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HORMAD1 expression is usually restricted to germline cells, but it becomes mis-expressed in epithelial cells in ~60% of triple-negative breast cancers (TNBCs), where it is associated with elevated genomic instability (1). HORMAD1 expression in TNBC is bimodal with HORMAD1-positive TNBC representing a biologically distinct disease group. Identification of HORMAD1-driven genetic dependencies may uncover novel therapies for this disease group. To study HORMAD1-driven genetic dependencies, we generated a SUM159 cell line model with doxycycline-inducible HORMAD1 that replicated genomic instability phenotypes seen in HORMAD1-positive TNBC (1). Using small interfering RNA screens, we identified candidate genes whose depletion selectively inhibited the cellular growth of HORMAD1-expressing cells. We validated five genes (ATR, BRIP1, POLH, TDP1 and XRCC1), depletion of which led to reduced cellular growth or clonogenic survival in cells expressing HORMAD1. In addition to the translesion synthesis (TLS) polymerase POLH, we identified a HORMAD1-driven dependency upon additional TLS polymerases, namely POLK, REV1, REV3L and REV7. Our data confirms that out-of-context somatic expression of HORMAD1 can lead to genomic instability and reveals that HORMAD1 expression induces dependencies upon replication stress tolerance pathways, such as translesion synthesis. Our data also suggest that HORMAD1 expression could be a patient selection biomarker for agents targeting replication stress.

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.

Classical quantifiers (like "all," "some," and "none") express relationships between two sets, allowing us to make generalizations (like "no elephants fly"). Devices like these appear to be universal in human languages. Is the ubiquity of quantification due to a universal property of the human mind or is it attributable to more gradual convergence through cultural evolution? We investigated whether classical quantifiers are present in a new language emerging in isolation from other languages, Nicaraguan Sign Language (NSL). An observational study of historical data collected in the 1990s found evidence of potential quantifier forms. To confirm the quantificational meaning of these signs, we designed a production study that elicited, from three age cohorts of NSL signers (N = 17), three types of quantifiers: universal (all), existential (some), and negative (none). We find evidence for these classical quantifiers in the very first generation of signers, suggesting they may reflect a universal property of human cognition or a very rapid construction process.

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

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.

In July 2012 the American College of Obstetricians and Gynecologists (ACOG) published Committee Opinion No. 530, stating that postpartum sterilization should be considered an urgent surgical procedure. The purpose of this study was to determine whether active dissemination of the ACOG Committee Opinion and education of staff (our intervention) would result in higher rates of postpartum sterilizations.Two separate studies were conducted: (1) a retrospective chart review that examined postpartum sterilization rates prior to our intervention and (2) a prospective study that examined postpartum sterilization rates after the intervention.In the 3 months prior to the ACOG publication, 14 of 23 (61%) intended postpartum sterilization procedures were performed. During the 4 months after publication, 29 of 52 (55%) were completed (x² p=0.619). In the 5 months after education efforts, 69 of 79 (87%) were completed, showing a statistically significant improvement frot both prior time periods (x² p0.001).Patients who desired postpartum sterilization during the prospective study were 5 times less likely to have their procedure canceled than were patients in the retrospective study. The Committee Opinion had. no significant effect on. com- pletion rates. Education of staff and the ongoing study were instrumental in facilitating desired tubal sterilizations.

To describe the prevalence of aspiration in children with unilateral vocal fold paralysis who underwent objective assessment of swallow function.Retrospective chart review.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.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.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.4 Laryngoscope, 129:569-573, 2019.

During acquisition, children must learn both the meanings of words and how to interpret them in context. For example, children must learn the logical semantics of the scalar quantifier some and its pragmatically enriched meaning: ‘some but not all’. Some studies have shown that ‘scalar implicature’ – that some implies ‘some but not all’ – poses a challenge even to nine-year-olds, while others find success by age three. We asked whether reports of children's successes might be due to the computation of exclusion inferences (like contrast or mutual exclusivity) rather than scalar implicatures. We found that young children (N = 214; ages 4;0–7;11) sometimes compute symmetrical exclusion inferences rather than asymmetric scalar inferences. These data suggest that a stronger burden of evidence is required in studies of implicature; before concluding that children compute implicatures, researchers should first show that children exhibit sensitivity to asymmetric entailment in the task.

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.

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.

We tested the hypothesis that, when children learn to correctly count sets, they make a semantic induction about the meanings of their number words. We tested the logical understanding of number words in 84 children that were classified as "cardinal-principle knowers" by the criteria set forth by Wynn (1992). Results show that these children often do not know (1) which of two numbers in their count list denotes a greater quantity, and (2) that the difference between successive numbers in their count list is 1. Among counters, these abilities are predicted by the highest number to which they can count and their ability to estimate set sizes. Also, children's knowledge of the principles appears to be initially item-specific rather than general to all number words, and is most robust for very small numbers (e.g., 5) compared to larger numbers (e.g., 25), even among children who can count much higher (e.g., above 30). In light of these findings, we conclude that there is little evidence to support the hypothesis that becoming a cardinal-principle knower involves a semantic induction over all items in a child's count list.

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.

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.

Employing 1,3-dipolar cycloaddition for the synthesis of the 7a-azaindole nucleus, analogues of melatonin have been synthesized and tested against human and amphibian melatonin receptors. Introducing a phenyl substituent in position 2 of the heterocyclic moiety significantly increased binding affinity to both the MT1 and MT2 receptors. Shifting the methoxy group from position 5 to 2 of the 7a-azaindole ring led to a substantial reduction of MT1 binding when MT2 recognition was maintained. We theoretically investigated the hypothesis whether the 2-methoxy function of the azamelatonin analogue 27 is able to mimic the 5-methoxy group of the neurohormone by directing its 2-methoxy function toward the methoxy binding site. DFT calculations and experimental binding differences of analogue compounds indicate that the energy gained by forming the methoxy-specific hydrogen-bond interaction should exceed the energy required for adopting an alternative conformation.

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.