bluuebunny/crossref_metadata_2025_split huggingface dataset:
Disruption of Estrogenic and Androgenic Bioactivities in Human Fetuses Exposed to Maternal Smoking
Authors: Talia Chiara , Budin Clémence , Panagiotis Filis , van der Burg Bart , Connolly Lisa , Fowler Paul A 
Published: May 2021

Abstract
Endocrine disruptors (EDs) interfere with hormonal signalling and, given that multiple developmental processes are hormone-driven, the prenatal period is a window of increased sensitivity. Maternal smoking is a real-life model of in utero exposure to a complex mixture of EDs. Cigarette smoke contains of &gt;7,000 pollutants, including polycyclic aromatic hydrocarbons (PAHs), which are AhR ligands and cross-talk with the estrogen receptor (ER) system. Prenatal exposure to cigarette smoke is associated with adverse outcomes, including intrauterine growth restriction and increased risk of metabolic syndrome later in life. We aimed to evaluate ED effects associated with smoke exposure in human fetuses. Fetal tissues (plasma, n=48; placenta, n=30; liver, n=29) from elective terminations of normally progressing pregnancies, ranging from 10 to 20 gestation weeks, were collected (SAFeR and FEGO studies: REC 15/NS/0123, REC 04/S0802/21). PAHs and PAH-like compounds were extracted from placenta and fetal liver. Bioactivity levels in plasma, placenta and liver extracts were determined using ER and androgen receptor (AR) transactivation reporter gene assays. PAH burden was evaluated using the AhR-responsive DRhp-CALUX assay. Smoke exposure was associated with a 1.3-fold increase in plasma estrogenic activity. The developmental trajectory of androgenic activity was altered in plasma of smoke-exposed fetuses, with significant anti-androgenic activity in older fetuses (&gt;16 weeks of gestation). In males, plasma androgenic activity was positively associated with testes weight and anogenital distance. In contrast, placentas from smoking mothers had significantly increased androgenic potential. Furthermore, AhR-like activity was 2.9-fold higher in smoke-exposed placentas compared to controls, and 2.3-fold higher in female compared to male fetal livers. Overall, all bioactivity levels were higher in placentas compared to fetal liver. Prenatal exposure to cigarette smoke is associated with higher placental AhR activation, indicative of increased xenotoxicants burden. We also report that smoke-exposed fetuses showed increased circulating estrogenic activity and disrupted androgenic potential, across 10-20 weeks of gestation, in both fetal plasma and placenta. This demonstrates that EDs present in cigarette smoke are able to interfere with hormonal signalling and alter dynamic endocrine activity profiles, which are critical to ensure appropriate, sex-specific, development. These ED effects are likely to disturb placental function and reprogramme fetal development and thus impacting on life-long health.
Comprehensive and Cost-Effective Strategy for Genetic Screening of Congenital Adrenal Hyperplasia (CAH) in India
Authors: Ravichandran Lavanya , Korula Sophy , Asha H S , Varghese Deny , Parthiban R , Jabasteen J , Jebasingh Felix , Kapoor Nitin , Cherian Kripa Elizabeth , Mathai Sarah , Simon Anna , Paul Thomas V , Thomas Nihal , Chapla Aaron 
Published: May 2021

Abstract
Background: With substantial challenges in molecular analysis of 21 hydroxylase deficiency and lack of studies in extended panel of genes implicated in CAH, genetic diagnosis is largely unavailable and unaffordable in India. Therefore, we aim to develop a cost-effective screening strategy in CAH using Allele Specific PCR and Targeted Next-Generation Sequencing (NGS). Methods: Long range PCR and restriction digestion were utilized to specifically amplify the CYP21A2 gene whereas multiplex PCR was used to amplify CYP11B1, CYP17A1, CYP19A1 and POR genes. In house developed Allele Specific PCR (ASPCR) for 8 hotspot mutations in CYP21A2 gene and targeted NGS for five genes was carried out. The results were validated using Sanger sequencing and MLPA. Results: Of the 50 patients suspected for 21 hydroxylase deficiency, 64% (n=32) were of Salt Wasting phenotype (SW), 30% (n=15) with Simple Virilizing (SV) phenotype and 6% (n=3) of the study population were suspected for non-classical (NC) CAH. The mutation positive rate of ASPCR was 86% (n=43). Seven patients carried more than two biallelic mutations indicating smaller gene conversions. The predominant mutation identified among the study subjects was I2G splice variant in SW phenotype (38%) and I172N in the SV phenotype (41%). Based on the Long range PCR amplification and restriction digestion we identified one patient with large gene conversion and one patient with large 30kb deletion. These results were confirmed with MLPA.
Additionally, utilizing the Targeted NGS we identified five patients with CYP21A2 variants (two patients with novel variants c.1274G&gt;T, c.17_18delTG and two other variants in three patients - c.1451G&gt;C, c.143A&gt;G). We also identified a CYP19A1:c.1142A&gt;T gene variant in a patient who was initially suspected for 21 hydroxylase deficiency. Out of six patients with 11 beta hydroxylase deficiency four patients were positive for homozygous CYP11B1 variants (c.1201-1G&gt;A, c.1200 + 1delG, c.412C&gt;T) and two patients with compound heterozygous variants (c.1024C&gt;T and c.1012dupC, c.623G&gt;A and c.412C&gt;T).
Discussion: Utilizing the novel allele specific PCR followed by NGS we identified a total of 96% (48/50) of 21 hydroxylase deficiency patients with homozygous or compound heterozygous mutations and 2% (2/50) were positive for single heterozygous variant in CYP21A2 gene. ASPCR followed by multigene targeted NGS assay for genetic screening in CAH has shown to be a sensitive and specific strategy established in a clinical setting. To best of our knowledge this is the most cost-effective and comprehensive multigene screening carried out in India.
Heterodimerization and Subcellular Distribution of Melatonin and Cannabinoid Type 1 Receptors
Authors: Cui Yuanxu , Jockers Ralf , Lahuna Olivier 
Published: May 2021

Abstract
Membrane receptors belonging to the G proteins coupled receptors (GPCRs) form the largest family of proteins in the human genome with more than 800 members. Until recently GPCRs functions were thought to occur only at the plasma membrane after activation upon binding of their cognate ligand. However evidences show that many functional GPCRs are found in intracellular compartments opening new direction of research to understand their roles in a cellular context. Among these intracellular compartments mitochondria are the latest organelle in which some GPCRs were identified. Melatonin receptor type 1 (MT1) and cannabinoid receptor type 1 (CB1) were identified in mouse neuronal mitochondria where they were shown to exert an inhibitory action on cytochrome c release (MT1) or on the respiratory chain (CB1). Using several techniques my current results describe a new cross-talk between MT1 and CB1 receptors. Confocal analysis of immunofluorescence experiments of cells coexpressing both receptors showed a high degree of colocalisation. A combination of coimmunoprecipitation experiments performed on extracts of transfected HEK293T or HeLa cell lines and immunodetection of receptors by Western-blot revealed that MT1 and CB1 receptors can physically interact to form heterodimers in absence of ligand. Heterodimers formation was also confirmed by Proximity Ligation Assay (PLA) experiments in live HEK293T and HeLa cells. Confocal analysis revealed a colocalisation of PLA staining with the mitochondrial marker TOMM20. Experiments in relation with the functional role of MT1 and CB1 in mitochondria are ongoing.
Single Nucleus RNA-Sequencing Reveals Overall Conservation of Hypothalamic Cell Identities but Differential Expression of Specific Genes in the Magel2 Null Mouse Model of Prader-Willi Syndrome
Authors: Yu Hui , Low Malcolm James 
Published: May 2021

Abstract
Prader-Willi syndrome (PWS) is a genetic disorder affecting 1 in 10,000 to 30,000 live births. Diagnostic features of PWS including insatiable appetite and obesity are well-defined and many are associated with disruption of hypothalamic function. PWS is caused by sporadic or inherited loss of expression from the paternal allele of one or more maternally imprinted/silenced genes located in chromosomal region 15q11-q13 that encompasses five protein coding genes Mkrn3, Magel2, Necdin and Snurf-Snprn and a family of snoRNAs. Seminal studies have indicated that isolated Magel2 gene silencing plays a pivotal role in the development of many, but not all, clinical features of PWS. Magel2 is highly expressed in the hypothalamus and loss of function studies revealed substantial cellular and molecular changes in hypothalamic neurons located in the suprachiasmatic, paraventricular, supraoptic and arcuate nuclei and the lateral hypothalamus. In addition to neuronal alterations, loss of MAGEL2 increases the density and activation of microglia in adult hypothalamus. In the current study, we characterized global changes in hypothalamic gene expression and searched for novel cell populations associated with loss of Magel2 expression using single nucleus RNA sequencing. Single cell nuclei were isolated in two technical replicates per group from hypothalami of adult male and female Magel2-null (C57BL/6-Magel2tm1Stw/J) and wild type sibling mice for the 10X genomics scRNA-seq pipeline. A total of 63,470 cells divided approximately equally by sex and Magel2 genotype were analyzed. Unsupervised cell clustering identified 19 distinct clusters in males (10 neuronal and 9 non-neuronal) and 21 clusters in females (11 neuronal and 10 non-neuronal) based on their transcriptional profiles of signature genes. The percentages of total cells and the transcriptional profiles of each defined cluster from all four combinations of genotype and sex were nearly identical, indicating that loss-of-function of Magel2 does not alter overall cell cluster identities in the hypothalamus. However, a quantitative analysis of gene expression profiles from all individual clusters demonstrated upregulation of a set of genes including Fkbp5, Zbtb16, Htra1, 2900097C17Rik and 1700030F04Rik predominantly in oligodendrocytes and astrocytes in both sexes of Magel2 null mice. In contrast, the majority of down-regulated genes were found in neuronal cell clusters in both sexes of Magel2 null mice. Our current study is the first to characterize cellular and genetic changes in the whole hypothalamus due to the lack of MAGEL2. The data provide a valuable resource for elucidating the regulatory mechanisms of MAGEL2 for the pathogenesis of PWS and shed light on the discovery of new candidate targets for the potential treatment of PWS. This study is supported by the Foundation for Prader-Willi Research and NIH grant R01DK068400.
Oral Octreotide Capsules Lowered Incidence and Improved Severity of Acromegaly Symptoms Compared to Injectable Somatostatin Receptor Ligands—Results From the MPOWERED Trial
Authors: Biermasz Nienke , Fleseriu Maria , Dreval Akexander V , Pokramovich Yulia , Bondar Irina , Isaeva Elena , Molitch Mark E , Macut Djuro P , Leonova Nina , Raverot Gerald , Gilgun-Sherki Yossi , Ludlam William H , Patou Gary , Haviv Asi , Gordon Murray B , Urbanavicius Vaidotas , Knispelis Robertas , Melmed Shlomo , Strasburger Christian J 
Published: May 2021

Abstract
Background: Patients with acromegaly may have high symptom burden. The phase 3 MPOWERED trial assessed control of acromegaly by oral octreotide capsules (OOC; MYCAPSSA®) in comparison to injectable somatostatin receptor ligands (iSRLs) in patients responding to both OOC and iSRLs. iSRLs have been first-line medical treatment for patients with acromegaly for decades. OOC are newly approved in the US for patients previously controlled on iSRLs.
Methods: Eligibility criteria for MPOWERED included acromegaly diagnosis, biochemical control of acromegaly (insulin-like growth factor I &lt;1.3 × upper limit of normal; mean integrated growth hormone, &lt;2.5 ng/mL) and ≥6 months’ iSRL (octreotide, lanreotide) treatment. Eligible patients entered a 26-week Run-in phase to determine the effective OOC dose; responders at week 24 then entered a 36-week randomized controlled treatment (RCT) phase receiving OOC or iSRLs. Acromegaly symptom number and severity (mild to severe, 1-3) were collected. Total score was calculated by summating all severity scores (Acromegaly Index of Severity [AIS]). Symptom results were assessed using total AIS score and proportion of patients experiencing individual symptoms.
Results: At beginning of Run-in, average AIS score of 92 randomized patients was 4.52, representative of symptoms experienced while previously receiving iSRLs. After 26 weeks’ OOC treatment at end of Run-in, average AIS score was significantly reduced to 3.46 (P&lt;0.001). More than 80% of patients on OOC improved or maintained AIS score during Run-in compared to baseline. Over this 26-week period, there was a significant reduction in extremity swelling (P=0.01) and fatigue (P=0.03). During the RCT, of patients randomized to OOC (n=55), 73% maintained or improved AIS score, and 75% maintained or reduced overall number of active symptoms. In comparison, 68% of those randomized to iSRLs (n=37) maintained or improved AIS score, and 70% maintained or reduced overall number of active symptoms.
Conclusion: Results from MPOWERED show that patients receiving OOC had significant improvement in number and severity of acromegaly symptoms after switching from iSRLs. These findings validate previous results from a phase 3 study of OOC in acromegaly in which patients switching to OOC from iSRLs showed significant reduction in joint pain, extremity swelling, and fatigue.1
1Melmed S, et al. JCEM. 2015;100(4):1699-1708.
SIX3 Variant Causes Pituitary Stalk Interruption Syndrome and Combined Pituitary Hormone Deficiency
Authors: Bando Hironori , Brinkmeier Michelle , Gergics Peter , Fang Qing , Mortensen Amanda Helen , Ozel Ayse Bilge , Ma Qianyi , Li Jun , Reynaud Rachel , Castinetti Frederic , Brue Thierry Christian , Camper Sally Ann 
Published: May 2021

Abstract
The genetic basis for congenital hypopituitarism and related disorders is beginning to emerge, and over 30 causal genes have been identified. Mutations in some of these genes can also cause holoprosencephaly (HPE) or septo-optic dysplasia. SIX3 is a homeodomain protein expressed in the developing brain, pituitary gland, and eye. Heterozygous mutations in SIX3 cause variable HPE in humans and mice. We identified two children with neonatal GH and TSH deficiency and stalk interruption who were doubly heterozygous for rare, likely deleterious variants in SIX3 and POU1F1. Functional studies demonstrated that both variants are disruptive. We used Six3 and Pou1f1 loss of function mice to assess the genetic interaction between Six3 and Pou1f1. Six3 heterozygotes have variable pituitary gland dysmorphology, while Pou1f1 heterozygotes are normal. A significant portion of the Six3+/-; Pou1f1+/dw doubly heterozygous mice have a more pronounced pituitary phenotype than Six3+/-, supporting the possibility of digenic pituitary disease. To understand the role of SIX3 in pituitary and hypothalamic development, we used Prop1-cre and Nkx2.1-cre to delete Six3. Disruption of Six3 expression in Rathke’s pouch caused poor activation of Lhx3 expression and arrested anterior pituitary development. The Nkx2.1-cre, Six3flox/flox embryos had no evidence of infundibulum evagination and failed to induce FGF and BMP signaling, which normally drive expansion of Rathke’s pouch. By E11.5 cells in Rathke’s pouch underwent apoptosis. The Nkx2.1-cre, Six3flox/flox embryos failed to activate expression of Lhx2 and Tbx3 in the neural ectoderm. These embryos had elevated CCND1, MYCN, and Axin2 expression in the area of the presumptive infundibulum. This indicates that SIX3 is necessary to repress cell proliferation and Wnt/beta-catenin signals to promote formation of the pituitary stalk. Thus, Six3 has essential roles in both the neural and oral ectoderm for hypothalamic and pituitary development, respectively. Heterozygous loss of function variants in SIX3 could be a contributor to multiple pituitary hormone deficiencies in children, especially if there are associated craniofacial abnormalities or PSIS.
A Mouse Model of Shiftwork Reveals Sex-Specific Impairments in Circadian Behavior and Reproductive Tissue Timekeeping
Authors: Yaw Alexandra M , Mclane-Svoboda Autumn , Nguyen Duong , Duong Thu Van Quynh , Hoffmann Hanne Mette 
Published: May 2021

Abstract
Approximately 21 million Americans are employed in nighttime or rotating shiftwork, which is linked with numerous health risks, including reduced reproductive success. While there is a strong body of clinical evidence associating shift workers with deficits in fertility, very little is known about the mechanisms by which shiftwork produces reproductive dysfunction. One important feature of shiftwork is extensive exposure to light at night. Light is one of the strongest timing cues for the circadian system, where exposure to mistimed light (light at night) can disrupt timekeeping throughout the body, leading to a mistiming of physiological processes and resulting in disease development. Our goal is to determine if a light-based model of rotating shiftwork (shift-light), composed of an alternating 6 hour phase advance or delay every 4 days for 5-10 weeks, disrupts circadian rhythms in the reproductive hypothalamic-pituitary-gonadal (HPG) axis, leading to mis-timed tissue rhythms and reduced fertility. We hypothesize that shift-light disrupts wheel running behavior and underlying cellular circadian rhythms in the HPG axis, resulting in reduced reproductive function. Using the validated circadian Per2:luciferase reporter mice, we assessed wheel-running behavior and Per2:luciferase rhythms in tissue explants from males and females. Behavioral data revealed that both sexes adapt their wheel running to shift-light paradigm; however, females, but not males, displayed a significant deficit in their ability to entrain to phase advances following 4 shifts. This sex-specific disruption was supported by preliminary tissue explant Per2:luciferase rhythms, which suggest that shift-light alters tissue level circadian phase synchrony in female, but not male, HPG axis tissues. Importantly, females exhibited shortened estrous cycling during shift-light, suggesting the altered HPG axis synchrony could be directly impacting reproductive function. We are currently working to extend this work to determine how this desynchrony impacts hormone release, including luteinizing hormone and follicle stimulating hormone. Together, this work provides insight into how shiftwork may influence circadian rhythms in reproductive tissues and suggests that females may have increased vulnerability to reproductive deficits from shiftwork.
Canine Pituitary Organoids as 3D In Vitro Model for Cushing Disease
Authors: Sanders Karin , Ringnalda Femke C A , van de Wetering Marc L , Kooistra Hans S , Meij Björn P , Clevers Hans , Galac Sara 
Published: May 2021

Abstract
Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.