Barcoded overexpression screens in gut Bacteroidales identify genes with roles in carbon utilization and stress resistance.

A mechanistic understanding of host-microbe interactions in the gut microbiome is hindered by poorly annotated bacterial genomes. While functional genomics can generate large gene-to-phenotype datasets to accelerate functional discovery, their applications to study gut anaerobes have been limited. For instance, most gain-of-function screens of gut-derived genes have been performed in Escherichia coli and assayed in a small number of conditions. To address these challenges, we develop Barcoded Overexpression BActerial shotgun library sequencing (Boba-seq). We demonstrate the power of this approach by assaying genes from diverse gut Bacteroidales overexpressed in Bacteroides thetaiotaomicron. From hundreds of experiments, we identify new functions and phenotypes for 29 genes important for carbohydrate metabolism or tolerance to antibiotics or bile salts. Highlights include the discovery of a D-glucosamine kinase, a raffinose transporter, and several routes that increase tolerance to ceftriaxone and bile salts through lipid biosynthesis. This approach can be readily applied to develop screens in other strains and additional phenotypic assays.

Variability in Resource Utilization in the Evaluation and Management of Simple Febrile Seizures Inpatients in US Children's Hospitals.

OBJECTIVE: To characterize resource utilization in the evaluation and treatment of hospitalized simple febrile seizure (SFS) patients in US tertiary pediatric hospitals. METHODS: This is a retrospective cohort study using the Pediatric Health Information System from 2010 to 2015. Children 6 months to 5 years of age who were inpatients with a diagnosis of SFS. Children who had brain magnetic resonance imaging (MRI), electroencephalography (EEG), or received anticonvulsants were compared with those who did not have testing or anticonvulsant treatment. Hospital-level variation in the utilization rates of MRI, EEG, or treatment with anticonvulsants was also evaluated. RESULTS: In Pediatric Health Information System-participating institutions, 8.4% (n=3640) of children presenting to the emergency department with SFS were hospitalized. Among these SFS inpatients, 57.8% (n= 2104) did not receive further evaluation with MRI/EEG or treatment with anticonvulsants. There was evidence of wide inter-hospital variation in resource utilization rates. The median (interquartile range) utilization rate was 6.2% (3.0 to 11.0%) for MRI, 28.5% (16.0 to 46.3%) for EEG and 17.1% (10.9 to 22.3%) for treatment with anticonvulsants. CONCLUSION: No specific hospital-level factors were identified that contributed to the variation in resource utilization in the evaluation and management of hospitalized SFS patients.

Molecular basis for catabolism of the abundant metabolite trans-4-hydroxy-L-proline by a microbial glycyl radical enzyme.

The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration.

Epidemiology and Resource Utilization of Simple Febrile Seizure-associated Hospitalizations in the United States, 2003-2012.

BACKGROUND: Simple febrile seizure (SFS) affects 2% to 4% of children under 6 years of age. The purpose of this study is to examine the epidemiologic patterns and resource utilization of SFS-associated hospitalizations in children aged younger than 6 years of age in the United States. MATERIALS AND METHODS: This study is a serial, retrospective analysis of the Healthcare Cost and Utilization Project Kids' Inpatient Databases for the years 2003, 2006, 2009, and 2012. SFS-associated hospitalizations were identified based on International Classification of Diseases, Ninth Revision, Clinical Modification, diagnosis code 780.31. We calculated the proportion of hospitalizations in pediatric patients under 6 years of age due to SFS and all other nonbirth causes, the rate of SFS-associated hospitalizations per 100,000 population, the mean length of stay and inflation-adjusted hospital costs of SFS-associated hospitalizations, as well as patient demographics and hospital characteristics. RESULTS: From 2003 to 2012, the weighted proportion of hospitalizations due to SFS declined from 0.83% to 0.41% (P<0.01) and the annual rate of SFS-associated hospitalizations per 100,000 population decreased from 48.0 to 18.7 (P<0.01). However, use of computed tomography, electroencephalogram, and lumbar puncture in SFS-associated hospitalizations decreased significantly (all P<0.001), but the utilization rate of magnetic resonance imaging remained stable (P=0.53). The mean length of stay for SFS-associated hospitalizations decreased from 2.03 days in 2003 to 1.74 days in 2012, and the mean hospital costs (exclusive of professional payment) decreased from $3830 in 2003 to $3223 in 2012 (both P<0.001). CONCLUSIONS: SFS-associated hospitalizations and resource utilization in children under 6 years of age have decreased markedly in the United States, probably due to improved clinical adherence to the practice parameters set forth by the American Academy of Pediatrics for managing patients with SFS.

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity.

The discovery of enzymes responsible for previously unappreciated microbial metabolic pathways furthers our understanding of host-microbe and microbe-microbe interactions. We recently identified and characterized a new gut microbial glycyl radical enzyme (GRE) responsible for anaerobic metabolism of trans-4-hydroxy-l-proline (Hyp). Hyp dehydratase (HypD) catalyzes the removal of water from Hyp to generate Δ1-pyrroline-5-carboxylate (P5C). This enzyme is encoded in the genomes of a diverse set of gut anaerobes and is prevalent and abundant in healthy human stool metagenomes. Here, we discuss the roles HypD may play in different microbial metabolic pathways as well as the potential implications of this activity for colonization resistance and pathogenesis within the human gut. Finally, we present evidence of anaerobic Hyp metabolism in sediments through enrichment culturing of Hyp-degrading bacteria, highlighting the wide distribution of this pathway in anoxic environments beyond the human gut.

Analysis of MRI Utilization in Pediatric Patients.

INTRODUCTION: Although animal studies have consistently demonstrated long-term neurocognitive deficits following early anesthetic exposure under certain conditions, risk in human children remains unknown. Magnetic resonance imaging (MRI) in young patients often requires anesthesia to facilitate image acquisition. We studied MRI utilization in a pediatric population, and associated use of anesthesia for ambulatory MRI. MATERIALS AND METHODS: Data were obtained from the New York State Inpatient Database and State Ambulatory Surgery and Services Database on MRI performed in children under the age of 18 years from 2005 to 2011. Demographic characteristics of children who underwent inpatient or ambulatory MRI were evaluated. A subset of ambulatory MRI patients was assessed to evaluate associated use of anesthesia. RESULTS: Overall, 2% of pediatric inpatient discharges (55,036/2,779,507), and 0.5% of pediatric ambulatory encounters (4670/943,520), had MRI performed. Inpatient MRI utilization did not change over time. Ambulatory MRI utilization decreased by 55.2%, from 8.07 per 1000 encounters in 2005 to 2006 to 3.62 per 1000 encounters in 2007 to 2011 (P<0.001). Associated use of anesthesia for ambulatory MRI in children aged below 1 year, 1 to below 6 years, 6 to below 12 years, and 12 to below 18 years, was 91.1%, 83.6%, 74.6%, and 47.5%, respectively. CONCLUSIONS: Significant percentages of MRI in children are associated with use of anesthesia, particularly in younger children, a population potentially more vulnerable to anesthetic neurotoxicity. Discussion of risks and benefits of anesthesia for MRI in children among physicians, patients, and families is recommended.

Use of Anesthesia for Imaging Studies and Interventional Procedures in Children.

Ongoing investigation from the Pediatric Anesthesia NeuroDevelopment Assessment (PANDA) study hopes to examine the long-term effect on cognitive and language development of a single anesthetic exposure in children undergoing inguinal hernia repair. The fifth PANDA Symposium, held in April 2016, continued the mission of previous symposia to examine evidence from basic science and clinical studies on potential neurotoxic effects of anesthetics on developing brain. At the 2016 Symposium, a panel of specialists from nonsurgical pediatric disciplines including anesthesiology, radiology, neurology, gastroenterology, oncology, cardiology, and critical care reviewed use of anesthesia in their practices, including how concern over possible neurodevelopmental effects of early childhood anesthetic exposure has changed discussion with patients and families regarding risks and benefits of imaging studies and interventional procedures involving sedation or anesthesia. This paper summarizes presentations from nonsurgical pediatric specialists at the 2016 PANDA Symposium.

Truncal blocks for perioperative pain management: a review of the literature and evolving techniques.

As the American healthcare system continues to evolve and reimbursement becomes tied to value-based incentive programs, perioperative pain management will become increasingly important. Regional anesthetic techniques are only one component of a successful multimodal pain regimen. In recent years, the use of peripheral and paraneuraxial blocks to provide chest wall and abdominal analgesia has gained popularity. When used within a multimodal regimen, truncal blocks may provide similar analgesia when compared with other regional anesthetic techniques. While there are other reviews that cover this topic, our review will also highlight the emerging role for serratus plane blocks, pectoral nerve blocks and quadratus lumborum blocks in providing thoracic and abdominal analgesia.

Skeletal muscle differentiation evokes endogenous XIAP to restrict the apoptotic pathway.

Myotube apoptosis occurs normally during muscle development and aging but it can lead to destruction of skeletal muscle in neuromuscular diseases. Therefore, understanding how myotube apoptosis is regulated is important for developing novel strategies for treatment of muscle loss. We investigated the regulation of apoptosis in skeletal muscle and report a striking increase in resistance to apoptosis following differentiation. We find mitotic C2C12 cells (myoblast-like cells) are sensitive to cytosolic cytochrome c microinjection. However, differentiated C2C12 cells (myotube-like cells) and primary myotubes are markedly resistant. This resistance is due to endogenous X-linked inhibitor of apoptotic protein (XIAP). Importantly, the selective difference in the ability of XIAP to block myotube but not myoblast apoptosis is not due to a change in XIAP but rather a decrease in Apaf-1 expression. This decrease in Apaf-1 links XIAP to caspase activation and death. Our findings suggest that in order for myotubes to die, they may degrade XIAP, functionally inactivate XIAP or upregulate Apaf-1. Importantly, we identify a role for endogenous Smac in overcoming XIAP to allow myotube death. However, in postmitotic cardiomyocytes, where XIAP also restricts apoptosis, endogenous Smac was not capable of overcoming XIAP to cause death. These results show that as skeletal muscle differentiate, they become resistant to apoptosis because of the ability of XIAP to regulate caspase activation. The increased restriction of apoptosis in myotubes is presumably important to ensure the long term survival of these postmitotic cells as they play a vital role in the physiology of organisms.

Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues.

Brain tumors are typically resistant to conventional chemotherapeutics, most of which initiate apoptosis upstream of mitochondrial cytochrome c release. In this study, we demonstrate that directly activating apoptosis downstream of the mitochondria, with cytosolic cytochrome c, kills brain tumor cells but not normal brain tissue. Specifically, cytosolic cytochrome c is sufficient to induce apoptosis in glioblastoma and medulloblastoma cell lines. In contrast, primary neurons from the cerebellum and cortex are remarkably resistant to cytosolic cytochrome c. Importantly, tumor tissue from mouse models of both high-grade astrocytoma and medulloblastoma display hypersensitivity to cytochrome c when compared with surrounding brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in the tumor tissue compared with low Apaf-1 levels in the adjacent brain tissue. These differences in Apaf-1 abundance correlate with differences in the levels of E2F1, a previously identified activator of Apaf-1 transcription. ChIP assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to the expression of Apaf-1 in brain tumors. Together, these results demonstrate an unexpected sensitivity of brain tumors to postmitochondrial induction of apoptosis. Moreover, they raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancer cells while sparing the surrounding brain parenchyma.