A distinct transcriptome characterizes neural crest-derived cells at the migratory wavefront during enteric nervous system development.

Enteric nervous system development relies on intestinal colonization by enteric neural crest-derived cells (ENCDCs). This is driven by a population of highly migratory and proliferative ENCDCs at the wavefront, but the molecular characteristics of these cells are unknown. ENCDCs from the wavefront and the trailing region were isolated and subjected to RNA-seq. Wavefront-ENCDCs were transcriptionally distinct from trailing ENCDCs, and temporal modelling confirmed their relative immaturity. This population of ENCDCs exhibited altered expression of ECM and cytoskeletal genes, consistent with a migratory phenotype. Unlike trailing ENCDCs, the wavefront lacked expression of genes related to neuronal or glial maturation. As wavefront ENCDC genes were associated with migration and developmental immaturity, the genes that remain expressed in later progenitor populations may be particularly pertinent to understanding the maintenance of ENCDC progenitor characteristics. Dusp6 expression was specifically upregulated at the wavefront. Inhibiting DUSP6 activity prevented wavefront colonization of the hindgut, and inhibited the migratory ability of post-colonized ENCDCs from midgut and postnatal neurospheres. These effects were reversed by simultaneous inhibition of ERK signaling, indicating that DUSP6-mediated ERK inhibition is required for ENCDC migration in mouse and chick.

Association Between Antibiotic Redosing Before Incision and Risk of Incisional Site Infection in Children With Appendicitis.

OBJECTIVE: To evaluate whether redosing antibiotics within an hour of incision is associated with a reduction in incisional surgical site infection (iSSI) in children with appendicitis. BACKGROUND: Existing data remain conflicting as to whether children with appendicitis receiving antibiotics at diagnosis benefit from antibiotic redosing before incision. METHODS: This was a multicenter retrospective cohort study using data from the Pediatric National Surgical Quality Improvement Program augmented with antibiotic utilization and operative report data obtained though supplemental chart review. Children undergoing appendectomy at 14 hospitals participating in the Eastern Pediatric Surgery Network from July 2016 to June 2020 who received antibiotics upon diagnosis of appendicitis between 1 and 6 hours before incision were included. Multivariable logistic regression was used to compare odds of iSSI in those who were and were not redosed with antibiotics within 1 hour of incision, adjusting for patient demographics, disease severity, antibiotic agents, and hospital-level clustering of events. RESULTS: A total of 3533 children from 14 hospitals were included. Overall, 46.5% were redosed (hospital range: 1.8%-94.4%, P <0.001) and iSSI rates were similar between groups [redosed: 1.2% vs non-redosed: 1.3%; odds ratio (OR) 0.84, (95%,CI, 0.39-1.83)]. In subgroup analyses, redosing was associated with lower iSSI rates when cefoxitin was used as the initial antibiotic (redosed: 1.0% vs nonredosed: 2.5%; OR: 0.38, (95% CI, 0.17-0.84)], but no benefit was found with other antibiotic regimens, longer periods between initial antibiotic administration and incision, or with increased disease severity. CONCLUSIONS: Redosing of antibiotics within 1 hour of incision in children who received their initial dose within 6 hours of incision was not associated with reduction in risk of incisional site infection unless cefoxitin was used as the initial antibiotic.

RET overactivation leads to concurrent Hirschsprung disease and intestinal ganglioneuromas.

Appropriately balanced RET signaling is of crucial importance during embryonic neural crest cell migration, proliferation and differentiation. RET deficiency, for example, leads to intestinal aganglionosis (Hirschsprung disease), whereas overactive RET can lead to multiple endocrine neoplasia (MEN) syndromes. Some RET mutations are associated with both intestinal aganglionosis and MEN-associated tumors. This seemingly paradoxical occurrence has led to speculation of a 'Janus mutation' in RET that causes overactivation or impairment of RET activity depending on the cellular context. Using an intestinal catenary culture system to test the effects of GDNF-mediated RET activation, we demonstrate the concurrent development of distal colonic aganglionosis and intestinal ganglioneuromas. Interestingly, the tumors induced by GDNF stimulation contain enteric neuronal progenitors capable of reconstituting an enteric nervous system when transplanted into a normal developmental environment. These results suggest that a Janus mutation may not be required to explain co-existing Hirschsprung disease and MEN-associated tumors, but rather that RET overstimulation alone is enough to cause both phenotypes. The results also suggest that reprogramming tumor cells toward non-pathological fates may represent a possible therapeutic avenue for MEN-associated neoplasms.

Loss of the polarity protein PAR3 activates STAT3 signaling via an atypical protein kinase C (aPKC)/NF-κB/interleukin-6 (IL-6) axis in mouse mammary cells.

PAR3 suppresses tumor growth and metastasis in vivo and cell invasion through matrix in vitro. We propose that PAR3 organizes and limits multiple signaling pathways and that inappropriate activation of these pathways occurs without PAR3. Silencing Pard3 in conjunction with oncogenic activation promotes invasion and metastasis via constitutive STAT3 activity in mouse models, but the mechanism for this is unknown. We now show that loss of PAR3 triggers increased production of interleukin-6, which induces STAT3 signaling in an autocrine manner. Activation of atypical protein kinase C ι/λ (aPKCι/λ) mediates this effect by stimulating NF-κB signaling and IL-6 expression. Our results suggest that PAR3 restrains aPKCι/λ activity and thus prevents aPKCι/λ from activating an oncogenic signaling network.

Agrin Inhibition in Enteric Neural Stem Cells Enhances Their Migration Following Colonic Transplantation.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced.

Implementation and Evaluation of an Academic Development Rotation for Surgery Residents.

OBJECTIVE: To describe the design, implementation, and evaluation of a two-week rotation intended to enhance junior surgical residents' preparation for their dedicated professional development time (PDT) and academic careers. DESIGN: As part of a multifaceted effort to promote residents' academic development, we designed a two-week, nonclinical "Academic Development Block" (ADB) rotation for postgraduate year (PGY)-2 and -3 residents. During this rotation, residents meet with clinical, research, and peer mentors and work on academic activities, with relevant deliverables specific to each class year. We analyzed feedback from postrotation surveys and interviews, which were inductively coded and thematically analyzed, and data on resident grant applications and earnings before and after implementation. SETTING: The general surgery residency program at a major urban, university-affiliated academic medical center. ADBs were first implemented in 2021. PARTICIPANTS: A total of 39 PGY-2 and PGY-3 residents rotated through the program with 51 ADBs over the first two years of implementation. RESULTS: Surveys indicated overwhelmingly positive perceptions on the value of ADBs, including the amount of structure and resources available. Free-response and interview themes indicated appreciation for time to meet with mentors, develop ideas, and complete academic work. Residents believed the ADB rotation accelerated their transition into PDT and was a marker of institutional commitment. Areas for improvement pertained to the timing of ADBs and pairing of mentors. Both cohorts who participated in at least 1 ADB had higher proportions of residents who successfully applied for grants and a greater amount of total funding awarded compared to all 4 of the most recent cohorts prior to implementation. CONCLUSIONS: A short academic development rotation protected from clinical responsibilities is a well-regarded intervention to help residents refine their career goals and prepare for their PDT. Similar initiatives may be of interest to residency programs seeking to foster their residents' academic career development.

Mature enteric neurons have the capacity to reinnervate the intestine with glial cells as their guide.

Here, we establish that plasticity exists within the postnatal enteric nervous system by demonstrating the reinnervation potential of post-mitotic enteric neurons (ENs). Employing BAF53b-Cre mice for selective neuronal tracing, the reinnervation capabilities of mature postnatal ENs are shown across multiple model systems. Isolated ENs regenerate neurites in vitro, with neurite complexity and direction influenced by contact with enteric glial cells (EGCs). Nerve fibers from transplanted ENs exclusively interface and travel along EGCs within the muscularis propria. Resident EGCs persist after Cre-dependent ablation of ENs and govern the architecture of the myenteric plexus for reinnervating ENs, as shown by nerve fiber projection tracing. Transplantation and optogenetic experiments in vivo highlight the rapid reinnervation potential of post-mitotic neurons, leading to restored gut muscle contractile activity within 2 weeks. These studies illustrate the structural and functional reinnervation capacity of post-mitotic ENs and the critical role of EGCs in guiding and patterning their trajectories.

A call for a unified and multimodal definition of cellular identity in the enteric nervous system.

The enteric nervous system (ENS) is a tantalizing frontier in neuroscience. With the recent emergence of single cell transcriptomic technologies, this rare and poorly understood tissue has begun to be better characterized in recent years. A precise functional mapping of enteric neuron diversity is critical for understanding ENS biology and enteric neuropathies. Nonetheless, this pursuit has faced considerable technical challenges. By leveraging different methods to compare available primary mouse and human ENS datasets, we underscore the urgent need for careful identity annotation, achieved through the harmonization and advancements of wet lab and computational techniques. We took different approaches including differential gene expression, module scoring, co-expression and correlation analysis, unbiased biological function hierarchical clustering, data integration and label transfer to compare and contrast functional annotations of several independently reported ENS datasets. These analyses highlight substantial discrepancies stemming from an overreliance on transcriptomics data without adequate validation in tissues. To achieve a comprehensive understanding of enteric neuron identity and their functional context, it is imperative to expand tissue sources and incorporate innovative technologies such as multiplexed imaging, electrophysiology, spatial transcriptomics, as well as comprehensive profiling of epigenome, proteome, and metabolome. Harnessing human pluripotent stem cell (hPSC) models provides unique opportunities for delineating lineage trees of the human ENS, and offers unparalleled advantages, including their scalability and compatibility with genetic manipulation and unbiased screens. We encourage a paradigm shift in our comprehension of cellular complexity and function in the ENS by calling for large-scale collaborative efforts and research investments.

Resolving Resident Colonic Muscularis Macrophage Diversity and Plasticity During Colitis.

BACKGROUND: Immune cell populations in the intestinal muscularis propria during colitis are poorly resolved. Maintaining homeostasis in this niche is critical, highlighted by the poorer prognosis of inflammatory bowel disease associated with muscularis propria inflammation. METHODS: This study utilizes single-cell RNA sequencing to survey the immune cell populations within the muscularis propria of normal colon and dextran sodium sulfate-induced colitis. Findings are validated by immunohistochemistry, flow cytometry and cell-lineage tracing in vivo, and in vitro assays with muscularis macrophages (MMφ). RESULTS: In naïve conditions, transcriptional duality is observed in MMφs with 2 major subpopulations: conventional resident Cx3cr1+ MMφs and Lyve1+ MMφs. The Lyve1+ population is phagocytic and expresses several known MMφ markers in mouse and human, confirming their identity as a bona fide MMφ subset. Single-cell transcriptomics indicate that resident MMφs are retained during colitis and exhibit plasticity toward an inflammatory profile. Lyve1+ MMφs, which express anti-inflammatory marker CD163, are absent during colitis, as confirmed by flow cytometry. In contrast, lineage tracing finds that resident Cx3cr1+ MMφs remain during colitis and are not completely replaced by the inflammatory infiltrating monocytes. In vitro studies provide biological evidence of the plasticity of resident Cx3cr1+ MMφs in response to lipopolysaccharide (LPS), mirroring transcriptional observations in vivo of their inflammatory plasticity. Potential markers for colitic MMφs, validated in animal models and in individuals with ulcerative colitis, are identified. CONCLUSIONS: Our findings contribute to the understanding of the immune system in the muscularis propria niche during colitis by resolving the heterogeneity and origins of colitic MMφs.

Involvement of the muscularis propria accompanies a poorer prognosis in IBD. This study characterizes muscularis macrophage subpopulations during colitis, highlighting the loss of anti-inflammatory LYVE-1+ macrophages and inflammatory plasticity in resident CX3CR1+ macrophages, providing insights into prognostic and therapeutic targets.

Primary payer status is associated with mortality and resource utilization for coronary artery bypass grafting.

BACKGROUND: Medicaid and uninsured populations are a significant focus of current healthcare reform. We hypothesized that outcomes after coronary artery bypass grafting (CABG) in the United States is dependent on primary payer status. METHODS AND RESULTS: From 2003 to 2007, 1,250,619 isolated CABG operations were evaluated using the Nationwide Inpatient Sample (NIS) database. Patients were stratified by primary payer status: Medicare, Medicaid, uninsured, and private insurance. Hierarchical multiple regression models were applied to assess the effect of primary payer status on postoperative outcomes. Unadjusted mortality for Medicare (3.3%), Medicaid (2.4%), and uninsured (1.9%) patients were higher compared with private insurance patients (1.1%, P<0.001). Unadjusted length of stay was longest for Medicaid patients (10.9 ± 0.04 days) and shortest for private insurance patients (8.0 ± 0.01 days, P<0.001). Medicaid patients accrued the highest unadjusted total costs ($113 380 ± 386, P<0.001). Importantly, after controlling for patient risk factors, income, hospital features, and operative volume, Medicaid (odds ratio, 1.82; P<0.001) and uninsured (odds ratio, 1.62; P<0.001) payer status independently conferred the highest adjusted odds of in-hospital mortality. In addition, Medicaid payer status was associated with the longest adjusted length of stay and highest adjusted total costs (P<0.001). CONCLUSIONS: Medicaid and uninsured payer status confers increased risk adjusted in-hospital mortality for patients undergoing coronary artery bypass grafting operations. Medicaid was further associated with the greatest adjusted length of stay and total costs despite risk factors. Possible explanations include delays in access to care or disparate differences in health maintenance.

Differentiated neuroblastoma cells remain epigenetically poised for de-differentiation to an immature state.

Neuroblastoma is the most common extracranial solid tumor of childhood and accounts for a significant share of childhood cancer deaths. Prior studies utilizing RNA sequencing of bulk tumor populations showed two predominant cell states characterized by high and low expression of neuronal genes. Although cells respond to treatment by altering their gene expression, it is unclear whether this reflects shifting balances of distinct subpopulations or plasticity of individual cells. Using mouse and human neuroblastoma cell lines lacking MYCN amplification, we show that the antigen CD49b (also known as ITGA2) distinguishes these subpopulations. CD49b expression marked proliferative cells with an immature gene expression program, whereas CD49b-negative cells expressed differentiated neuronal marker genes and were non-cycling. Sorted populations spontaneously switched between CD49b expression states in culture, and CD49b-negative cells could generate rapidly growing, CD49b-positive tumors in mice. Although treatment with the chemotherapy drug doxorubicin selectively killed CD49b-positive cells in culture, the CD49b-positive population recovered when treatment was withdrawn. We profiled histone 3 (H3) lysine 27 acetylation (H3K27ac) to identify enhancers and super enhancers that were specifically active in each population and found that CD49b-negative cells maintained the priming H3 lysine 4 methylation (H3K4me1) mark at elements that were active in cells with high expression of CD49b. Improper maintenance of primed enhancer elements might thus underlie cellular plasticity in neuroblastoma, representing potential therapeutic targets for this lethal tumor.

Single-cell multiome sequencing clarifies enteric glial diversity and identifies an intraganglionic population poised for neurogenesis.

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to simultaneously assess transcription and chromatin accessibility in EGCs undergoing spontaneous neurogenesis in culture, as well as small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. A subset of in vivo EGCs, highly enriched within the myenteric ganglia and that persist into adulthood, have a gene expression program and chromatin state consistent with neurogenic potential. These results clarify the mechanisms underlying EGC potential for neuronal fate transition.

Applications of Single-Cell Sequencing Technology to the Enteric Nervous System.

With recent technical advances and diminishing sequencing costs, single-cell sequencing modalities have become commonplace. These tools permit analysis of RNA expression, DNA sequence, chromatin structure, and cell surface antigens at single-cell resolution. Simultaneous measurement of numerous parameters can resolve populations including rare cells, thus revealing cellular diversity within organs and permitting lineage reconstruction in developing tissues. Application of these methods to the enteric nervous system has yielded a wealth of data and biological insights. We review recent papers applying single-cell sequencing tools to the nascent neural crest and to the developing and mature enteric nervous system. These studies have shown significant diversity of enteric neurons and glia, suggested paradigms for neuronal specification, and revealed signaling pathways active during development. As technology evolves and multiome techniques combining two or more of transcriptomic, genomic, epigenetic, and proteomic data become prominent, we anticipate these modalities will become commonplace in ENS research and may find a role in diagnostic testing and personalized therapeutics.

Ednrb -/- mice with hirschsprung disease are missing Gad2-expressing enteric neurons in the ganglionated small intestine.

Hirschsprung disease is most often characterized by aganglionosis limited to the distal colon and rectum, and mice lacking the Endothelin receptor type B (Ednrb) faithfully recapitulate this phenotype. However, despite the presence of enteric ganglia in the small intestine, both human patients and Ednrb-/- mice suffer from dysmotility and altered gastrointestinal function, thus raising the possibility of enteric nervous system (ENS) abnormalities proximal to the aganglionic region. We undertook the present study to determine whether abnormalities with the ENS in ganglionated regions may account for abnormal gastrointestinal function. We performed single-cell RNA sequencing on ENS cells from the small intestine of Ednrb-/- mice and compared the results to a published single-cell dataset. Our results identified a missing population of neurons marked by the enzyme Gad2, which catalyzes the production of γ-Aminobutyric acid (GABA), in the small intestine of Ednrb-/- animals. This result was confirmed by immunostaining enteric ganglia from Ednrb-/- mice and their wild-type littermates. These data show for the first time that ganglionated regions of the Hirschsprung gut lack a neuronal subpopulation, which may explain the persistent gastrointestinal dysfunction after surgical correction of Hirschsprung disease.

Tamoxifen administration alters gastrointestinal motility in mice.

BACKGROUND: Tamoxifen is widely used for Cre-estrogen receptor-mediated genomic recombination in transgenic mouse models to mark cells for lineage tracing and to study gene function. However, recent studies have highlighted off-target effects of tamoxifen in various tissues and cell types when used for induction of Cre recombination. Despite the widespread use of these transgenic Cre models to assess gastrointestinal (GI) function, the effect of tamoxifen exposure on GI motility has not been described. METHODS: We examined the effects of tamoxifen on GI motility by measuring total GI transit, gastric emptying, small intestinal transit, and colonic contractility in wild-type adult mice. KEY RESULTS: We observed a significant delay in total GI transit in tamoxifen-treated mice, with unaltered gastric emptying, accelerated small intestinal transit, and abnormal colonic motility. CONCLUSION: Our findings highlight the importance of considering GI motility alterations induced by tamoxifen when designing protocols that utilize tamoxifen as a Cre-driver for studying GI function.

Acute pain consult and management is associated with improved mortality in rib fracture patients.

INTRODUCTION: Traumatic rib fractures result in significant patient morbidity and mortality, which increases with patient age and number of rib fractures. A dedicated acute pain service (APS) providing expertize in multimodal pain management may reduce these risks and improve outcomes. We aimed to test the hypothesis that protocolized APS consultation decreases mortality and morbidity in traumatic rib fracture patients. METHODS: This is a retrospective observational, propensity-matched cohort study of adult patients with trauma with rib fractures from 2012 to 2015, at a single, large level 1 trauma center corresponding to introduction and incorporation of APS consultation into the institutional rib fracture pathway. Using electronic medical records and trauma registry data, we identified adult patients presenting with traumatic rib fractures. Patients with hospital length of stay (LOS) ≥2 days were split into two cohorts based on presence of APS consult using 1:1 propensity matching of age, gender, comorbidities and injury severity. The primary outcome was difference in hospital mortality. Secondary outcomes included LOS and pulmonary morbidity. RESULTS: 2486 patients were identified, with a final matched cohort of 621 patients receiving APS consult and 621 control patients. The mortality rate was 1.8% among consult patients and 6.6% among control patients (adjusted OR 0.25, 95% CI 0.13 to 0.50; p=0.001). The average treatment effect of consult on mortality was 4.8% (95% CI 1.2% to 8.5%;. p<0.001). APS consultation was associated with increased intensive care unit (ICU) LOS (1.19 day; 95% CI 0.48 to 1.90; p=0.001) and hospital LOS (1.61 days; 95% CI 0.81 to 2.41 days; p<0.001). No difference in pulmonary complications was observed. DISCUSSION: An APS consult in rib fracture patients is associated with decreased mortality and no difference in pulmonary complications yet increased ICU and hospital LOS.

Schwann cells in the subcutaneous adipose tissue have neurogenic potential and can be used for regenerative therapies.

Stem cell therapies for nervous system disorders are hindered by a lack of accessible autologous sources of neural stem cells (NSCs). In this study, neural crest-derived Schwann cells are found to populate nerve fiber bundles (NFBs) residing in mouse and human subcutaneous adipose tissue (SAT). NFBs containing Schwann cells were harvested from mouse and human SAT and cultured in vitro. During in vitro culture, SAT-derived Schwann cells remodeled NFBs to form neurospheres and exhibited neurogenic differentiation potential. Transcriptional profiling determined that the acquisition of these NSC properties can be attributed to dedifferentiation processes in cultured Schwann cells. The emerging population of cells were termed SAT-NSCs because of their considerably distinct gene expression profile, cell markers, and differentiation potential compared to endogenous Schwann cells existing in vivo. SAT-NSCs successfully engrafted to the gastrointestinal tract of mice, migrated longitudinally and circumferentially within the muscularis, differentiated into neurons and glia, and exhibited neurochemical coding and calcium signaling properties consistent with an enteric neuronal phenotype. These cells rescued functional deficits associated with colonic aganglionosis and gastroparesis, indicating their therapeutic potential as a cell therapy for gastrointestinal dysmotility. SAT can be harvested easily and offers unprecedented accessibility for the derivation of autologous NSCs from adult tissues. Evidence from this study indicates that SAT-NSCs are not derived from mesenchymal stem cells and instead originate from Schwann cells within NFBs. Our data describe efficient isolation procedures for mouse and human SAT-NSCs and suggest that these cells have potential for therapeutic applications in gastrointestinal motility disorders.

Optimal timing for Soave primary pull-through in short-segment Hirschsprung disease: A meta-analysis.

BACKGROUND: The optimal age for endorectal pull-through (ERPT) surgery in infants with short-segment Hirschsprung disease varies, with a trend toward earlier surgery. However, it is unclear if the timing of surgery impacts functional outcomes. We undertook the present study to determine the optimal timing of ERPT in infants with short-segment Hirschsprung disease. METHODS: The NCBI PubMed database was searched for English-language manuscripts published between 2000 and 2019 analyzing functional outcomes for patient following the initial Soave ERPT for short-segment Hirschsprung disease. Raw data from these studies was obtained from the corresponding author for each manuscript. We combined data from these papers with our own institutional data and performed a meta-analysis. RESULTS: A total of 780 infants were included in our meta-analysis. Constipation occurred in 1.0-31.7%, soiling 1.3-26.0%, anastomotic stricture 0.0-14.6%, and anastomotic leak 0.0-3.4%. Regarding age at ERPT, younger infants at the time of initial corrective surgery had higher rates of soiling, stricture, and leak. On sub-group analysis, patients <2.5 months at their initial corrective surgery had higher rates of soiling (25.9% vs. 11.4%, p<0.01), as well as stricture (10.0% vs 1.7%, p<0.01) and leak (5.5% vs 1.3%, p<0.01). CONCLUSION: While age at Soave endorectal pull-through for short-segment Hirschsprung disease has decreased over time, functional outcomes associated with this trend have only recently been examined. Our findings suggest that patients <2.5 months old at the time of endorectal pull-through may have worse functional outcomes, emphasizing the need to consider further study of the timing of surgery in this population.

Association of Gangrenous, Suppurative, and Exudative Findings With Outcomes and Resource Utilization in Children With Nonperforated Appendicitis.

Importance: The clinical significance of gangrenous, suppurative, or exudative (GSE) findings is poorly characterized in children with nonperforated appendicitis. Objective: To evaluate whether GSE findings in children with nonperforated appendicitis are associated with increased risk of surgical site infections and resource utilization. Design, Setting, and Participants: This multicenter cohort study used data from the Appendectomy Targeted Database of the American College of Surgeons Pediatric National Surgical Quality Improvement Program, which were augmented with operative report data obtained by supplemental medical record review. Data were obtained from 15 hospitals participating in the Eastern Pediatric Surgery Network (EPSN) research consortium. The study cohort comprised children (aged ≤18 years) with nonperforated appendicitis who underwent appendectomy from July 1, 2015, to June 30, 2020. Exposures: The presence of GSE findings was established through standardized, keyword-based audits of operative reports by EPSN surgeons. Interrater agreement for the presence or absence of GSE findings was evaluated in a random sample of 900 operative reports. Main Outcomes and Measures: The primary outcome was 30-day postoperative surgical site infections (incisional and organ space infections). Secondary outcomes included rates of hospital revisits, postoperative abdominal imaging, and postoperative length of stay. Multivariable mixed-effects regression was used to adjust measures of association for patient characteristics and clustering within hospitals. Results: Among 6133 children with nonperforated appendicitis, 867 (14.1%) had GSE findings identified from operative report review (hospital range, 4.2%-30.2%; P < .001). Reviewers agreed on presence or absence of GSE findings in 93.3% of cases (weighted κ, 0.89; 95% CI, 0.86-0.92). In multivariable analysis, GSE findings were associated with increased odds of any surgical site infection (4.3% vs 2.2%; odds ratio [OR], 1.91; 95% CI, 1.35-2.71; P < .001), organ space infection (2.8% vs 1.1%; OR, 2.18; 95% CI, 1.30-3.67; P = .003), postoperative imaging (5.8% vs 3.7%; OR, 1.70; 95% CI, 1.23-2.36; P = .002), and prolonged mean postoperative length of stay (1.6 vs 0.9 days; rate ratio, 1.43; 95% CI, 1.32-1.54; P < .001). Conclusions and Relevance: In children with nonperforated appendicitis, findings of gangrene, suppuration, or exudate are associated with increased surgical site infections and resource utilization. Further investigation is needed to establish the role and duration of postoperative antibiotics and inpatient management to optimize outcomes in this cohort of children.

Top ten strategies to enhance grant-writing success.

Mastering the art of grant writing is one of the most essential skills to obtaining research funding. Given the importance of pursuing high-quality surgical research and supporting the academic goals of surgeon-scientists, ensuring that surgeons have the necessary skills to write effective and successful grants is of paramount importance. In this article, we present 10 strategies for composing a strong research grant application. These strategies apply to federal or nongovernmental funding agencies and are broadly relevant to basic, translational, and clinical investigators. We believe these recommendations can help surgeon-scientists be effective grant writers and compete successfully for research funding.