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Laparoscopic appendectomy for pediatric acute appendicitis is commonly performed by pediatric surgeons. A single incision approach has been investigated, but the lack of technical uniformity has resulted in conflicting data. We hypothesized that an initial single incision laparoscopic-assisted extracorporeal appendectomy (SILEA) approach is associated with similar complication rates as compared to the standard three-incision laparoscopic appendectomy (TILA).Approximately 1300 laparoscopic appendectomies were retrospectively reviewed for acute appendicitis over a 5 year period. Patients were split into TILA or SILEA cohorts. Propensity score matching identified 102 matched cases in both cohorts. Case and control cohort comparisons were then analyzed.Successful SILEA was associated with no difference in post-operative complication rate. SILEA was associated with decreased postoperative narcotic dosage and shorter operative time than TILA.An initial SILEA approach is safe and associated with similar complication rates as TILA. Based on this data, the authors advocate adoption of the an initial SILEA approach for uncomplicated, freely mobile, acute appendicitis with seamless conversion to TILA if the appendix is not amendable to SILEA.Level III.

Intestinal rotation abnormality (IRA) predisposes to lethal midgut volvulus. An understanding of intestinal development illustrates the process of normal intestinal rotation and fixation. An appreciation of the clinical presentation and consequences of missed IRA will enhance clinical suspicion and timely evaluation. Selecting the appropriate imaging modality to diagnose IRA requires an understanding of the benefits and limitations of each. The Ladd's procedure continues to be the appropriate surgical treatment for IRA with or without volvulus. Laparoscopy has emerged as an option for the diagnosis and treatment of IRA. Populations in which IRA is always associated, but a Ladd's procedure rarely required, include patients with congenital diaphragmatic hernia and abdominal wall defects. Prevalence of IRA is higher in children with congenital heart disease and heterotaxy syndrome; asymptomatic patients require multidisciplinary consideration of the risks and benefits of screening for IRA, whether a Ladd's procedure is required, and the timing thereof. [ Pediatr Ann . 2016;45(7):e247–e250.]

Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by β-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by β-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK(1)R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nM, 10 min) induced interaction of NK(1)R and β-arrestin at the plasma membrane, and the SP-NK(1)R-β-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK(1)R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H(+)ATPase inhibitor bafilomycin A(1), which prevent endosomal SP degradation, suppressed NK(1)R recycling by >50%. Preincubation of neurones with SP (10 nM, 5 min) desensitized Ca(2+) transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK(1)R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK(1)R-β-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK(1)R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating β-arrestin-mediated endosomal signalling.

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca2+ concentration ([Ca2+]i) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca2+]i, pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.

Activated G protein-coupled receptors traffic to endosomes and are sorted to recycling or degradative pathways. Endosomes are also a site of receptor signaling of sustained and pathophysiologically important processes, including inflammation. However, the mechanisms of endosomal sorting of receptors and the impact of disease on trafficking have not been fully defined. We examined the effects of inflammation on the subcellular distribution and trafficking of the substance P (SP) neurokinin 1 receptor (NK1R) in enteric neurons. We studied NK1R trafficking in enteric neurons of the mouse colon using immunofluorescence and confocal microscopy. The impact of inflammation was studied in IL10−/−-piroxicam and trinitrobenzenesulfonic acid colitis models. NK1R was localized to the plasma membrane of myenteric and submucosal neurons of the uninflamed colon. SP evoked NK1R endocytosis and recycling. Deletion of β-arrestin2, which associates with the activated NK1R, accelerated recycling. Inhibition of endothelin-converting enzyme-1 (ECE-1), which degrades endosomal SP, prevented recycling. Inflammation was associated with NK1R endocytosis in myenteric but not submucosal neurons. Whereas the NK1R in uninflamed neurons recycled within 60 min, NK1R recycling in inflamed neurons was delayed for >120 min, suggesting defective recycling machinery. Inflammation was associated with β-arrestin2 upregulation and ECE-1 downregulation, which may contribute to the defective NK1R recycling. We conclude that inflammation evokes redistribution of NK1R from the plasma membrane to endosomes of myenteric neurons through enhanced SP release and defective NK1R recycling. Defective recycling may be secondary to upregulation of β-arrestin2 and downregulation of ECE-1. Internalized NK1R may generate sustained proinflammatory signals that disrupt normal neuronal functions.

In previous studies, we have found that negatively charged, but not uncharged, amino acids and sugars block sea urchin fertilization. These studies were developed from modeling work in non-living systems using derivatized agarose beads that suggested that charge-charge bonding may control at least some adhesive interactions. In the present study, the effects of positively charged, negatively charged and uncharged molecules were examined in the sea urchin sperm-egg system in over 300 individual trials. The results indicate that depending on the specific molecules utilized, both sperm and egg are exquisitely sensitive to charged but not uncharged molecules and to pH changes in sea water caused by some of the charged molecules. It is shown that egg activation, as well as sperm motility and sperm-egg interactions, can be affected by charged molecules. One compound, fructose-1-phosphate blocked fertilization in S. purpuratus sea urchins but not in Lytechinus pictus sea urchins. These findings indicate that charge alone cannot explain all the results. In this case, the presence of a ketone instead of an aldehyde group indicates that species-specific components may control fertilization reactions. The present study is a comprehensive survey of the effects of charge, pH and molecular structure on the fertilization activation continuum in a model system of sea urchins.

DegS (HhoB), a putative serine protease related to DegP/HtrA, regulates the basal and induced activity of the essential Escherichia coli sigma factor sigma (E), which is involved in the cellular response to extracytoplasmic stress. DegS promotes the destabilization of the sigma (E)-specific anti-sigma factor RseA, thereby releasing sigma (E) to direct gene expression. We demonstrate that degS is an essential E. coli gene and show that the essential function of DegS is to provide the cell with sigma (E) activity. We also show that the putative active site of DegS is periplasmic and that DegS requires its N-terminal transmembrane domain for its sigma (E)-related function.

The substance P neurokinin 1 receptor (NK1R) regulates motility, secretion, inflammation and pain in the intestine. The distribution of the NK1R is a key determinant of the functional effects of substance P in the gut. Information regarding the distribution of NK1R in subtypes of mouse enteric neurons is lacking and is the focus of the present study. NK1R immunoreactivity (NK1R-IR) is examined in whole-mount preparations of the mouse distal colon by indirect immunofluorescence and confocal microscopy. The distribution of NK1R-IR within key functional neuronal subclasses was determined by using established neurochemical markers. NK1R-IR was expressed by a subpopulation of myenteric and submucosal neurons; it was mainly detected in large multipolar myenteric neurons and was colocalized with calcitonin gene-related peptide, neurofilament M, choline acetyltransferase and calretinin. The remaining NK1R-immunoreactive neurons were positive for nitric oxide synthase. NK1R was expressed by most of the submucosal neurons and was exclusively co-expressed with vasoactive intestinal peptide, with no overlap with choline acetyltransferase. Treatment with substance P resulted in the concentration-dependent internalisation of NK1R from the cell surface into endosome-like structures. Myenteric NK1R was mainly expressed by intrinsic primary afferent neurons, with minor expression by descending interneurons and inhibitory motor neurons. Submucosal NK1R was restricted to non-cholinergic secretomotor neurons. These findings highlight key differences in the neuronal distribution of NK1R-IR between the mouse, rat and guinea-pig, with important implications for the functional role of NK1R in regulating intestinal motility and secretion.

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.

Background & Aims Transient receptor potential ankyrin (TRPA) 1, an excitatory ion channel expressed by sensory neurons, mediates somatic and visceral pain in response to direct activation or noxious mechanical stimulation. Although the intestine is routinely exposed to irritant alimentary compounds and inflammatory mediators that activate TRPA1, there is no direct evidence for functional TRPA1 receptors on enteric neurons, and the effects of TRPA1 activation on intestinal function have not been determined. We characterized expression of TRPA1 by enteric neurons and determined its involvement in the control of intestinal contractility and transit. Methods TRPA1 expression was characterized by reverse-transcription polymerase chain reaction and immunofluorescence analyses. TRPA1 function was examined by Ca 2+ imaging and by assays of contractile activity and transit. Results We detected TRPA1 messenger RNA in the mouse intestine and TRPA1 immunoreactivity in enteric neurons. The cecum and colon had immunoreactivity for neuronal TRPA1, but the duodenum did not. TRPA1 immunoreactivity was also detected in inhibitory motoneurons and descending interneurons, cholinergic neurons, and intrinsic primary afferent neurons. TRPA1 activators, including cinnamaldehyde, allyl isothiocyanate (AITC), and 4-hydroxynonenal, increased [Ca 2+ ] i in myenteric neurons. These were reduced by a TRPA1 antagonist (HC-030031) or deletion of Trpa1 . TRPA1 activation inhibited contractility of the segments of colon but not stomach or small intestine of Trpa1 +/+ but not Trpa1 −/− mice; this effect was reduced by tetrodotoxin or N G -nitro-l-arginine methyl ester. Administration of AITC by gavage did not alter gastric emptying or small intestinal transit, but luminal AITC inhibited colonic transit via TRPA1. Conclusions Functional TRPA1 is expressed by enteric neurons, and activation of neuronal TRPA1 inhibits spontaneous neurogenic contractions and transit of the colon.

Background & Aims Opioids and opiates inhibit gastrointestinal functions via μ, δ, and κ receptors. Although agonists of the δ opioid receptor (DOR) suppress motility and secretion, little is known about the localization and regulation of DOR in the gastrointestinal tract. Methods We studied mice in which the gene that encodes the enhanced green fluorescent protein (eGFP) was inserted into Oprd1 , which encodes DOR, to express an approximately 80-kilodalton product (DOReGFP). We used these mice to localize DOR and to determine how agonists regulate the subcellular distribution of DOR. Results DOReGFP was expressed in all regions but was confined to enteric neurons and fibers within the muscularis externa. In the submucosal plexus, DOReGFP was detected in neuropeptide Y–positive secretomotor and vasodilator neurons of the small intestine, but rarely was observed in the large bowel. In the myenteric plexus of the small intestine, DOReGFP was present in similar proportions of excitatory motoneurons and interneurons that expressed choline acetyltransferase and substance P, and in inhibitory motoneurons and interneurons that contained nitric oxide synthase. DOReGFP was present mostly in nitrergic myenteric neurons of colon. DOReGFP and μ opioid receptors often were co-expressed. DOReGFP-expressing neurons were associated with enkephalin-containing varicosities, and enkephalin-induced clathrin- and dynamin-mediated endocytosis and lysosomal trafficking of DOReGFP. DOReGFP replenishment at the plasma membrane was slow, requiring de novo synthesis, rather than recycling. Conclusions DOR localizes specifically to submucosal and myenteric neurons, which might account for the ability of DOR agonists to inhibit gastrointestinal secretion and motility. Sustained down-regulation of DOReGFP at the plasma membrane of activated neurons could induce long-lasting tolerance to DOR agonists.

Background Bile acids (BAs) regulate cells by activating nuclear and membrane-bound receptors. G protein coupled bile acid receptor 1 (GpBAR1) is a membrane-bound G-protein-coupled receptor that can mediate the rapid, transcription-independent actions of BAs. Although BAs have well-known actions on motility and secretion, nothing is known about the localization and function of GpBAR1 in the gastrointestinal tract. Methods We generated an antibody to the C-terminus of human GpBAR1, and characterized the antibody by immunofluorescence and Western blotting of HEK293-GpBAR1-GFP cells. We localized GpBAR1 immunoreactivity (IR) and mRNA in the mouse intestine, and determined the mechanism by which BAs activate GpBAR1 to regulate intestinal motility. Key Results The GpBAR1 antibody specifically detected GpBAR1-GFP at the plasma membrane of HEK293 cells, and interacted with proteins corresponding in mass to the GpBAR1-GFP fusion protein. GpBAR1-IR and mRNA were detected in enteric ganglia of the mouse stomach and small and large intestine, and in the muscularis externa and mucosa of the small intestine. Within the myenteric plexus of the intestine, GpBAR1-IR was localized to �50% of all neurons and to >80% of inhibitory motor neurons and descending interneurons expressing nitric oxide synthase. Deoxycholic acid, a GpBAR1 agonist, caused a rapid and sustained inhibition of spontaneous phasic activity of isolated segments of ileum and colon by a neurogenic, cholinergic and nitrergic mechanism, and delayed gastrointestinal transit. Conclusions & Inferences G protein coupled bile acid receptor 1 is unexpectedly expressed in enteric neurons. Bile acids activate GpBAR1 on inhibitory motor neurons to release nitric oxide and suppress motility, revealing a novel mechanism for the actions of BAs on intestinal motility.