Context Central precocious puberty (CPP) has strong genetic and epigenetic influences. MECP2, an X-linked gene, encodes a DNA methylation reader with a role in gene transcription regulation. MECP2 loss-of-function mutations are linked to neurodevelopmental disorders, particularly with Rett syndrome, a severe disorder that may be associated with early puberty. MECP2 variants were identified in girls with CPP with or without neurodevelopmental disorders.Objective This work aimed to investigate potential MECP2 variants in boys with idiopathic CPP at 3 academic hospitals.Methods MECP2 DNA sequencing was performed in 10 boys with idiopathic CPP to screen for coding variants (5 exome; 5 Sanger sequencing). There were no mutations in other CPP-causing genes (MKRN3 and DLK1). MECP2 protein levels of wild-type and identified variants were assessed in a gonadotropin-releasing hormone neuronal cell line.Results We identified 2 hemizygous missense MECP2 variants in 2 unrelated boys. Patient 1 had very early sporadic CPP, speech delay, and autism. He harbored a p.Val312Ile variant, an extremely rare variant located in a critical MECP2 domain. Patient 2 had sporadic CPP with no apparent neurodevelopmental disorders. He harbored a rare variant, p.Arg366Cys. Both MECP2 variants were inherited from mothers with normal puberty. Neither patient had features of Rett syndrome. Both variants had reduced MECP2 protein levels, suggesting a potential deleterious effect.Conclusion MECP2 rare variants were identified in boys with sporadic CPP, expanding this association as previously described in girls. Our findings provide additional evidence for a role of MECP2, an epigenetic factor, in hypothalamic control of pubertal timing.

Mutations in the MECP2 gene, encoding the epigenetic reader Methyl-CpG binding protein 2, are the main cause of Rett syndrome, a rare neurodevelopmental disorder. Besides severe symptoms such as profound intellectual disability, loss of speech and motor skills, and epilepsy, loss of function of MECP2 has been associated with pubertal dysregulation, but the biological mechanisms leading to this remain unclear. Using a mouse model of Rett, in which males are hemizygous and females heterozygous for Mecp2 loss of function mutation, we assessed the onset and progression of puberty, together with increase in body weight and onset of neurological symptoms in post-weaning mice until puberty. In brain samples of young adult mice, we analysed hypothalamic Gonadotropin releasing hormone (GnRH) neurons by immunofluorescent labelling, and in plasma samples we measured circulating GnRH, LH, and testosterone concentrations. Finally, we analysed testosterone-dependent arginine-vasopressin circuits. In our mouse model we found delayed puberty in Mecp2 CD1-null males, associated with a reduced rate of weight gain, but with puberty onset occurring at a lower body weight than in wildtype controls. Despite later puberty onset, Mecp2CD1-null male mice were found to have an increased number of GnRH neurons, but displayed lower levels of circulating reproductive hormones. Consequently, Mecp2CD1-null males have deficient testosterone-dependent arginine-vasopressin innervation. In female Mecp2CD1-heterozygous mice, we found no overall significant differences in pubertal development or GnRH neurons. The lack of significant alterations in females might be related to a later onset of neurological symptoms due to heterozygosity. Our data supports that MECP2 is essential for typical pubertal development, with complete loss of Mecp2 in a male murine model resulting in abnormalities of pubertal timing related to lower body weight, with an observed increase in hypothalamic GnRH neurons.

Background Mutations in the MECP2 gene, encoding the epigenetic reader Methyl-CpG binding protein 2, are the main cause of Rett syndrome, a rare neurodevelopmental disorder. Besides severe symptoms such as profound intellectual disability, loss of speech and motor skills and epilepsy, loss of function of MECP2 has been associated with pubertal dysregulation, but the biological mechanisms leading to this remain unclear. Methods We first carried out a patient survey to assess pubertal timing in a sample of Spanish patients with Rett syndrome. Second, using a mouse model of Rett, in which males are hemizygous and females heterozygous for Mecp2 loss of function mutation, we assessed the onset and progression of puberty, together with increase in body weight and onset of neurological symptoms in post-weaning mice until puberty. In brain samples of young adult mice, we analysed hypothalamic Gonadotropin releasing hormone (GnRH) neurons by immunofluorescent labelling, and in plasma samples measured circulating GnRH and testosterone concentrations. Finally, we analysed testosterone dependent arginine-vasopressin circuits. Results Our data in patients are in agreement with previous reports showing that a subset of female patients with Rett syndrome experience a delayed timing of menarche. Further, in our mouse model we found delayed puberty in Mecp2 CD1 -null males, associated with a reduced rate of weight gain, but with puberty onset occurring at a lower body weight than in wildtype controls. Despite later puberty onset, Mecp2CD1 -null male mice were found to have an increased number of GnRH neurons, but displayed lower levels of circulating reproductive hormones. Consequently, Mecp2CD1- null males have deficient testosterone-dependent arginine-vasopressin innervation. In female Mecp2CD1 - heterozygous mice, we found no overall significant differences in pubertal development or GnRH neurons, albeit in a subset of mice with early neurological symptoms, we found lower body weight, and a trend to delayed vaginal opening but precocious first oestrous, attributable to variable phenotypic penetrance. Conclusions Our data supports that MECP2 is essential for typical pubertal development, with complete loss of Mecp2 in a male murine model resulting in abnormalities of pubertal timing with an observed increase in hypothalamic GnRH neurons. ### Competing Interest Statement The authors have declared no competing interest. FinRett, 2022 Conselleria de Educación, Cultura, Universidades y Empleo, Generalitat Valenciana, CIAICO/2023/027, ACIF/2022/387, ACIF/2024/402 Wellcome Trust, 222049/Z/20/Z Barts Charity, MGU0552 British Society of Neuroendocrinology Society for Endocrinology

Glucocorticoids (GCs) are widely recognized for their potent anti-inflammatory and analgesic effects. Although they can cause an array of side effects when delivered systemically these are generally avoided when delivered locally at disease sites such as the eyes, lungs and joints. Glucocorticoid formulations for local use range from crystals and particles through to non-biodegradable implants. In many formulations burst release means that their effectiveness does not persist for more than a few weeks. Novel delivery methods that achieve prolonged delivery of GCs along with sequential degradation of the polymer vehicle has the potential to enhance the effectiveness of these drugs and achieve better control of disease. In this study we use a soft lithography method to produce polymer microchamber array films (MCAs) containing crystals of GCs. We demonstrate that the rate of glucocorticoid release can be adjusted through the choice of polymer used in the manufacture of films with rapid release observed with PLGA 50/50 over the course of 9 weeks and the longest duration of release observed with PLA films which continued beyond a year. Importantly, these release studies do not show evidence of burst release and all films displayed a significant duration of zero order release kinetics. Observations of film degradation were made through changes in their size, microscopic appearance and liberation of lactic acid from the films during the course of experiments demonstrated the association with GC release kinetics. These flexible films can be rolled into fibers with little change in release kinetics and the rolled MCAs can also be injected in vivo through a syringe needle to a delivery site. We envisage that this study could lead to an innovative approach to achieve prolonged release of GCs from biodegradable formulations at disease sites.

The neuroendocrine control of puberty and reproduction is fascinatingly complex, with up- and down-regulation of key reproductive hormones during fetal, infantile, and later childhood periods that determine the correct function of the hypothalamic-pituitary-gonadal axis and the timing of puberty. Neuronal development is a vital element of these processes, and multiple conditions of disordered puberty and reproduction have their etiology in abnormal neuronal migration or function. Although there are numerous documented cases across multiple conditions wherein patients have both neurodevelopmental disorders and pubertal abnormalities, this has mostly been described ad hoc and the associations are not clearly documented. In this review, we aim to describe the overlap between these two groups of conditions and to increase awareness to ensure that puberty and reproductive function are carefully monitored in patients with neurodevelopmental conditions, and vice versa. Moreover, this commonality can be explored for clues about the disease mechanisms in these patient groups and provide new avenues for therapeutic interventions for affected individuals.

Introduction: Precocious puberty is defined as the appearance of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys. Central precocious puberty (CPP) is a rare condition that is diagnosed when premature activation of the hypothalamic-pituitary-gonadal axis is detected, in association with precocious breast development or testicular growth. Idiopathic CPP is historically considered to be the most common form, but in recent years defects in a small but growing number of genes regulating the timing of puberty have been identified in an increasing proportion of cases of CPP. Delta-like non-canonical Notch ligand 1 (DLK1) is understood to be one of the key genes involved in the etiology of CPP, although its mechanistic role is not yet fully understood. Case Presentation: We identified a novel de novo variant of DLK1 (c.835C>T; p.Gln279*) in an 8-year-old girl of Bangladeshi origin. She presented with an advanced Tanner staging of B4P4A2, significantly advanced bone age (BA, 13 years), a near-adult proportioned uterus, with a history of menarche at the age of 7.4 years. Diagnosis was confirmed by raised basal luteinizing hormone concentration. She was found to have truncal obesity associated with abnormal fasting insulin levels and mildly elevated cholesterol levels. These findings are consistent with previous literature describing an association between patients with DLK1 deficiency and an impaired metabolic profile. The patient was treated for 2 years with GnRH agonists with ongoing biochemical follow-up into adolescence. Conclusion: This case illustrates the susceptibility to metabolic derangement for patients with mutations in DLK1 and the need for ongoing monitoring after puberty. Our summary of previously identified DLK1 variants and their metabolic consequences demonstrates the frequency of obesity, lipid abnormalities, and insulin dysregulation in this patient cohort in childhood and beyond. This knowledge can guide future clinical practice for patients with CPP patients due to DLK1 deficiency.

Background Identification of genetic causes of central precocious puberty have revealed epigenetic mechanisms as regulators of human pubertal timing. MECP2, an X-linked gene, encodes a chromatin-associated protein with a role in gene transcription. MECP2 loss-of-function mutations usually cause Rett syndrome, a severe neurodevelopmental disorder. Early pubertal development has been shown in several patients with Rett syndrome. The aim of this study was to explore whether MECP2 variants are associated with an idiopathic central precocious puberty phenotype. Methods In this translational cohort study, participants were recruited from seven tertiary centres from five countries (Brazil, Spain, France, the USA, and the UK). Patients with idiopathic central precocious puberty were investigated for rare potentially damaging variants in the MECP2 gene, to assess whether MECP2 might contribute to the cause of central precocious puberty. Inclusion criteria were the development of progressive pubertal signs (Tanner stage 2) before the age of 8 years in girls and 9 years in boys and basal or GnRH-stimulated LH pubertal concentrations. Exclusion criteria were the diagnosis of peripheral precocious puberty and the presence of any recognised cause of central precocious puberty (CNS lesions, known monogenic causes, genetic syndromes, or early exposure to sex steroids). All patients included were followed up at the outpatient clinics of participating academic centres. We used high-throughput sequencing in 133 patients and Sanger sequencing of MECP2 in an additional 271 patients. Hypothalamic expression of Mecp2 and colocalisation with GnRH neurons were determined in mice to show expression of Mecp2 in key nuclei related to pubertal timing regulation. Findings Between Jun 15, 2020, and Jun 15, 2022, 404 patients with idiopathic central precocious puberty (383 [95%] girls and 21 [5%] boys; 261 [65%] sporadic cases and 143 [35%] familial cases from 134 unrelated families) were enrolled and assessed. We identified three rare heterozygous likely damaging coding variants in MECP2 in five girls: a de novo missense variant (Arg97Cys) in two monozygotic twin sisters with central precocious puberty and microcephaly; a de novo missense variant (Ser176Arg) in one girl with sporadic central precocious puberty, obesity, and autism; and an insertion (Ala6_Ala8dup) in two unrelated girls with sporadic central precocious puberty. Additionally, we identified one rare heterozygous 3 & PRIME;UTR MECP2 insertion (36_37insT) in two unrelated girls with sporadic central precocious puberty. None of them manifested Rett syndrome. Mecp2 protein colocalised with GnRH expression in hypothalamic nuclei responsible for GnRH regulation in mice. Interpretation We identified rare MECP2 variants in girls with central precocious puberty, with or without mild neurodevelopmental abnormalities. MECP2 might have a role in the hypothalamic control of human pubertal timing, adding to the evidence of involvement of epigenetic and genetic mechanisms in this crucial biological process.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes.

Nanoengineered vehicles have the potential to deliver cargo drugs directly to disease sites, but can potentially be cleared by immune system cells or lymphatic drainage. In this study we explore the use of magnetism to hold responsive particles at a delivery site, by incorporation of superparamagnetic iron oxide nanoparticles (SPIONs) into layer-by-layer (LbL) microcapsules. Microcapsules with SPIONs were rapidly phagocytosed by cells but did not trigger cellular ROS synthesis within 24 hours of delivery nor affect cell viability. In a non-directional cell migration assay, SPION containing microcapsules significantly inhibited movement of phagocytosing cells when placed in a magnetic field. Similarly, under flow conditions, a magnetic field retained SPION containing microcapsules at a physiologic wall shear stress of 0.751 dyne cm-2. Even when the SPION content was reduced to 20%, the majority of microcapsules were still retained. Dexamethasone microcrystals were synthesised by solvent evaporation and underwent LbL encapsulation with inclusion of a SPION layer. Despite a lower iron to volume content of these structures compared to microcapsules, they were also retained under shear stress conditions and displayed prolonged release of active drug, beyond 30 hours, measured using a glucocorticoid sensitive reporter cell line generated in this study. Our observations suggest use of SPIONs for magnetic retention of LbL structures is both feasible and biocompatible and has potential application for improved local drug delivery.

C-type natriuretic peptide (CNP) is the most conserved member of the mammalian natriuretic peptide family, and is implicated in the endocrine regulation of growth, metabolism and reproduction. CNP is expressed throughout the body, but is particularly abundant in the central nervous system and anterior pituitary gland. Pituitary gonadotropes are regulated by pulsatile release of gonadotropin releasing hormone (GnRH) from the hypothalamus, to control reproductive function. GnRH and CNP reciprocally regulate their respective signalling pathways in αT3-1 gonadotrope cells, but effects of pulsatile GnRH stimulation on CNP expression has not been explored. Here, we examine the sensitivity of the natriuretic peptide system in LβT2 and αT3-1 gonadotrope cell lines to continuous and pulsatile GnRH stimulation, and investigate putative CNP target genes in gonadotropes. Multiplex RT-qPCR assays confirmed that primary mouse pituitary tissue express Nppc,Npr2 (encoding CNP and guanylyl cyclase B (GC-B), respectively) and Furin (a CNP processing enzyme), but failed to express transcripts for Nppa or Nppb (encoding ANP and BNP, respectively). Pulsatile, but not continuous, GnRH stimulation of LβT2 cells caused significant increases in Nppc and Npr2 expression within 4 h, but failed to alter natriuretic peptide gene expression in αT3-1 cells. CNP enhanced expression of cJun, Egr1, Nr5a1 and Nr0b1, within 8 h in LβT2 cells, but inhibited Nr5a1 expression in αT3-1 cells. Collectively, these data show the gonadotrope natriuretic peptide system is sensitive to pulsatile GnRH signalling, and gonadotrope transcription factors are putative CNP-target genes. Such findings represent additional mechanisms by which CNP may regulate reproductive function.

Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCG beta-subunit (LHB), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates LHB promoter activity. Here, studies of the LHB proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equine chorionic girdle (ChG) and surrounding tissues. Luciferase assays demonstrated endogenous activity of the LHB promoter in BeWo choriocarcinoma cells with greatest activity by a proximal 335 bp promoter fragment. Transactivation studies in COS7 cells using an equine GCM1 expression vector showed GCM1 could transactivate the proximal 335 bp LHB promoter. Chromatin immunoprecipitation using primary ChG trophoblast cells showed GCM1 to preferentially bind to the most proximal GCM1-binding site over site 2. Mutation of site 1 but not site 2 resulted in a loss of endogenous promoter activity in BeWo cells and failure of GCM1 to transactivate the promoter in COS-7 cells. Together, these data show that GCM1 binds to site 1 in the LHB promoter but also requires the upstream segment of the LHB promoter between -119 bp and -335 bp of the translation start codon for activity. GCM1 binding partners, ETV1, ETV7, HOXA13, and PITX1, were found to be differentially expressed in the ChG between days 27 and 34 and are excellent candidates for this role. In conclusion, GCM1 was demonstrated to drive the LHB promoter, through direct binding to a predicted GCM1-binding site, with requirement for another factor(s) to bind the proximal promoter to exert this function. Based on these findings, we hypothesize that ETV7 and HOXA13 act in concert with GCM1 to initiate LHB transcription between days 30 and 31, with ETV1 partnering with GCM1 to maintain transcription.

Equine chorionic girdle trophoblast cells play important endocrine and immune functions critical in supporting pregnancy. Very little is known about the genes and pathways that regulate chorionic girdle trophoblast development. Our aim was to identify genes and signalling pathways active in vivo in equine chorionic girdle trophoblast within a critical 7-days window. We exploited the late implantation of the equine conceptus to obtain trophoblast tissue. An Agilent equine 44K microarray was performed using RNA extracted from chorionic girdle and chorion (control) from equine pregnancy days 27, 30, 31 and 34 (n = 5), corresponding to the initiation of chorionic girdle trophoblast proliferation, differentiation and migration. Data were analysed using R packages limma and maSigPro, Ingenuity Pathway Analysis and DAVID and verified using qRT-PCR, promoter analysis, western blotting and migration assays. Microarray analysis showed gene expression (absolute log FC >2, FDR-adjusted P < 0.05) was rapidly and specifically induced in the chorionic girdle between days 27 and 34 (compared to day 27, day 30 = 116, day 31 = 317, day 34 = 781 genes). Pathway analysis identified 35 pathways modulated during chorionic girdle development (e.g. FGF, integrin, Rho GTPases, MAPK) including pathways that have limited description in mammalian trophoblast (e.g. IL-9, CD40 and CD28 signalling). Rho A and ERK/MAPK activity was confirmed as was a role for transcription factor ELF5 in regulation of the CGB promoter. The purity and accessibility of chorionic girdle trophoblast proved to be a powerful resource to identify candidate genes and pathways involved in early equine placental development.

Chromatin immunoprecipitation (ChIP) has become a widely used methodology for assessment of protein/DNA interactions. The technique allows the analysis of direct binding of transcription factors to gene promoters, identification of histone modifications, and localization of DNA modifying enzymes. Antibodies conjugated to agarose beads can be utilized to immunoprecipitate specific proteins, cross-linked to sheared chromatin regions to which they are bound endogenously. With downstream applications including quantitative real-time polymerase chain reaction (qRT-PCR), genome-wide sequencing (ChIP-seq), microarray analysis (ChIP-chip), and mass spectrometry (ChIP-MS), the technique enables comprehensive assessment of protein/DNA interactions. Here I describe ChIP, followed by qRT-PCR, to assess direct binding of a single protein to multiple predicted binding sites within a gene promoter.

Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family that are secreted by trophoblast cells. PSGs may modulate immune, angiogenic and platelet responses during pregnancy. Until now, PSGs are only found in species that have a highly invasive (hemochorial) placentation including humans, mice and rats. Surprisingly, analyzing the CEACAM gene family of the horse, which has a non-invasive epitheliochorial placenta, with the exception of the transient endometrial cups, we identified equine CEACAM family members that seem to be related to PSGs of rodents and primates. We identified seven genes that encode secreted PSG-like CEACAMs Phylogenetic analyses indicate that they evolved independently from an equine CEACAM1-like ancestor rather than from a common PSG-like ancestor with rodents and primates. Significantly, expression of PSG-like genes (CEACAM44, CEACAM48, CEACAM49 and CEACAM55) was found in non-invasive as well as invasive trophoblast cells such as purified chorionic girdle cells and endometrial cup cells. Chorionic girdle cells are highly invasive trophoblast cells that invade the endometrium of the mare where they form endometrial cups and are in close contact with maternal immune cells. Therefore, the microenvironment of invasive equine trophoblast cells has striking similarities to the microenvironment of trophoblast cells in hemochorial placentas, suggesting that equine PSG-like CEACAMs and rodent and primate PSGs have undergone convergent evolution. This is supported by our finding that equine PSG-like CEACAM49 exhibits similar activity to certain rodent and human PSGs in a functional assay of platelet-fibrinogen binding. Our results have implications for understanding the evolution of PSGs and their functions in maternal-fetal interactions.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

TGFβ superfamily proteins, acting via SMAD (Sma- and Mad-related protein)2/3 pathways, regulate placental function; however, the role of SMAD1/5/8 pathway in the placenta is unknown. This study investigated the functional role of bone morphogenetic protein (BMP)4 signaling through SMAD1/5 in terminal differentiation of primary chorionic gonadotropin (CG)-secreting trophoblast. Primary equine trophoblast cells or placental tissues were isolated from day 27–34 equine conceptuses. Detected by microarray, RT-PCR, and quantitative RT-PCR, equine chorionic girdle trophoblast showed increased gene expression of receptors that bind BMP4. BMP4 mRNA expression was 20- to 60-fold higher in placental tissues adjacent to the chorionic girdle compared with chorionic girdle itself, suggesting BMP4 acts primarily in a paracrine manner on the chorionic girdle. Stimulation of chorionic girdle-trophoblast cells with BMP4 resulted in a dose-dependent and developmental stage-dependent increase in total number and proportion of terminally differentiated binucleate cells. Furthermore, BMP4 treatment induced non-CG-secreting day 31 chorionic girdle trophoblast cells to secrete CG, confirming a specific functional response to BMP4 stimulation. Inhibition of SMAD2/3 signaling combined with BMP4 treatment further enhanced differentiation of trophoblast cells. Phospho-SMAD1/5, but not phospho-SMAD2, expression as determined by Western blotting was tightly regulated during chorionic girdle trophoblast differentiation in vivo, with peak expression of phospho-SMAD1/5 in vivo noted at day 31 corresponding to maximal differentiation response of trophoblast in vitro. Collectively, these experiments demonstrate the involvement of BMP4-dependent pathways in the regulation of equine trophoblast differentiation in vivo and primary trophoblast differentiation in vitro via activation of SMAD1/5 pathway, a previously unreported mechanism of TGFβ signaling in the mammalian placenta.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)