggesting that ongoing G-protein-coupled receptor activity does not give rise to tonic GluD1R currents. Further, the tonic GluD1R current is unaffected by the addition of external glycine or D-serine, which influences GluD2R current at millimolar concentrations. Instead, GqPCR-stimulated and tonic GluD1R currents are regulated by physiological levels of external calcium. In current-clamp recordings, block of GluD1R channels hyperpolarizes the membrane by ~7 mV at subthreshold potentials, reducing excitability. Thus, GluD1R carries a G-protein-independent tonic current that contributes to subthreshold neuronal excitation in the dorsal raphe nucleus.

Spatiotemporal Control of Noradrenaline-Dependent Synaptic Transmission in Mouse Dorsal Raphe Serotonin Neurons.

Activity of dorsal raphe neurons is controlled by noradrenaline afferents. In this brain region, noradrenaline activates Gαq-coupled α1-adrenergic receptors (α1-AR), causing action potential (AP) firing and serotonin release. In vitro, electrical stimulation elicits vesicular noradrenaline release and subsequent activation of α1-AR to produce an EPSC (α1-AR-EPSC). The duration of the α1-AR-EPSC (∼27 s) is much longer than that of most other synaptic currents, but the factors that govern the spatiotemporal dynamics of α1-AR are poorly understood. Using an acute brain slice preparation from adult male and female mice and electrophysiological recordings from dorsal raphe neurons, we found that the time course of the α1-AR-EPSC was slow, but highly consistent within individual serotonin neurons. The amount of noradrenaline released influenced the amplitude of the α1-AR-EPSC without altering the time constant of decay suggesting that once released, extracellular noradrenaline was cleared efficiently. Reuptake of noradrenaline via noradrenaline transporters was a primary means of terminating the α1-AR-EPSC, with little evidence for extrasynaptic diffusion of noradrenaline unless transporter-dependent reuptake was impaired. Taken together, the results demonstrate that despite slow intrinsic signaling kinetics, noradrenaline-dependent synaptic transmission in the dorsal raphe is temporally and spatially controlled and noradrenaline transporters are critical regulators of serotonin neuron excitability. Given the functionally distinct types of neurons intermingled in the dorsal raphe nucleus and the unique roles of these neural circuits in physiological responses, transporters may preserve independence of each synapse to encode a long-lasting but discrete signal.SIGNIFICANCE STATEMENT The dorsal raphe nucleus is the predominant source of serotonin in the brain and is controlled by another monoamine, noradrenaline. In this brain region, noradrenaline activates G-protein-coupled α1-adrenergic receptors (α1-AR) causing action potential (AP) firing and serotonin release. Despite high interest in pharmacotherapies to enhance serotonin signaling, the factors that govern noradrenaline α1-AR signaling have received little attention. Here, we show using mouse brain slices that the time course of α1-AR signaling is slow, persisting for tens of seconds. Despite slow intrinsic signaling kinetics, noradrenaline-dependent synaptic transmission in the dorsal raphe is controlled temporally and spatially by efficient noradrenaline transporter-dependent clearance of extracellular noradrenaline. Thus, noradrenaline transporters are critical regulators of serotonin neuron excitability.

Using Computer Vision to Automate Hand Detection and Tracking of Surgeon Movements in Videos of Open Surgery.

Open, or non-laparoscopic surgery, represents the vast majority of all operating room procedures, but few tools exist to objectively evaluate these techniques at scale. Current efforts involve human expert-based visual assessment. We leverage advances in computer vision to introduce an automated approach to video analysis of surgical execution. A state-of-the-art convolutional neural network architecture for object detection was used to detect operating hands in open surgery videos. Automated assessment was expanded by combining model predictions with a fast object tracker to enable surgeon-specific hand tracking. To train our model, we used publicly available videos of open surgery from YouTube and annotated these with spatial bounding boxes of operating hands. Our model's spatial detections of operating hands significantly outperforms the detections achieved using pre-existing hand-detection datasets, and allow for insights into intra-operative movement patterns and economy of motion.

Plant-necrotroph co-transcriptome networks illuminate a metabolic battlefield.

A central goal of studying host-pathogen interaction is to understand how host and pathogen manipulate each other to promote their own fitness in a pathosystem. Co-transcriptomic approaches can simultaneously analyze dual transcriptomes during infection and provide a systematic map of the cross-kingdom communication between two species. Here we used the Arabidopsis-B. cinerea pathosystem to test how plant host and fungal pathogen interact at the transcriptomic level. We assessed the impact of genetic diversity in pathogen and host by utilization of a collection of 96 isolates infection on Arabidopsis wild-type and two mutants with jasmonate or salicylic acid compromised immunities. We identified ten B. cinereagene co-expression networks (GCNs) that encode known or novel virulence mechanisms. Construction of a dual interaction network by combining four host- and ten pathogen-GCNs revealed potential connections between the fungal and plant GCNs. These co-transcriptome data shed lights on the potential mechanisms underlying host-pathogen interaction.

Novel Technique for Laparoscopic Common Bile Duct Exploration Using Endovascular Instrumentation.

Treatment of choledocholithiasis is sometimes a two-step process involving both surgeons and gastroenterologists. Common bile duct (CBD) exploration can be performed at the same time as cholecystectomy but often requires the use of rigid tools, increasing the risk of CBD damage. Here, we report the case of a 64-year-old man who presented with epigastric pain and a positive Murphy's sign. Ultrasonography revealed cholecystitis with cholelithiasis. Gangrenous cholecystitis was visualized upon surgical exploration, and an intraoperative cholangiogram diagnosed likely choledocholithiasis. Cholecystectomy was completed, and CBD exploration was performed by the manipulation of endovascular equipment using a trans-cystic approach through to the ampulla of Vater, and the patient made a complete recovery without complications. The substantial flexibility, gentleness, and durability of endovascular instruments allow for minimal tension on structures during the removal of gallstones from the CBD, providing safe, definitive treatment for choledocholithiasis during cholecystectomy.

Role of the highly conserved G68 residue in the yeast phosphorelay protein Ypd1: implications for interactions between histidine phosphotransfer (HPt) and response regulator proteins.

BACKGROUND: Many bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins function as key components of these pathways. HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H + 4 position relative to the phosphorylatable histidine is found in a significant number of annotated HPt protein sequences. Previous reports demonstrated that substitutions at this position result in diminished phosphotransfer activity between HPt proteins and their cognate signaling partners. RESULTS: We report the analysis of partner binding interactions and phosphotransfer activity of the prototypical HPt protein Ypd1 from Saccharomyces cerevisiae using a set of H + 4 (G68) substituted proteins. Substitutions at this position with large, hydrophobic, or charged amino acids nearly abolished phospho-acceptance from the receiver domain of its upstream signaling partner, Sln1 (Sln1-R1). An in vitro binding assay indicated that G68 substitutions caused only modest decreases in affinity between Ypd1 and Sln1-R1, and these differences did not appear to be large enough to account for the observed decrease in phosphotransfer activity. The crystal structure of one of these H + 4 mutants, Ypd1-G68Q, which exhibited a diminished ability to participate in phosphotransfer, shows a similar overall structure to that of wild-type. Molecular modelling suggests that the highly conserved active site residues within the receiver domain of Sln1 must undergo rearrangement to accommodate larger H + 4 substitutions in Ypd1. CONCLUSIONS: Phosphotransfer reactions require precise arrangement of active site elements to align the donor-acceptor atoms and stabilize the transition state during the reaction. Any changes likely result in an inability to form a viable transition state during phosphotransfer. Our data suggest that the high degree of evolutionary conservation of residues with small side chains at the H + 4 position in HPt proteins is required for optimal activity and that the presence of larger residues at the H + 4 position would cause alterations in the positioning of active site residues in the partner response regulator.

Digital Imaging Combined with Genome-Wide Association Mapping Links Loci to Plant-Pathogen Interaction Traits.

Plant resistance to generalist pathogens with broad host ranges, such as Botrytis cinerea (Botrytis), is typically quantitative and highly polygenic. Recent studies have begun to elucidate the molecular genetic basis of plant-pathogen interactions using commonly measured traits, including lesion size and/or pathogen biomass. However, with the advent of digital imaging and high-throughput phenomics, there are a large number of additional traits available to study quantitative resistance. In this study, we used high-throughput digital imaging analysis to investigate previously poorly characterized visual traits of plant-pathogen interactions related to disease resistance using the Arabidopsis (Arabidopsis thaliana)/Botrytis pathosystem. From a large collection of visual lesion trait measurements, we focused on color, shape, and size to test how these aspects of the Arabidopsis/Botrytis interaction are genetically related. Through genome-wide association mapping in Arabidopsis, we show that lesion color and shape are genetically separable traits associated with plant disease resistance. Moreover, by employing defined mutants in 23 candidate genes identified from the genome-wide association mapping, we demonstrate links between loci and each of the different plant-pathogen interaction traits. These results expand our understanding of the functional mechanisms driving plant disease resistance.

Bowel endometriosis: diagnosis and management.

The most common location of extragenital endometriosis is the bowel. Medical treatment may not provide long-term improvement in patients who are symptomatic, and consequently most of these patients may require surgical intervention. Over the past century, surgeons have continued to debate the optimal surgical approach to treating bowel endometriosis, weighing the risks against the benefits. In this expert review we will describe how the recommended surgical approach depends largely on the location of disease, in addition to size and depth of the lesion. For lesions approximately 5-8 cm from the anal verge, we encourage conservative surgical management over resection to decrease the risk of short- and long-term complications.

Plastic Transcriptomes Stabilize Immunity to Pathogen Diversity: The Jasmonic Acid and Salicylic Acid Networks within the Arabidopsis/Botrytis Pathosystem.

To respond to pathogen attack, selection and associated evolution has led to the creation of plant immune system that are a highly effective and inducible defense system. Central to this system are the plant defense hormones jasmonic acid (JA) and salicylic acid (SA) and crosstalk between the two, which may play an important role in defense responses to specific pathogens or even genotypes. Here, we used the Arabidopsis thaliana-Botrytis cinerea pathosystem to test how the host's defense system functions against genetic variation in a pathogen. We measured defense-related phenotypes and transcriptomic responses in Arabidopsis wild-type Col-0 and JA- and SA-signaling mutants, coi1-1 and npr1-1, individually challenged with 96 diverse B. cinerea isolates. Those data showed genetic variation in the pathogen influences on all components within the plant defense system at the transcriptional level. We identified four gene coexpression networks and two vectors of defense variation triggered by genetic variation in B. cinerea This showed that the JA and SA signaling pathways functioned to constrain/canalize the range of virulence in the pathogen population, but the underlying transcriptomic response was highly plastic. These data showed that plants utilize major defense hormone pathways to buffer disease resistance, but not the metabolic or transcriptional responses to genetic variation within a pathogen.

Epistasis × environment interactions among Arabidopsis thaliana glucosinolate genes impact complex traits and fitness in the field.

Despite the growing number of studies showing that genotype × environment and epistatic interactions control fitness, the influences of epistasis × environment interactions on adaptive trait evolution remain largely uncharacterized. Across three field trials, we quantified aliphatic glucosinolate (GSL) defense chemistry, leaf damage, and relative fitness using mutant lines of Arabidopsis thaliana varying at pairs of causal aliphatic GSL defense genes to test the impact of epistatic and epistasis × environment interactions on adaptive trait variation. We found that aliphatic GSL accumulation was primarily influenced by additive and epistatic genetic variation, leaf damage was primarily influenced by environmental variation and relative fitness was primarily influenced by epistasis and epistasis × environment interactions. Epistasis × environment interactions accounted for up to 48% of the relative fitness variation in the field. At a single field site, the impact of epistasis on relative fitness varied significantly over 2 yr, showing that epistasis × environment interactions within a location can be temporally dynamic. These results suggest that the environmental dependency of epistasis can profoundly influence the response to selection, shaping the adaptive trajectories of natural populations in complex ways, and deserves further consideration in future evolutionary studies.

The Quantitative Basis of the Arabidopsis Innate Immune System to Endemic Pathogens Depends on Pathogen Genetics.

The most established model of the eukaryotic innate immune system is derived from examples of large effect monogenic quantitative resistance to pathogens. However, many host-pathogen interactions involve many genes of small to medium effect and exhibit quantitative resistance. We used the Arabidopsis-Botrytis pathosystem to explore the quantitative genetic architecture underlying host innate immune system in a population of Arabidopsis thaliana. By infecting a diverse panel of Arabidopsis accessions with four phenotypically and genotypically distinct isolates of the fungal necrotroph B. cinerea, we identified a total of 2,982 genes associated with quantitative resistance using lesion area and 3,354 genes associated with camalexin production as measures of the interaction. Most genes were associated with resistance to a specific Botrytis isolate, which demonstrates the influence of pathogen genetic variation in analyzing host quantitative resistance. While known resistance genes, such as receptor-like kinases (RLKs) and nucleotide-binding site leucine-rich repeat proteins (NLRs), were found to be enriched among associated genes, they only account for a small fraction of the total genes associated with quantitative resistance. Using publically available co-expression data, we condensed the quantitative resistance associated genes into co-expressed gene networks. GO analysis of these networks implicated several biological processes commonly connected to disease resistance, including defense hormone signaling and ROS production, as well as novel processes, such as leaf development. Validation of single gene T-DNA knockouts in a Col-0 background demonstrate a high success rate (60%) when accounting for differences in environmental and Botrytis genetic variation. This study shows that the genetic architecture underlying host innate immune system is extremely complex and is likely able to sense and respond to differential virulence among pathogen genotypes.

Natural genetic variation in Arabidopsis thaliana defense metabolism genes modulates field fitness.

Natural populations persist in complex environments, where biotic stressors, such as pathogen and insect communities, fluctuate temporally and spatially. These shifting biotic pressures generate heterogeneous selective forces that can maintain standing natural variation within a species. To directly test if genes containing causal variation for the Arabidopsis thaliana defensive compounds, glucosinolates (GSL) control field fitness and are therefore subject to natural selection, we conducted a multi-year field trial using lines that vary in only specific causal genes. Interestingly, we found that variation in these naturally polymorphic GSL genes affected fitness in each of our environments but the pattern fluctuated such that highly fit genotypes in one trial displayed lower fitness in another and that no GSL genotype or genotypes consistently out-performed the others. This was true both across locations and within the same location across years. These results indicate that environmental heterogeneity may contribute to the maintenance of GSL variation observed within Arabidopsis thaliana.

A prospective, multi-institutional study of pediatric all-terrain vehicle crashes.

BACKGROUND: Pediatric all-terrain vehicle (ATV) injuries have been increasing annually for more than a decade. The purpose of this study was to prospectively evaluate crash circumstances and clinical outcomes resulting from pediatric ATV crashes. METHODS: Three pediatric trauma centers prospectively collected data from patients during their hospitalization for injuries sustained in ATV crashes from July 2007 through June 2012. Patients completed a 35-item questionnaire describing the crash circumstances (ATV engine size, safety equipment use, and training/experience). Clinical data (injuries, surgical procedures, etc.) were collected for each patient. RESULTS: Eighty-four patients were enrolled, with a mean (SD) age of 13.0 (3.1) years, and were predominantly male (n = 55, 65%). Injuries were musculoskeletal (42%), central nervous system (39%), abdominal (20%), thoracic (16%), and genitourinary (4%). Multisystem injuries were prevalent (27%), and two patients died. Thirty-three patients (43%) required operative intervention. Most children were riding for recreation (96%) and ignored ATV manufacturers' recommendation that children younger than 16 years ride ATVs with smaller (≤90 cc) engines (71%). Dangerous riding practices were widespread: no helmet (70%), no adult supervision (56%), double riding (50%), riding on paved roads (23%), and nighttime riding (16%). Lack of helmet use was significantly associated with head injury (53% vs. 25%, p = 0.03). Rollover crashes were most common (44%), followed by collision with a stationary object (25%) or another vehicle (12%). Half (51%) of children said that they would ride an ATV again. CONCLUSION: These data demonstrate a relationship between dangerous ATV riding behaviors and severe injuries in children who crash. Children younger than 16 years should not operate ATVs, and legislation that effectively restricts ATV use in children is urgently needed. LEVEL OF EVIDENCE: Epidemiologic study, level III.

Repair of esophageal atresia with tracheoesophageal fistula via thoracotomy: a contemporary series.

BACKGROUND: A recent series detailing thoracoscopic repair of esophageal atresia with tracheoesophageal fistula (EA/TEF) reported lower complication rates compared with historic controls. This study provides a contemporary cohort of patients repaired via thoracotomy for comparison with the recent large multi-institutional thoracoscopic series. METHODS: Records of patients with EA/TEF between 1993 and 2008 were reviewed. Attention was focused on demographics and complications including anastomotic leak, recurrent fistulae, stricture formation, and need for fundoplication. RESULTS: Seventy-two patients underwent repair of EA/TEF via thoracotomy. Complication rates in the current series compared with the thoracoscopic series were anastomotic leak, 2.7% versus 7.6%; recurrent fistulae, 2.7% versus 1.9%; stricture, 5.5% versus 3.8%; and need for fundoplication, 12% versus 24%. Differences in complication rates did not reach statistical significance. Two children in this cohort developed mild scoliosis attributed to congenital vertebral anomalies, neither of whom required intervention. CONCLUSIONS: Thoracoscopic repair of EA/TEF yielded complication rates similar to this contemporary series; however, trends toward increased anastomotic leaks and greater need for fundoplication were noted. No musculoskeletal sequelae were directly attributable to thoracotomy.

Should we be concerned about jejunoileal atresia during repair of duodenal atresia?

INTRODUCTION: During repair for duodenal atresia, it has been emphasized that inspection of the small bowel to identify a second atresia is required. The laparoscopic approach for repair of duodenal atresia has been criticized for its limitation to perform this step. Given that duodenal atresia and jejunoileal atresias do not share common embryologic origins, we question the validity of this concern. Therefore, we conducted a multicenter retrospective review of duodenal atresia patients to quantify the incidence of jejunoileal atresia in this population. METHODS: After institutional review board approval (IRB #07-12-187X), a retrospective review was conducted on all patients who have undergone duodenal atresia repair at seven institutions over the past 7-12 years. Demographics and the presence or absence of a jejunoileal atresia were recorded. RESULTS: Four hundred eight patients with duodenal atresia were identified. The mean gestaational age was 36.3 ± 2.9 weeks, and the mean weight was 2.5 ± 0.8 kg. Mean age at operation was 19 days (range, 1-1314). There was a 28% incidence of trisomy 21. Two patients (0.5%) were identified as having a second intestinal atresia, and both were type IIIb. One patient was diagnosed at the time of duodenal atresia repair; the other was a delayed diagnosis. Both patients did well after repair. CONCLUSIONS: In this, the largest series of duodenal atresia patients compiled to date, the rate of a concomitant jejunoileal atresia is less than 1%. This low incidence is not high enough to mandate extensive inspection of the entire bowel in these patients, and a second atresia should not be a concern during laparoscopic repair of duodenal atresia.

Early repair of congenital diaphragmatic hernia on extracorporeal membrane oxygenation.

BACKGROUND: Timing of repair of congenital diaphragmatic hernia (CDH) in babies that require stabilization on extracorporeal membrane oxygenation (ECMO) remains controversial. Although many centers delay operation until physiologic stabilization has occurred or ECMO is no longer needed, we repair soon after ECMO has been initiated. The purpose of this study is to determine if our approach has achieved acceptable morbidity and mortality. METHODS: Charts of live-born babies with CDH treated at our institution between 1993 and 2007 were retrospectively reviewed. Data were then compared with The Congenital Diaphragmatic Hernia Study Group and Extracorporeal Life Support Organization registries. RESULTS: Forty-eight (39%) patients required ECMO Thirty-four of these 48 neonates were cannulated before operative repair. Venoarterial ECMO was used exclusively. The mean (SD) time of repair from cannulation was 55 (21) hours. Survival for this subset of patients was 71%. Three patients (8.8%) who underwent repair on ECMO experienced surgical site hemorrhage that required intervention. CONCLUSION: Early repair of CDH in neonates on ECMO can be accomplished with acceptable rates of morbidity and mortality.

Diminishing role of contrast enema in simple meconium ileus.

PURPOSE: Contrast enema is the initial study of choice for simple meconium ileus to confirm diagnosis and to relieve obstruction. Despite favorable historically published results, our clinical impression suggests decreased effectiveness of the contrast enema resulting in more surgical interventions in contemporary practice. METHODS: A retrospective multiinstitutional review for a 12-year period was conducted for neonates diagnosed with meconium ileus by contrast enema. The neonates were divided into 2 groups-historic group (HG = before 2002) and contemporary group (CG = after 2002). T test was used for comparison of continuous variables and chi(2) for categorical data. RESULTS: Thirty-seven total patients were identified (21 females and 16 males). Obstruction was relieved in 8 neonates (22% overall success rate). Average enema attempt per patient was decreased in the CG group compared to HG (1.4 vs 1.9). The success rate in the CG group was 5.5% (1/18) compared to 39% (7/18) in HG. CONCLUSIONS: In this review, success of contrast enema for relief of meconium ileus has significantly decreased over time. These findings may be because of reluctance to repeat enemas, change in radiologist experience, or use of contrast agent. As a result, higher rates of operative intervention are now observed. In stable patients, surgeons should recommend repeat enemas before exploration.

Probiotic fortified diet reduces bacterial colonization and translocation in a long-term neonatal rabbit model.

BACKGROUND: Probiotic fortified diet reduces bacterial colonization and translocation in a short-term neonatal rabbit model when continuously challenged with pathogen. The purpose of this study was to determine if live probiotic diet could remain effective at decreasing colonization/translocation of pathogens in a long-term neonatal rabbit model without ill effects of the probiotic outside the gastrointestinal (GI) tract. METHODS: Rabbit pups were born via cesarean delivery 1 day preterm and assigned to 2 diets: a newly formulated diet (controls) vs the same diet fortified with the live probiotic Lactoccocus lactis. Enterobacter cloacae was added to both preparations before each feed. Pups were gavage fed twice daily, and weights were recorded. Rabbits were sacrificed on day 7, and organs were harvested and cultured for target organism growth. RESULTS: The probiotic fortified diet resulted in a significant decrease in Enterobacter translocation to the liver and decreased colonization in the stomach and lungs. There was no evidence of Lactococccus translocation or colonization outside of the GI tract. CONCLUSION: This probiotic fortified diet was effective at decreasing pathogenic bacteria colonization and translocation in a long-term neonatal model. The addition of L lactis to the diet resulted in appropriate growth without any colonization or translocation of the probiotic outside of the GI tract.

Return of the surgeon in the diagnosis of pyloric stenosis.

BACKGROUND: The diagnosis of pyloric stenosis (PS) by physical examination is a lost art that has been replaced by radiology-performed ultrasound (US). The purpose of this study is to demonstrate that the diagnosis of PS can be made solely upon the surgeons US evaluation. METHODS: Surgical ultrasonographers included 2 senior general surgery residents and 2 pediatric surgery residents without prior formal US experience. These surgeons underwent proctored training in the use of US for PS. Measurements including channel length and muscle thickness were recorded at bedside. A positive examination included muscle thickness more than 4 mm and channel length more than 16 mm. Patients with positive results underwent pyloromyotomy. Negative results were confirmed with a repeat US through the radiology department, and infants without PS were subsequently referred for appropriate medical management. RESULTS: Thirty-two consecutive patients with suspected PS were evaluated using surgeon-performed ultrasonography. All examinations were diagnostically accurate. There were no false-positive or false-negative result. Seven patients (22%) were correctly determined to be negative for PS. The remaining 25 infants underwent successful pyloromyotomy with resolution of symptoms. CONCLUSION: Surgeons who have undergone focused training to perform US for PS can diagnose the condition without confirmatory testing by a radiologist.