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Background Esophageal growth using the Foker process (FP) for long-gap esophageal atresia (LGEA) has evolved over time. Methods Contemporary LGEA patients treated from 2014–2020 were compared to historical controls (2005 to Results 102 contemporary LGEA patients (type A 50%, B 18%, C 32%; 36% prior anastomotic attempt; 20 with esophagostomy) underwent either primary repair (n=23), jejunal interposition (JI; n = 14), or Foker process (FP; n = 65; 49 primary [p], 16 rescue [r]). The contemporary p-FP cohort experienced significantly fewer leaks on traction (4% vs 22%), bone fractures (2% vs 22%), anastomotic leak (12% vs 37%), and Foker failure (FP→JI; 0% vs 15%), when compared to historical p-FP patients (n = 27), all p ≤ 0.01. Patients who underwent a completely (n = 11) or partially (n = 11) minimally invasive FP experienced fewer median days paralyzed (0 vs 8 vs 17) and intubated (9 vs 15 vs 25) compared to open FP patients, respectively (all p ≤ 0.03), with equivalent leak rates (18% vs 9% vs 26%, p = 0.47). At one-year post-FP, most patients (62%) are predominantly orally fed. Conclusion With continued experience and technical refinements, the Foker process has evolved with improved outcomes, less morbidity and maximal esophageal preservation.

OBJECTIVES To determine if children with laryngeal penetration on videofluoroscopic swallow study (VFSS) who received feeding interventions (thickened liquids, change in liquid flow rate, and/or method of liquid delivery) had improved symptoms and decreased hospitalizations compared with those without intervention. METHODS We performed a retrospective cohort study of children under 2 years with laryngeal penetration on VFSS at our institution in 2015 to determine initial and follow-up VFSS findings, symptom improvement at follow-up, and hospitalization risk before and after VFSS. Proportions were compared with Fisher exact test and hospitalizations with paired t tests. RESULTS We evaluated 137 subjects with age 8.93 ± 0.59 months who had laryngeal penetration without aspiration on VFSS. Fifty-five percent had change in management, with 40% receiving thickening and 15% a change in flow rate. There was significant improvement in symptoms for children that had feeding intervention and this improvement was the greatest with thickening (OR 41.8, 95% CI 12.34-141.69, P

Objectives/hypothesis To describe the prevalence of aspiration in children with unilateral vocal fold paralysis who underwent objective assessment of swallow function. Study design Retrospective chart review. Methods A study of patients presenting to our institution with unilateral vocal fold paralysis in 2015 was conducted. All patients were diagnosed using flexible laryngoscopy. Patients were included if they underwent at least one modified barium swallow (MBS) study for evaluation of their swallowing function due to recurrent respiratory issues and/or feeding difficulty. Results Twenty-eight patients diagnosed with unilateral vocal fold paralysis underwent an MBS study at our institution in 2015. Median age at the time of MBS study was 1.7 years (interquartile range: 0.4-4.3). Twenty-six patients (92.9%) had dysphagia. Sixteen patients were found to aspirate on MBS study. All patients who aspirated did so without overt signs (silent aspiration). Eighteen patients had congenital heart disease (64.3%) and nine had a history of prematurity (32.1%). Eight patients (28.6%) presented with developmental delays. Conclusions Patients who present with unilateral vocal fold paralysis and recurrent respiratory and/or feeding issues may be affected by prominent issues such as swallowing dysfunction and silent aspiration. Clinicians should be aware of this risk and evaluate patients for any signs of feeding or swallowing difficulties. Level of evidence 4 Laryngoscope, 129:569-573, 2019.

Please view the video clip (Video 1) showing the results of a videofluoroscopic swallow study (also known as a modified barium swallow study). Video 1. Click here to view the video. The most likely clinical scenario of the infant in this video is a: The videofluoroscopic swallow study (VFSS), also known as a modified barium swallow study , is a form of instrumental assessment of swallow function. It is conducted in the radiology department by a speech-language pathologist in collaboration with a radiologist; thus, the patient needs to be stable to allow transfer to this location. The infant’s caregivers and …

Objective To determine whether the use of acid suppression and thickened feeds impact laryngomalacia outcomes in infants, including supraglottoplasty risk, time to supraglottoplasty, and hospitalization risk. Study design We performed a retrospective cohort study to compare risk and time with supraglottoplasty and frequency and duration of hospitalizations for infants diagnosed with laryngomalacia at Boston Children's Hospital between January 1 and December 31, 2017. The primary outcomes were supraglottoplasty requirement, time to supraglottoplasty, and hospitalization risk. Multivariate analyses were performed to determine predictors of supraglottoplasty and hospitalization risk after adjusting for laryngomalacia severity and comorbidities in addition to propensity score adjustment. Kaplan–Meier curves were created to determine the impact of acid suppression use on time to supraglottoplasty. Results In total, 236 subjects with mean age 62.6 ± 4 days were included in the analysis; 55% were treated with acid suppression. Subjects treated with acid suppression had a greater risk of supraglottoplasty (hazard ratio 3.36, 95% CI 1.36-8.29, P = .009), shorter time to supraglottoplasty (5.64 ± 0.92 vs 7.98 ± 1.92 months, P = .006), and increased respiratory hospitalization risk (relative risk 1.97, 95% CI 1.01-3.85, 0.047), even after adjustment for covariates. Subjects receiving thickening had fewer respiratory hospitalization nights and longer time to supraglottoplasty (9.3 ± 1.7 vs 4.56 ± 0.73 months, P = .004), even after adjustment. Conclusions Acid suppression use does not reduce the frequency of supraglottoplasty and related hospitalizations compared with untreated subjects. However, patients treated with thickening have decreased hospitalization and longer time to supraglottoplasty, suggesting that thickening of feeds may be a preferred intervention over acid suppression.

Importance Thickening of fluids is a common strategy for feeding patients with oropharyngeal dysphagia but has known risks and should be stopped once it is safe to do so. Weaning children from thickened fluids safely can be challenging, and novel methods are required. Objective To describe the use of a systematic weaning process (SWP) for children who received thickened liquids owing to oropharyngeal dysphagia and identified risk of aspiration. Design, Setting, and Participants Retrospective case series (2010 to 2015) at a tertiary care center of 50 children with documented aspiration by clinical swallowing assessment, airway evaluation, and videofluoroscopic swallow study with at least 4 months of follow-up. All patients were initially receiving thickened fluids. A 10% reduction in thickness was made every 2 weeks based on clinical symptoms. Caregivers progressed to the next incremental level if there were no signs or symptoms of aspiration. Main Outcomes and Measures Number of patients weaned to a thin-fluid diet. Results Of 50 children (32 [64%] male; median [interquartile range] age, 0.7 [1.0] y at presentation and 1.8 [1.3] y at start of wean) using the SWP, 44 (88%) were able to reduce the amount of thickener used. A successful wean from thickened fluids to thin fluids was completed in 39 (78%). The mean (SD) duration of a successful wean was 0.9 (0.6) years. Five patients tolerated a reduction in thickener but not a full wean to thin fluids. For 6 patients, weaning failed and they continued to receive thickened fluids. Of those whose weaning failed, 2 patients developed pneumonia. Of the 39 successfully weaned patients, 14 (36%) experienced a temporary stall but eventually tolerated thin fluids. Only 2 (5%) developed pneumonia while all other successfully weaned patients (n = 37 [95%]) did not experience any substantial respiratory issues. Overall, 46 (92%) of children required 2 or fewer videofluoroscopic swallow study evaluations. Conclusions and Relevance Patients with oropharyngeal dysphagia and aspiration should be gradually weaned off of thickened fluids. The SWP uses small incremental steps to gradually reduce the amount of thickener. Using this method, most children tolerated a reduction in thickeners and a thin-fluid diet. The SWP presents a safe and effective way of gradually returning children to a more normal diet.

Importance Laryngomalacia is the most common laryngeal anomaly and is commonly associated with stridor in children, but the recurrent respiratory and/or feeding difficulties associated with this condition may pose a threat to the well-being of the affected child. Objective To describe the prevalence of aspiration in pediatric patients with laryngomalacia who present with recurrent respiratory issues and/or feeding difficulty. Design, Setting, and Participants This retrospective review of medical records involved 142 pediatric patients. These patients received a laryngomalacia diagnosis, presented with recurrent respiratory and/or feeding difficulties, and underwent a modified barium swallow (MBS) study at a tertiary referral children’s hospital from January 1, 2015, to December 31, 2015. Each patient was assessed for aspiration and swallowing discoordination. Data collection and analysis were performed from December 1, 2016, to September 30, 2017. Main Outcomes and Measures Demographic data, presenting symptoms, medical comorbidities, and feeding and dysphagia details were collected and analyzed. Speech-language pathologists reviewed all MBS reports to identify the presence and type of aspiration (ie, silent vs nonsilent) with different textures and consistencies of food and liquid. Results A total of 395 patients were diagnosed with laryngomalacia in 2015. One hundred forty-two of these patients (35.9%) presented with recurrent respiratory issues and/or feeding difficulties and were referred for MBS study for further evaluation. Ninety-two (64.8%) were male, with a median (interquartile range) age at the time of MBS study of 7.9 (3.2-20.5) months. Among these patients, 128 (90.1%) had swallowing dysfunction documented during the MBS study. Aspiration was identified in 60 patients (42.3%), and silent aspiration was documented in 59 (98.3%) of these 60 patients. Epilepsy or seizures (risk difference [RD], 11%; 95% CI, 5%-17%), laryngeal cleft (RD, 8%; 95% CI, 3%-13%), and premature birth (RD, 15%; 95% CI, 5%-25%) were statistically significantly associated with abnormal MBS findings. Conclusions and Relevance Swallowing dysfunction and aspiration were commonly found in pediatric patients with laryngomalacia and recurrent feeding and/or respiratory issues; these children should undergo an MBS study for dysphagia and silent aspiration.

Bilingualism is common throughout the world, and bilingual children regularly develop into fluently bilingual adults. In contrast, children with cochlear implants (CIs) are frequently encouraged to focus on a spoken language to the exclusion of sign language. Here, we investigate the spoken English language skills of 5 children with CIs who also have deaf signing parents, and so receive exposure to a full natural sign language (American Sign Language, ASL) from birth, in addition to spoken English after implantation. We compare their language skills with hearing ASL/English bilingual children of deaf parents. Our results show comparable English scores for the CI and hearing groups on a variety of standardized language measures, exceeding previously reported scores for children with CIs with the same age of implantation and years of CI use. We conclude that natural sign language input does no harm and may mitigate negative effects of early auditory deprivation for spoken language development.

The American College of Obstetricians and Gynecologists (ACOG) and the IADPSG (International Association of Diabetes and Pregnancy Study Groups) proposed distinct approaches to diagnosing gestational diabetes mellitus (GDM). We sought to analyze these paradigms: (1) ACOG 2-step approach where screening is followed by diagnostic testing, (2) IADPSG 1-step diagnostic testing.We reviewed data from pregnant women (24-28 wks) screened for GDM over two periods: (1) November 2011-May 2012 (2) November 2012-May 2013. Period 1: 2-step approach (screening 1-h glucose challenge test (GCT) followed by a diagnostic 3-h 100-g glucose tolerance test (GTT) when abnormal (≥140 mg/dl)). Period 2: an abnormal value after a 2-h 75-g GTT result was diagnostic of GDM. We compared the incidence of GDM and perinatal outcomes using either approach.Out of 471 patients screened by ACOG 2-step approach, 72 (15.3%) had an abnormal 1-h screening and underwent the 3-h diagnostic GTT, and 26 (5.5%) developed GDM. The 1-step approach resulted in 53 (15.96%) with GDM of a total 332 evaluated. There was no statistically significant difference in perinatal outcomes between the two cohorts. Maternal weight at the start and the end of pregnancy was greater for patients diagnosed by the ACOG 2-step approach.Adopting 1-step approach (ADA) to diagnose GDM resulted in a 3-fold increase in prevalence of GDM with no differences in perinatal outcomes.

The sympathetic nervous system (SNS) arises from neural crest (NC) cells during embryonic development and innervates the internal organs of vertebrates to modulate their stress response. NRP1 and NRP2 are receptors for guidance cues of the class 3 semaphorin (SEMA) family and are expressed in partially overlapping patterns in sympathetic NC cells and their progeny. By comparing the phenotypes of mice lacking NRP1 or its ligand SEMA3A with mice lacking NRP1 in the sympathetic versus vascular endothelial cell lineages, we demonstrate that SEMA3A signalling through NRP1 has multiple cell-autonomous roles in SNS development. These roles include neuronal cell body positioning, neuronal aggregation and axon guidance, first during sympathetic chain assembly and then to regulate the innervation of the heart and aorta. Loss of NRP2 or its ligand SEMA3F impaired sympathetic gangliogenesis more mildly than loss of SEMA3A/NRP1 signalling, but caused ectopic neurite extension along the embryonic aorta. The analysis of compound mutants lacking SEMA3A and SEMA3F or NRP1 and NRP2 in the SNS demonstrated that both signalling pathways cooperate to organise the SNS. We further show that abnormal sympathetic development in mice lacking NRP1 in the sympathetic lineage has functional consequences, as it causes sinus bradycardia, similar to mice lacking SEMA3A., Highlights ► NRP1 and NRP2 are co-expressed in sympathetic neurons and their precursors. ► SEMA3A signals through NRP1 to regulate sympathetic gangliogenesis and axon guidance. ► SEMA3F signals through NRP2 to regulate sympathetic axon guidance. ► SEMA3A/NRP1 and SEMA3F/NRP2 act synergistically in sympathetic nervous system patterning. ► Abnormal sympathetic innervation of the heart leads to bradycardia in NRP1 mutants.

Gonadotropin-releasing hormone (GnRH) neurons are neuroendocrine cells that are born in the nasal placode during embryonic development and migrate through the nose and forebrain to the hypothalamus, where they regulate reproduction. Many molecular pathways that guide their migration have been identified, but little is known about the factors that control the survival of the migrating GnRH neurons as they negotiate different environments. We previously reported that the class 3 semaphorin SEMA3A signals through its neuropilin receptors, NRP1 and NRP2, to organise the axons that guide migrating GnRH neurons from their birthplace into the brain. By combining analysis of genetically altered mice with in vitro models, we show here that the alternative neuropilin ligand VEGF164 promotes the survival of migrating GnRH neurons by co-activating the ERK and AKT signalling pathways through NRP1. We also demonstrate that survival signalling relies on neuronal, but not endothelial, NRP1 expression and that it occurs independently of KDR, the main VEGF receptor in blood vessels. Therefore, VEGF164 provides survival signals directly to developing GnRH neurons, independently of its role in blood vessels. Finally, we show that the VEGF164-mediated neuronal survival and SEMA3A-mediated axon guidance cooperate to ensure that migrating GnRH neurons reach the brain. Thus, the loss of both neuropilin ligands leads to an almost complete failure to establish the GnRH neuron system.

Septic arthropathy leads to rapid joint destruction, impairment, and disability.Staphylococcuscan be particularly virulent to bone and joints leading to adverse obstetric events. At 28 of weeks gestation, a patient presented with pyelonephritis and progressive left shoulder pain. Magnetic resonance imaging indicated early clavicular destruction and acromial involvement. Glenohumeral joint aspiration producedStaphylococcus aureus. The patient then had premature rupture of membranes and progressed rapidly to preterm delivery. Placental pathology revealed chorioamnionitis and microabscesses. Treatment of the infected joint required further surgical drainage and bone resection as well as extended antibiotics. It is important to remember that joint pain in pregnancy may indicate infective arthritis, and pyelonephritis can be a source of such an infection. Evaluation includes magnetic resonance imaging and consultation for joint aspiration. Prompt recognition and treatment are necessary to prevent joint destruction.

Kallmann syndrome (KS) is a genetic disease characterized by hypogonadotropic hypogonadism and impaired sense of smell. The genetic causes underlying this syndrome are still largely unknown, but are thought to be due to a developmental defect in the migration of gonadotropin-releasing hormone (GnRH) neurons. Understanding the causes of the disease is hampered by lack of appropriate mouse models. GnRH neurons are hypothalamic cells that centrally control reproduction in mammals by secreting the GnRH decapeptide into the portal blood vessels of the pituitary to stimulate the production of gonadotropins. During development, these cells are born in the nasal placode outside the brain and migrate in association with olfactory/vomeronasal axons to reach the forebrain and position themselves in the hypothalamus. By combining the analysis of genetically altered mice with in vitro models, we demonstrate here that a secreted guidance cue of the class 3 semaphorin family, SEMA3A, is essential for the development of the GnRH neuron system: loss of SEMA3A signalling alters the targeting of vomeronasal nerves and the migration of GnRH neurons into the brain, resulting in reduced gonadal size. We found that SEMA3A signals redundantly through both its classical receptors neuropilin (NRP) 1 and, unconventionally, NRP2, while the usual NRP2 ligand SEMA3F is dispensable for this process. Strikingly, mice lacking SEMA3A or semaphorin signalling through both NRP1 and NRP2 recapitulate the anatomical features of a single case of KS analysed so far, and may therefore be used as genetic models to elucidate the pathogenesis of KS.

BACKGROUND Although vertebral hemangiomas are found in 10% of the population, they rarely become symptomatic. We describe management of an enlarging cervical vertebral hemangioma during pregnancy. CASE A primiparous woman at 33 weeks of gestation presented with neck pain and paresthesias. She had a known cervical hemangioma. Imaging revealed a C6-C7 hemangioma with extradural extension. Her symptoms fluctuated, and she was delivered by cesarean at 36 weeks of gestation. Improvement occurred after delivery, and radiographic regression of the hemangioma was demonstrated. CONCLUSION Hormonal and vascular changes of pregnancy may exacerbate spinal hemangiomas. Depending on the location, these may lead to profound neurologic disability and management depends on the degree of myelopathy. Surgical intervention may be necessary to avoid neurologic injury, but amelioration after delivery is possible.

A series of beta-substituted and beta,beta-disubstituted N-acyl 5-methoxy-1-methyltryptamines and 5-methoxytryptamines have been prepared as melatonin analogues to investigate the nature of the binding site of the melatonin receptor. The affinity of analogues was determined in a radioligand binding assay using cloned human MT(1) and MT(2) receptor subtypes expressed in NIH 3T3 cells. Agonist and antagonist potency of all analogues was measured using the pigment aggregation response of a clonal line of Xenopus laevis melanophores. beta-Methylmelatonin (17a) and beta,beta-dimethylmelatonin (17b), though showing a slight decrease in binding at human receptors, show an increase in potency on Xenopus. N-Butanoyl 5-methoxy-1-methyl-beta,beta-trimethylenetryptamine (12c) is an antagonist at human MT(1) receptors but an agonist at MT(2), while N-butanoyl 5-methoxy-1-methyl-beta,beta-tetramethylenetryptamine (13c) is an antagonist at MT(1) but had no action at MT(2) and is one of the first examples of an MT(1) selective antagonist.

International audience; Individuals with an inherited deficiency in gonadotropin-releasing hormone (GnRH) have impaired sexual reproduction. Previous genetic linkage studies and sequencing of plausible gene candidates have identified mutations associated with inherited GnRH deficiency, but the small number of affected families and limited success in validating candidates have impeded genetic diagnoses for most patients. Using a combination of exome sequencing and computational modeling, we have identified a shared point mutation in semaphorin 3E (SEMA3E) in 2 brothers with Kallmann syndrome (KS), which causes inherited GnRH deficiency. Recombinant wild-type SEMA3E protected maturing GnRH neurons from cell death by triggering a plexin D1-dependent (PLXND1-dependent) activation of PI3K-mediated survival signaling. In contrast, recombinant SEMA3E carrying the KS-associated mutation did not protect GnRH neurons from death. In murine models, lack of either SEMA3E or PLXND1 increased apoptosis of GnRH neurons in the developing brain, reducing innervation of the adult median eminence by GnRH-positive neurites. GnRH neuron deficiency in male mice was accompanied by impaired testes growth, a characteristic feature of KS. Together, these results identify SEMA3E as an essential gene for GnRH neuron development, uncover a neurotrophic function for SEMA3E in the developing brain, and elucidate SEMA3E/PLXND1/PI3K signaling as a mechanism that prevents GnRH neuron deficiency.

Introduction: The mammary primordium forms during embryogenesis as a result of inductive interactions between its constitutive tissues, the mesenchyme and epithelium, and represents the earliest evidence of commitment to the mammary lineage. Previous studies of embryonic mouse mammary epithelium indicated that, by mid-gestation, these cells are determined to a mammary cell fate and that a stem cell population has been delimited. Mammary mesenchyme can induce mammary development from simple epithelium even across species and classes, and can partially restore features of differentiated tissue to mouse mammary tumours in co-culture experiments. Despite these exciting properties, the molecular identity of embryonic mammary cells remains to be fully characterised. \ud Methods: Here, we define the transcriptome of the mammary primordium and the two distinct cellular compartments that comprise it, the mammary primordial bud epithelium and mammary mesenchyme. Pathway and network analysis was performed and comparisons of embryonic mammary gene expression profiles to those of both postnatal mouse and human mammary epithelial cell sub-populations and stroma were made. \ud Results: Several of the genes we have detected in our embryonic mammary cell signatures were previously shown to regulate mammary cell fate and development, but we also identified a large number of novel candidates. Additionally, we determined genes that were expressed by both embryonic and postnatal mammary cells, which represent candidate regulators of mammary cell fate, differentiation and progenitor cell function that could signal from mammary lineage inception during embryogenesis through postnatal development. Comparison of embryonic mammary cell signatures with those of human breast cells identified potential regulators of mammary progenitor cell functions conserved across species. \ud Conclusions: These results provide new insights into genetic regulatory mechanisms of mammary development, particularly identification of novel potential regulators of mammary fate and mesenchymal-epithelial cross-talk. Since cancers may represent diseases of mesenchymal-epithelial communications, we anticipate these results will provide foundations for further studies into the fundamental links between developmental, stem cell and breast cancer biology.

Neuropilin 1 (NRP1) is a transmembrane glycoprotein that is essential for blood vessel development in vertebrates. Best known for its ability to bind members of the vascular endothelial growth factor (VEGF) and class 3 semaphorin families through its extracellular domain, it also has a highly conserved cytoplasmic domain, which terminates in a SEA motif that binds the PDZ protein synectin/GIPC1/NIP. Previous studies in zebrafish embryos and tissue culture models raised the possibility that the SEA motif of NRP1 is essential for angiogenesis. Here, we describe the generation of mice that express a form of NRP1 that lacks the cytoplasmic domain and, therefore, the SEA motif (Nrp1(cyto)(Δ)(/)(Δ) mice). Our analysis of pre- and perinatal vascular development revealed that vasculogenesis and angiogenesis proceed normally in these mutants, demonstrating that the membrane-anchored extracellular domain is sufficient for vessel growth. By contrast, the NRP1 cytoplasmic domain is required for normal arteriovenous patterning, because arteries and veins crossed each other at an abnormally high frequency in the Nrp1(cyto)(Δ)(/)(Δ) retina, as previously reported for mice with haploinsufficient expression of VEGF in neural progenitors. At crossing sites, the artery was positioned anteriorly to the vein, and both vessels were embedded in a shared collagen sleeve. In human eyes, similar arteriovenous crossings are risk factors for branch retinal vein occlusion (BRVO), an eye disease in which compression of the vein by the artery disrupts retinal blood flow, causing local tissue hypoxia and impairing vision. Nrp1(cyto)(Δ)(/)(Δ) mice may therefore provide a suitable genetic model to study the aetiology of BRVO.

The effects of GABAergic influences intrinsic to the hypothalamus on the secretion of somatostatin were studied using cultured fetal rat hypothalamic neurons. The existence of GABAergic neurons within the cultures was confirmed by immunocytochemistry. These neurons appeared to be actively secreting GABA as antagonism of GABAA receptors with bicuculline and picrotoxinin caused a dose-dependent increase in the release of immunoreactive somatostatin (SRIF), which was Ca(2+)-dependent. Although exogenous GABA inhibited SRIF secretion at concentrations of 10(-6) M and greater, muscimol, a GABAA agonist, inhibited SRIF release at 10(-8) M, whereas baclofen, a GABAB agonist, required concentrations two orders of magnitude greater to produce an effect. Phaclofen, a GABAB antagonist, was inactive (10(-8)-10(-4) M). A GABA uptake inhibitor, SKF 89976A, produced a dose-dependent inhibition of SRIF release. These results, therefore, support a role for intrahypothalamic GABA neurons in the regulation of SRIF secretion in the neonatal rat, predominantly via a type A receptor, and provide further evidence for a neuroendocrine role for GABA in controlling growth hormone secretion.

The peripheral nervous system (PNS) of higher vertebrates is segmented to align the spinal nerve roots with the vertebrae. This copatterning is set up during embryogenesis, when vertebrae develop from the sclerotome layer of the metameric somites, and PNS neurons and glia differentiate from neural crest cells (NCCs) that preferentially migrate into the anterior sclerotome halves. Previous analyses of mice deficient in the class 3 semaphorin (SEMA3) receptors neuropilin (NRP) 1 or 2 raised the possibility that each controlled a distinct aspect of trunk NCC migration. We now demonstrate that both pathways act sequentially in distinct NCC subpopulations and thereby cooperate to enforce segmental NCC migration. Specifically, SEMA3A/NRP1 signalling first directs one population of NCCs from the intersomitic path into the sclerotome, and SEMA3F/NRP2 signalling acts subsequently to restrict a second population to the anterior half of the sclerotome. NCC exclusion from either the posterior sclerotome or the intersomitic boundary is sufficient to enforce the separation of neighbouring NCC streams and the segregation of sensory NCC progeny into metameric dorsal root ganglia (DRG). By contrast, the combined loss of both guidance pathways leads to ectopic invasion of the intersomitic furrows and posterior sclerotome halves, disrupting metameric NCC streaming and DRG segmentation. Refereed/Peer-reviewed

Insomnia, which is severe enough to warrant treatment, occurs in approximately 10% of the general population. It is associated with a range of adverse consequences for human health, economic productivity and quality of life. In animal and human studies, administration of melatonin has been reported to promote sleep, although there has been controversy regarding its effectiveness. The present study used a chronically implanted radiotelemetry transmitter to record electroencephalogram (EEG) and electromyogram (EMG) to enable discrimination of wake (W), nonrapid eye movement (NREM) sleep and rapid eye movement (REM) sleep in un-restrained rats. The acute action of melatonin and ramelteon, a melatonin agonist recently approved for long-term treatment of insomnia in the USA, was examined. Radioligand binding assays on recombinant human MT(1)/MT(2) receptors showed that both the melatonin and ramelteon were both high affinity, nonsubtype selective ligands. Both compounds acted as potent full agonists on a cellular model of melatonin action, the pigment aggregation response in Xenopus laevis melanophores. Both melatonin and ramelteon (10 mg/kg, i/p), administered close to the mid-point of the dark phase of the L:D cycle, significantly reduced NREM sleep latency (time from injection to the appearance of NREM sleep). Both the drugs also produced a short-lasting increase in NREM sleep duration, but the NREM power spectrum was unaltered. Neither drug altered REM latency, REM sleep duration nor power spectrum during REM sleep. In conclusion, ramelteon administration, like melatonin, exerted an acute, short-lasting sleep-promoting effect in the rat, the model most commonly used to evaluate the activity of novel hypnotic drugs.

The synthesis of a series of new N-OMe fluoro-indoles with melatoninergic activity in the Xenopus melanophore assay is described. All of the 4-F substituted compounds, 22a-e and 25a,b, were antagonists on the clonal Xenopus melanophore line. Conversely, the 5-F substituted analogs (15a-e) did not share the same pharmacological profile, as two of them, compounds 15d (R=c-C(3)H(5)) and 15e (R=c-C(4)H(7)), exhibited a weak agonistic and partial agonistic activity, respectively, whilst the other three (15a-c) were all agonists. It seems that in this case the nature of the response (agonist or antagonist activity) is solely dependent on the shape of the R group.

A series of new C-3 and N1-substituted 4-fluorotryptamides have been prepared and tested for their ability to activate pigment granule aggregation in Xenopus laevis melanophores and bind to the recombinant human MT(1) and MT(2) melatonin receptor subtypes expressed in NIH 3T3 cells. Planar sp(2) geometry at C-3-betaC seems to decrease the population of the preferred conformation as it renders 4-fluoroindoles 4b-d weaker antagonists than their C-3-betaC-unsubstituted congeners 3a-e. This effect is not preclusively linked with the C-3 region, as the same geometry around N1 (compounds 5a-c) similarly leads to weak antagonistic action. Last, the new C-3 substituted 4-fluorotryptamides presented herein are substantially more potent than their respective N-OMe functionalized congeners, previously reported.

A series of 7-substituted melatonin and 1-methylmelatonin analogues were prepared and tested against human and amphibian melatonin receptors. 7-Substituents reduced the agonist potency of all the analogues in the Xenopus laevis melanophore assay, 7-bromomelatonin (5d) and N-butanoyl 7-bromo-5-methoxytryptamine (5f) being the most active compounds, but both were 42-fold less potent than melatonin (1). Whereas all the analogues bind with lower affinity at the human MT(1) receptor than melatonin, 5d, 5f and N-propanoyl 7-bromo-5-methoxytryptamine (5e) show a similar binding affinity to melatonin at the MT(2) receptor and consequently show some MT(2) selectivity. These results suggest that the receptor pocket around C-7 favours binding by an electronegative group, suggesting an electropositive region in this area of the receptor.

A series of new 2,3-dihydro-1H-pyrrolo[3,2,1-ij]quinolin-6-alkanamides, with and without alkyl and cycloalkyl moieties in the beta-position of the alkanamido side chain, have been prepared and tested for their ability to activate pigment granule aggregation in Xenopus laevis melanophores and bind to the recombinant human MT(1) and MT(2) melatonin receptor subtypes expressed in NIH 3T3 cells. An increase of the spacer's length in the side chain by a methylene unit (from 17d to 21d) leads to a six-fold decrease in antagonistic activity. On the other hand, the introduction of two methyl groups in the beta-position of the side chain of 17a induces agonist potency (compound 24), implying thus that the two beta-methyl groups are not only tolerated by the receptor, but constitute functional probes in its dynamic agonist-antagonist conformational equilibrium. The presence of more bulky beta-substituents, regardless of the size of the R group, compounds 24a,b, seems to lead to antagonism and to a noteworthy MT(2) subtype selectivity. Last, the new N1-C7 annulated derivatives presented herein are substantially more potent than their respective N1-C2 annulated counterparts, previously reported.

A series of new N -acyl 8,9-dihydro-4-methoxy-7 H -2-benzo[ de ]quinolinalkanamines have been prepared and tested for their ability to activate pigment granule aggregation in Xenopus laevis melanophores and bind to the recombinant human MT 1 and MT 2 melatonin receptor subtypes expressed in NIH 3T3 cells. Compounds with a single methylene spacer in the side chain ( 7 ) have no agonist activity, but are weak antagonists in the Xenopus melanophore assay, irrespectively of the size or shape of the R substituent (R = CH 3 to c-C 4 H 7 ). In contrast, compounds with two ( 8 ) or three ( 9 ) methylene spacers show partial agonist activity, though this does vary with the nature of the R substituent. Interestingly, the cyclopropane and cyclobutane R substituents, which are usually linked with antagonism, render the cyclopropanecarboxamido analog 9d and its cyclobutanecarboxamido congener 9e weak agonists. It seems, therefore, that in these compounds the R substituent constitutes a functional probe in the dynamic agonist–antagonist conformational equilibrium. One of the new molecules, antagonist 8c , exhibits a noteworthy MT 2 subtype selectivity (13-fold), whereas the acetamido analog 9a (with a three methylene units spacer) also acts as an antagonist and is the only analog exhibiting MT 1 selectivity (>10-fold). In contrast to the analogous N 1–C7 annulated indole derivatives, recently reported, the new C1–C8 condensed isoquinolines are not all pure antagonists. Despite their modest receptor affinity at the binding site these compounds demonstrate that the nature of the response (agonist or antagonist activity) is dependent, in this case, on both the side chain spacer’s length and the size and shape of the R group.

Melatonin (5-methoxy N-acetyltryptamine) is a hormone synthesized and released from the pineal gland at night, which acts on specific high affinity G-protein coupled receptors to regulate various aspects of physiology and behaviour, including circadian and seasonal responses, and some retinal, cardiovascular and immunological functions. In amphibians, such as Xenopus laevis, another role of melatonin is in the control of skin coloration through an action on melanin-containing pigment granules (melanosomes) in melanophores. In these cells, very low concentrations of melatonin activate the Mel(1c) receptor subtype triggering movement of granules toward the cell centre thus lightening skin colour. Mel(1c) receptor activation reduces intracellular cAMP via a pertussis toxin-sensitive inhibitory G-protein (Gi), but how this and other intracellular signals regulate pigment movement is not yet fully understood. However, melanophores have proven an excellent model for the study of the molecular mechanisms which coordinate intracellular transport. Melanosome transport is reversible and involves both actin- (myosin V) and microtubule-dependent (kinesin II and dynein) motors. Melanosomes retain both kinesin and dynein during anterograde and retrograde transport, but the myosin V motor seems to be recruited to melanosomes during dispersion, where it assists kinesin II in dominating dynein thus driving net dispersion. Recent work suggests an important role for dynactin in coordinating the activity of the opposing microtubule motors. The melanophore pigment aggregation response has also played a vital role in the ongoing effort to devise specific melatonin receptor antagonists. Much of what has been learnt about the parts of the melatonin molecule required for receptor binding and activation has come from detailed structure-activity data using novel melatonin ligands. Work aiming to devise ligands specific for the distinct melatonin receptor subtypes stands poised to deliver selective agonists and antagonists which will be valuable tools in understanding the role of this enigmatic hormone in health and disease.

Analogues of melatonin (1) and of N-acetyl 5-ethoxytryptamine (3) in which the oxygen atoms are replaced by sulfur have been prepared and tested against human and amphibian melatonin receptors. All sulfur analogues show a decreased binding affinity at human MT1 and MT2 receptors and a reduced potency as melatonin agonists on the Xenopus melanophore assay. The 5-methoxy oxygen of melatonin is significantly more important for receptor binding than the amide oxygen. N-Acetyl 5-ethoxytryptamine shows a decrease in both binding affinity and potency in comparison with melatonin. In this series, replacing either the ethoxy or amide oxygen by sulfur has a similar but smaller effect on both binding affinity and potency. Using K(B)(H) values from Abraham's equations we have assessed the possibility of estimating EC50 values for sulfur isosteres from the EC50 values of their oxygen analogues.

SUMMARY The activity of 17β-hydroxysteroid dehydrogenase (17βHSD) was measured in human breast tumours and in normal breast tissue from premenopausal, perimenopausal and postmenopausal women. Enzyme activity was higher in tumour tissue than in normal tissue from the same breast and under the conditions of the assay the oxidation of oestradiol was higher than the reduction of oestrone. The physiological status of the women in the study did not relate to the activity of the enzyme in either normal or tumour tissue although fibroadenomas had less activity than adenocarcinomas. In postmenopausal women tumour tissue oestrogens were 2–3 fold higher than in normal tissue from the same breast. Furthermore, tumour tissue concentrations of oestradiol tended to be higher than those of oestrone although in normal tissue the two oestrogens were present in similar concentrations. In plasma from the same women oestrone was the predominant oestrogen. There appears to be no direct relationship between 17βHSD activity and oestrogen concentrations but the enzyme may play a part in determining the balance between oestrone and oestradiol according to substrate and cofactor availability.

Fertility critically depends by a small number of hypothalamic neurons secreting the neurohormone GnRH. During development GnRH neurons migrate from the nasal placode to the hypothalamus by following the migratory path formed by the vomeronasal axons. Developmental defects of this process can cause congenital GnRH deficiency (GD), characterized by absent/delayed puberty and consequent infertility. The underlying mutated loci are for the majority of GD cases unknown, partially because of a poor understanding of the molecular mechanisms that control the development of these crucial neuroendocrine cells. Here we provide evidence of the importance of class 3 semaphorins and their receptors in this process. Specifically, I will explain how two different semaphorins play distinct roles during the migration of GnRH neurons. Thus, semaphorin3A affects the migration of GnRH neurons in the nasal compartment, via the co-receptors Neuropilin-1 and 2, whereas semaphorin3E via its receptor plexind1 controls the survival of GnRH neurons once positioned in the hypothalamus. Accordingly, disrupted semaphorin signalling may be involved in the aethiopathogenesis of genetic diseases characterized by GnRH deficiency.