bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Mayer-Rokitansky-Kuster-Hauser (MRKH) Patient Families by Whole Genome Sequencing
Authors: Dougherty Michael P , Chorich Lynn P , Layman Lawrence Clarke
Published: May 2021

Introduction: MRKH is a characterized by the congenital absence of the uterus and vagina in 46,XX individuals. A subset of these patients also has associated renal, skeletal, cardiac and/or auditory defects. Familial cases suggest a genetic component, but to date only pathogenic variants in WNT4 and HNF1B have been confirmed. We hypothesize that de novo heterozygous variants in candidate genes will be present in some patients with MRKH.

Methods: DNAs from 30 quads (an MRKH proband and three relatives) were subjected to whole genome sequencing (WGS), and heterozygous variants in coding regions with < 0.02 frequency were filtered by two different methods. In the first approach, variants were filtered by 1) top consequence variant (splice site, stop-gain, frameshift, and missense, respectively); 2) impact score; 3) mapping quality; 4) cytobands; 5) intolerance; 6) de novo variants; and 7) plausibility based on familial genotype. The second approach considered only heterozygous variants found in the proband and absent in all other family members, which were then filtered by top consequence (splice donor and acceptor sites, stop-gain, frameshift).

Results: Five pedigrees were excluded for inadequate sequence in one or more individuals. 55,033 variants in coding regions with < 2% frequency were identified in the 25 remaining quads for analysis. Using the first approach, 42 candidate gene variants in 32 genes were identified - 12 splice variants, 10 stop-gains, 15 frameshift variants and 5 missense variants. Of these, MUC22 contained 2 missense variants from different families. Additionally, DICER1 had multiple splice variants and is essential for mouse urogenital tract development. In the second approach, 39 candidate genes were identified—6 splice variants in 6 genes, 18 stop-gains in 17 genes, and 17 frameshift variants in 16 genes. Zinc finger genes (ZNF418, ZNF646, ZNF135, and ZNF772) comprised the most frequent class of the 39 genes. Two genes (MIR4436A and ZNF418) contained attractive variants in two different families.

Conclusion: WGS has been shown to improve detection of gene variants in coding regions, more so than whole exome sequencing (WES). We previously performed WES on 111 MRKH probands without family members and analyzed variants in candidate genes suggested by mouse and preliminary human studies. Interestingly, in this study, only three genes overlapped with previously suspected candidate genes. Here, we identified new candidates based upon potential deleteriousness. These candidate genes will be studied further in our families to determine their role in Mullerian development.
Human Placenta MicroRNA Differences Between First and Third Trimester
Authors: Gonzalez Tania L , Eisman Laura E , Joshi Nikhil , Flowers Amy E , Wu Di , Wang Yizhou , Santiskulvong Chintda , Tang Jie , Buttle Rae A , Sauro Erica Joan A , Clark Ekaterina L , Jefferies Caroline , Chan Jessica , Lin Yayu , Williams John , Pisarska Margareta Danuta
Published: May 2021

MiRNAs are widespread regulators of gene expression, and altered miRNA expression in the placenta may be involved in abnormal placentation and related pregnancy-associated diseases. It is essential to understand miRNA expression changes across gestation before miRNAs can be used as biomarkers or prognostic indicators. Using next-generation sequencing, we characterize the normative human placenta miRNA transcriptome in both the first and third trimester using leftover chorionic villus sampling tissue from prenatal tests (N=113) as well as placentae collected at delivery (N=47). We identified 2503 miRNAs, including 1647 with stable expression across gestation (p>=0.05). There were 775 significantly differentially expressed miRNAs (FDR<0.05), with 402 upregulated in first trimester and 373 upregulated in third trimester. We also examine expression of the placenta-specific miRNA clusters on chromosomes 14 and 19 which are important in pregnancy. We identified predicted targets with high confidence scores or experimental verification, then used these to identify enriched canonical biological pathways. Our study identified canonical pathways consistently targeted across gestation, including pathways regulating senescence, proliferation and growth factor signaling. We also identified differentially impacted pathways, including growth- and immune-mediated pathways. This work provides a rich atlas to direct functional studies investigating the epigenetic differences in first and third trimester placentae. We gratefully acknowledge support from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD091773) and the Ruth L. Kirschstein National Research Service Award (T32DK007770) of the National Institutes of Health.
Investigation of Epigenetic Control of DAX-1 Expression in Human Cell Lines
Authors: Riedstra Caroline P , Tzagarakis-Foster Christina
Published: May 2021

Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X chromosome, gene 1 (DAX-1 or NR0B1) is an orphan nuclear hormone receptor implicated in Adrenal Hypoplasia Congenita (AHC) and Dosage Sensitive Sex Reversal (DSS). In both instances, DAX-1 plays a key role in growth and development by modulating hormone function. In DSS, mutations on the X-chromosome lead to duplication of the region containing DAX-1, resulting in sex reversal, and in AHC, mutations in the DAX-1 gene diminish development of adrenal tissue which leads to a reduction in adrenal hormone production. Expressed predominantly in tissues such as the testes, ovaries, breast, adrenal cortex, and lung, DAX-1 may serve as an indicator of aberrant growth. Here we hypothesize that DAX-1 is epigenetically regulated, specifically in cancer cells, thereby reducing its expression. We surveyed various human cancer cells in order to determine whether inhibiting DNA methylating enzymes released epigenetic control of the DAX-1 gene, resulting in an increase in expression. By implementing molecular techniques, such as bisulfite sequencing, we determined the precise methylation sites in the DAX-1 gene. Additionally, we carried out methylation specific restriction enzyme analysis to differentiate degrees of methylation between lung, breast, liver, cervical, and adrenal carcinoma cell lines. Following confirmation of the precise methylation sites, we utilized chromatin immunoprecipitation (ChIP) in order to identify the modifying proteins present on the DAX-1 CpG islands. In conjunction with these experimental techniques, we implemented a bioinformatics approach to analyze methylation in the promoter region of the DAX-1 gene across tissue sample data acquired from The Cancer Genome Atlas Program. The results of this research could lead to a translational application of understanding where this orphan NHR fits into the development and progression of cancer. As a quickly growing field, cancer epigenetics is a key player in the ongoing pursuit for identifying biomarkers that may be pertinent in future therapeutic applications.
Molecular Logic of Hypothalamus Development
Authors: Kim Thomas
Published: May 2021

The hypothalamus is a central regulator of physiological homeostasis. During development, multiple transcription factors coordinate the patterning and specification of hypothalamic nuclei. However, the molecular mechanisms controlling hypothalamic patterning and cell fate specification are poorly understood. To identify genes that control these processes, we have previously used single-cell RNA sequencing (scRNA-Seq) to profile mouse hypothalamic gene expression across multiple developmental time points and established database HyDD (Hypothalamus Developmental Database). We next used HyDD to characterize multiple mutant lines targetting key transcription factors that came out from our scRNA-Seq database (Nkx2.2, Dlx1/2, Isl1, Foxd1, Lhx2), and was able to comprehensively characterize mutants that have altered hypothalamic patterning. Our phenotype result supports a modified columnar model of organization for the diencephalon, where prethalamus and hypothalamus are situated in adjacent dorsal and ventral domains of the anterior diencephalon. Furthermore, using our mouse hypothalamus as a guideline, we are comparing scRNA-Seq dataset of developing chicken, zebrafish and human hypothalamus, to identify evolutionarily conserved and divergent region-specific gene regulatory networks. Lastly, we are improving mouse HyDD, in order to characterize adult hypothalamus neuronal subtypes.
Regulation of Melanocortin-5 Receptor Pharmacology by Two Isoforms of MRAP2 in Ricefield Eel (Monopterus albus)
Authors: Liu Ting , Yi Ti-Lin , Yang Dai-Qin , Tao Ya-Xiong
Published: May 2021

The melanocortin-5 receptor (MC5R) has been implicated in the regulation of exocrine gland secretion, immune regulation, and muscle fatty acid oxidation. However, its function in fish is not well established. Melanocortin-2 receptor accessory protein 2 (MRAP2) can modulate trafficking, ligand binding, and signaling of melanocortin receptors. Ricefield eel (Monopterus albus) is an economically and evolutionarily important fish widely distributed in tropics and subtropics. To explore potential interaction between eel MC5R and MRAP2, herein we cloned ricefield eel mc5r, mrap2X1 and mrap2X2. Eel mc5r consisted of a 1056 bp open reading frame encoding a protein of 351 amino acids. Eel mrap2X1 consisted of a 708 bp open reading frame encoding a protein of 235 amino acids, while eel mrap2X2consisted of a 567 bp open reading frame encoding a protein of 188 amino acids. Multiple sequence alignment showed that maMRAP2X1 and maMRAP2X2 shared 90.43% identity. Interestingly, maMRAP2X2 lost the transmembrane domain. Phylogenetic analysis showed that maMC5R and maMRAP2s were closely related to piscine MC5Rs and MRAP2s, respectively. The maMC5R was further demonstrated to be a functional receptor and could be modulated by maMRAP2s in pharmacological studies. Three agonists, [Nle4, D-Phe7]-alpha-melanocyte stimulating hormone (NDP-MSH), alpha-MSH, and adrenocorticotropin (ACTH), could bind to maMC5R and induce intracellular cAMP production dose-dependently. Compared to human MC5R (hMC5R), maMC5R displayed a significantly decreased Bmax but higher binding affinity to alpha-MSH or ACTH. No significant difference in constitutive activity was observed between hMC5R and maMC5R. When stimulated with α-MSH and ACTH, maMC5R showed significantly lower EC50 and Rmax than that of hMC5R. Eel MRAP2s had no effect on cell surface and total expression of maMC5R, whereas they significantly increased Bmax. Only maMRAP2X2 significantly decreased the binding affinity of ACTH. Both maMRAP2X1 and maMRAP2X2 significantly reduced Rmax but did not affect EC50 in response to alpha-MSH or ACTH stimulation. The availability of maMC5R pharmacological characteristics and the modulation by maMRAP2s will facilitate the investigation of its function in regulating diverse physiological processes in ricefield eel.
Laminin-α4 Is Uniquely Upregulated in Subcutaneous White Adipose Tissue in Murine and Human Models of Obesity
Authors: Goddi Anna , Carmona Alanis , Schroedl Liesl , Piron Matthew J , White Jeremy M , Porras Maria Gonzalez , Brey Eric M , Brady Matthew J , Cohen Ronald
Published: May 2021

As research into the adipocyte microenvironment has advanced, it is becoming more widely accepted that the extracellular matrix (ECM) contributes to adipocyte dysfunction. The majority of current published work focuses on the role of collagens in metabolic disease while less emphasis has been placed on the contribution of laminins, an important component of the adipocyte basement membrane. Laminins are trimeric ECM proteins composed of α, β, and γ chains. The α chains contain sites which can interact with cell surface receptors and is considered the driver of tissue-specific expression and specialized signaling. Our group has shown that the laminin-α4 (LAMA4) chain, which is highly expressed in mature adipocytes, plays a role in adipocyte function and thermogenesis in mice (1). In this study we investigate the relationship between laminin α chain expression and obesity by assessing gene expression of LAMA1-5 in subcutaneous white adipose tissue (sWAT) from mice fed chow (RCD) and 45% high fat diet (HFD) for 8 weeks. Expression of LAMA2 and LAMA4 was significantly increased in the HFD sWAT compared to chow (6.1 fold, p=0.01 and 4.9 fold, p=0.001 respectively), however LAMA4 displayed a much stronger positive correlation with weight (R2=0.697) than did LAMA2 (R2=0.382). In order to validate the relevance of these findings in human models of obesity, we evaluated gene expression of LAMA2, LAMA4, and LAMA5 in sWAT biopsies from non-diabetic adult females with obesity (class II or higher). sWAT samples from obese subjects exhibited 4.5 fold higher LAMA4 expression (p=0.0089) than samples from non-obese control subjects, suggesting that the LAMA4 chain may play an important role in human obesity. Lastly we examined changes in sWAT LAMA4 expression following a period of weight loss in obese mice and in human subjects after bariatric surgery, and found that LAMA4 expression levels remain largely unchanged in both cases. In this study we demonstrate the relationship between LAMA4 expression and obesity and present findings that can be extended to human models of obesity.

Reference: (1) Vaicik et al., Endocrinology. 2018 Jan;159(1):356–67.
Sox2 Is Required Independently in Both Stem and Differentiated Cells for Pituitary Tumour Genesis in P27 Null Mice
Authors: Moncho-Amor Veronica , Chakravarty Probir , Galichet Christophe , Lovell-Badge Robin , Rizzoti Karine
Published: May 2021

Tumour development can depend on cell intrinsic dysfunction, but, in some cases, extrinsic factors are important drivers. Here, we have established a genetically tractable model demonstrating that the same gene is relevant both cell-autonomously and non-cell-autonomously for tumorigenesis. P27, a cell cycle inhibitor, is also able to drive repression of the transcription factor Sox2. This interaction plays a crucial role during development of p27-/- pituitary tumours because loss of one copy of Sox2 impairs tumorigenesis (1). However, SOX2 is expressed in both endocrine and stem cells (SC), and its contribution to tumorigenesis in either cell type is unknown. We have thus explored the cellular origin and mechanisms underlying endocrine tumorigenesis in p27-/- pituitaries. We found that pituitary hyperplasia is associated with reduced cellular differentiation, in parallel with increased levels of SOX2 in stem and endocrine cells. Using conditional loss-of-function and lineage tracing approaches, we show that SOX2 is required cell-autonomously in p27-/- endocrine cells for these to give rise to tumours, and in SCs for promotion of tumorigenesis. This is supported by studies deleting the Sox2 regulatory region 2 (Srr2), the target of P27 repressive action. Single cell transcriptomic analysis further reveals that activation of a SOX2-dependent MAPK pathway in SCs is important for tumorigenesis. Altogether, our data highlight different aspects of the role of SOX2 following loss of p27, according to cellular context, and uncover an unexpected SOX2-dependent tumour-promoting role for SCs. Our results imply that targeting SCs, in addition to tumour cells, may represent an efficient anti-tumoral strategy in certain contexts.

(1) Li H, et al. (2012) p27(Kip1) directly represses Sox2 during embryonic stem cell differentiation. Cell Stem Cell 11(6):845-852.
The Use of Genetic Testing Panels for Dyslipidemia: A Quality Improvement Project at the McGill University Health Centre
Authors: Pasqua Melissa-Rosina , Pare Aurelie , Blank David , Gilfix Brian
Published: May 2021

Background: Genetic testing panels are used to identify the most common genetic causes of dyslipidemia, and the results of these panels can guide treatment and management. The objective of this quality improvement project was to assess the appropriateness of genetic testing panels requested by the McGill University Health Centre (MUHC). Methods: Genetic testing panels sent for analysis from January 2018 to December 2019 were identified. Ordering physician specialty, patient personal and family medical history, lipid panel results, and genetic testing results were collected. Then, validated Familial Hyperlipidemia (FH) scores (Simon-Broome Registry Criteria, Dutch Lipid Clinic Network Criteria, FH Canada criteria) were calculated for patients who underwent genetic testing for suspected FH. Results: There were 36 genetic test panels sent out for analysis during the study period, of which 24 were accessible for data analysis. Pathogenic mutations were identified in 7/24 (29%) of the analyzed panels. The 19/24 (79%) of the panels were requested by lipid specialists, and all of the panels positive for pathogenic mutations were requested by lipid specialists. Interestingly, 23/24 (94%) of the patients met the Canadian criteria for at least considering genetic testing, suggesting that most panels were appropriately requested. Only 3/24 (12%) of patients had insufficient criteria for FH by the Simon-Broome criteria, but all of these carried pathogenic mutations. Conclusion: These results suggest that at the MUHC, using the Canadian criteria identifies a greater number of patients for genetic testing and for appropriate diagnosis and treatment.
Mice Deficient for an Intestinal G Protein-Coupled Receptor Expression Have Increased Satiety During Rebound Hyperphagia
Authors: Yan Shijun , Ren Hongxia
Published: May 2021

Gut-derived hormones have been successfully developed as the therapeutic targets to combat the increasing prevalence of diabetes and obesity. G protein-coupled receptors (GPCRs) in the gastrointestinal (GI) tract are involved in maintaining glucose and energy homeostasis by regulating the release of gut hormones in response to luminal dietary nutrients as well as microbial metabolites. We identified that an orphan GPCR, Gpr17, was expressed in the intestinal epithelium and found that loss of intestinal Gpr17 expression increased gut incretin hormone secretion from enteroendocrine cells (EECs). However, it is unknown how Gpr17 ablation in the intestinal epithelium affects feeding behavior and satiety regulation. To address this question, we used genetic knockout approach to generate intestinal Gpr17-deficient mice and analyzed their feeding behavior. Here we show that intestinal Gpr17-deficient mice had similar growth curve, body composition, and ad libitum food intake compared with littermate controls. Interestingly, intestinal Gpr17-deficient mice responded to fasting-refeeding challenge with reduced fasting locomotor activity and less food intake after refeeding, suggesting increased satiety during the phase of rebound hyperphagia. Moreover, we performed fasting-refeeding challenge with Gpr17-deficient mice fed on high-fat diet (HFD), and our meal pattern analysis revealed that these mice had reduced meal duration of the first meal after refeeding. In conclusion, our genetic knockout studies in rodents showed that ablating intestinal Gpr17 increased satiety during rebound hyperphagia in the fasting-refeeding experimental paradigm. Intestinal Gpr17 could be developed as a therapeutic target to treat obesity by improving energy balance through gut hormone secretion and meal pattern control.
Pegvisomant as Monotherapy or Combination Therapy in Somatostatin Refractory Acromegaly
Authors: Hordejuk Dawid , Laws Edward Raymond , Kaiser Ursula B , Min Le
Published: May 2021

Background: Pegvisomant, a growth hormone antagonist, has been widely used as monotherapy or combination therapy with somatostatin (SST) analogs and/or dopamine agonists in acromegaly poorly controlled by SST analogs. Limited information is available to compare pegvisomant monotherapy, combination with SST analogs or dopamine agonists, and combination of all three agents.

Method: In this retrospective cohort study, we identified 23 patients with SST analog refractory acromegaly who received pegvisomant as monotherapy or in combination with SST analogs and/or dopamine agonists through the Research Patient Data Registry. We divided the patients into four groups: Group 1. pegvisomant alone (n=8); Group 2. pegvisomant plus a SST analog (pasireotide, octreotide or lanreotide) (n=8); Group 3. pegvisomant plus cabergoline (n=5) Group 4. Pegvisomant plus SST analog and dopamine agonist (n=2). We analyzed the changes in IGF-1, HbA1C, ALT and AST, blood pressure, and radiographic tumor size before and 6 months after treatment.

Results: In 6 months, the mean IGF-1 level (ng/ml) changed from baseline 482 to 290 and decreased by 40% (P = 0.050) in group 1, changed from baseline 623 to 291 and decreased by 53% (P= 0.003) in group 2, changed from baseline 579 to 367 and decreased by 36% (p = 0.100) in group 3, and decreased 47% from 609 to 326 (P= 0.100) in group 4.

The mean systolic blood pressure (mmHg) before and 6 months after treatment changed from 139 to 128 (p = 0.001) in group 1, changed from 130 to 126 (p = 0.553) in group 2, changed from 134 to 126 (p = 0.373) in group 3, and changed from 125 to 127 (p= 0.700) in group 4. Diastolic blood pressure (mmHg) changed from 82 to 76 (P = 0.110) in group 1, changed from 79 to 76 (p = 0.325) in group 2, changed from 80 to 74 (p=0.002) in group 3, and changed from 80 to 75 (p=0.126) in group 4.

There were no significant changes in ALT and AST and A1C before and 6 months after treatment in all groups. In terms of radiographic tumor size change before and 6 months after the treatment, there was no change in tumor size in 5 of 5 patients in group 1. In group 2, the tumor size in 4 of 7 remained unchanged but 3 of 7 patients had increased tumor sizes. In group 3, there was no change in tumor size in 3 of 3 patients. In group 4, there was no change in tumor size in 2 of 2 patients.

Conclusion: Our results suggest that in somatostain analog refractory acromegaly, combination pegvisomant and a SST analog significantly decreased IGF-1 level although decrease in IGF-1 in pegvisomant monotherapy almost reach statistical significance (P = 0.050). Although there was a trend in decrease of blood pressure in all groups, the decrease reached significant significance in systolic blood pressure in group 1 and diastolic blood pressure in group 3. Finally, except group 2, the tumor size remained unchanged.
Real-World Experience With Pasireotide Lar in Acromegaly Resistant to First-Generation Somatostatin Analogs: A Single Center 1-Year Observation
Authors: Stelmachowska-Banas Maria , Czajka-Oraniec Izabella Katarzyna , Tomasik Agnieszka , Zgliczynski Wojciech
Published: May 2021

Introduction: First generation somatostatin analogs (SSAs) are the treatment of choice in persistent acromegaly after transsphenoidal surgery. However, they are effective in 25% to 45 % of patients and a second generation SSA - pasireotide LAR may be a more effective alternative. Aim Our aim was to evaluate the impact of 1-year treatment with pasireotide LAR on disease control and on glucose metabolism in patients with acromegaly after debulking surgery resistant to first-generation SSAs. Material and methods In this single-center prospective study 29 consecutive patients with resistant acromegaly were treated with pasireotide LAR. The initial drug dose was 40 mg i.m. every 28 days. If patient did not achieve biochemical control (GH <2.5 ng/mL and IGF-1 ≤ULN for age and sex) at month 3, the dose was increased to 60 mg i.m. every 28 days. Assessment (GH, IGF-1, glucose, HbA1c) was performed at month 3, 6, 9 and 12. Results: In total, 22 patients (10 females, 12 males) completed a 1-year treatment. Mean age was 41.8±13.8 years. Twelve patients (54.5%) were ≤ 40 years old (including 6 (27.3%) patients of age ≤30 years). At baseline, mean IGF-1 level was 2.3 (SD 0.7) x ULN (age- and sex-specific) (583.9 ng/mL; SD 182.2) and mean GH concentration was 3.9 (SD 2.8) ng/mL. Both values decreased significantly after 1 year of treatment (P<0.001). Pasireotide LAR dose was increased to 60 mg in 16 (72.7%) patients and decreased to 20 mg in one patient patient due to worsening of diabetes control. The magnitude of mean GH level decrease was the largest within first 6 months (mean change from baseline: -1.75 ng/mL, 95% CI: -2.64, -0.85, P=0.0006). Mean IGF-1 level decreased rapidly within the first 3 months (mean change from baseline: -153.20 ng/mL, 95% CI: -203.20, -103.19, P<0.0001) and remained low during 12-month follow-up. GH level ≤ 1 ng/mL and ≤2.5 ng/mL was achieved by 7 (31.8%) and 17 (77.3%) patients, respectively. Six patients (27.3%) achieved normal IGF-1 level (IGF-1 ≤1 x ULN) (P=0.0275). IGF-1 ≤1.3 x ULN was observed in 11 (50.0%) of patients. Full biochemical control (GH ≤1 ng/mL and IGF-1 ≤1 x ULN) was achieved in 3 (13.6%) patients. Pasireotide LAR treatment resulted in significant increase of mean fasting glucose level: 119.2 (SD 17.3) vs. 107.5 (SD 13.9) mg/dL, P<0.001. The largest change was observed in first 3 months, and it remained stable until month 12. HbA1c level also increased significantly during first 3 months and stayed on similar level during follow-up (mean for month 12: 6.3 (SD 0.6) vs. 5.9 (SD 0.5) % at baseline, P<0.001). Conclusions: Pasireotide LAR is an effective treatment in most patients with persistent acromegaly after surgical debulking resistant to first generation SSAs. The largest increase of glycemia occurs during first 3 months of treatment and it remains stable afterwards.
FOXA2 as a Candidate Gene Responsible for Congenital Panhypopituitarism: A Review of the Literature
Authors: Correia Jorge César , Corral Laura Gómez , Kaygusuz Sare Betül , Gualtieri Angelica , Gaston-Massuet Carles
Published: May 2021

The pituitary gland produces hormones that regulate multiple functions including growth, metabolism, reproduction, and homeostasis. Thus, congenital hypopituitarism (CH) can have profound and widespread repercussions on physiological balance. Recent research identified the forkhead box A2 transcription factor (FOXA2) as a candidate gene responsible for CH. We sought to review the literature for mouse models and case reports describing FOXA2 mutations, to shed more light on the potential role of this gene in CH. Using a pretested search strategy, we searched the PubMed database, including in the review only original research articles reporting FOXA2 gene mutations in mouse models and human case reports. A total of 10 studies reporting different Foxa2 mutations in mouse models were included. These works described the involvement of Foxa2 in the regulation of murine organogenesis. Foxa2 was found to participate in the development of ventral midline structures and endodermal-derived organs. In addition, when mutated, it was found to determine defects in node, notochord and the neural tube, precursors of the pituitary gland. Foxa2 was also found to have important effects on glucose homeostasis and its deficiency is characterized by hyperinsulinemic hypoglycaemia. Regarding human case reports, a total of 5 cases describing nonsynonymous missense mutations of FOXA2 were identified. All mutations were localized in the DNA binding domain, which might regulate the expression of tissue-specific genes important for cell differentiation. Panhypopituitarism was a prominent feature among the cases as well as hypoglycaemia in infancy with abnormal glucose homeostasis later in life. Additional 6 cases describing patients with varying deletions of 20p11.2 that encompasses FOXA2 were selected. In addition to panhypopituitarism, the patients were found to have several other dysmorphic features, affecting the face as well as the cardiac, gastrointestinal and genital systems. Authors proposed a region of approximately 1.35 Mb that covers around 17 genes, among which FOXA2, as the critical region associated with hypopituitarism. However, the other genes also deleted from this region are associated with the development of the central nervous system and the pancreas and may be responsible of the observed phenotype of these patients. Considering this evidence, FOXA2 seems to be a strong candidate for CH. However, further research is required to elucidate its involvement in pituitary development, as well as the genetic cause that drive FOXA2 haploinsufficiency in determining CH.
Preclinical Weight Loss Efficacy of AM833 in Combination With Semaglutide in Rodent Models of Obesity
Authors: John Linu M , Kruse Thomas , Raun Kirsten
Published: May 2021

Weight management pharmacotherapies seldom induce body weight loss that is comparable to that produced by bariatric surgery. In this regard, combination therapies targeting multiple signaling pathways that regulate energy balance may provide a means to achieve greater weight loss efficacy while also allowing the use of lower doses of each drug, and thus mitigating potential gastrointestinal tolerability issues commonly observed with current therapeutics for weight management. Amylin and GLP-1 are peptide hormones that regulate appetite. Upon ingestion of a meal, amylin is co-secreted with insulin from pancreatic beta-cells, while GLP-1 is secreted from enteroendocrine cells in the intestine. Both native peptides have a short half-life and reduce food intake, delay gastric emptying and decrease glucagon levels. Amylin and GLP-1 analogues have been developed for the treatment of diabetes, as well as weight management. The long-acting once-weekly GLP-1 analogue, semaglutide is approved for the treatment of type 2 diabetes and is in clinical development for weight management. AM833 (NNC0174-0833) is a long-acting, once-weekly human amylin analogue that is also in clinical development for weight management. Here, we present the combined effect of AM833 and semaglutide on weight loss in rodent models of obesity. Diet-induced obese (DIO) rats and mice on a high energy diet were dosed subcutaneously once-daily for 24 or 28 days with sub-maximal doses of AM833, semaglutide or two modes of combination treatments. The first combination mode consisted of concurrent co-dosing of both drugs, while the second entailed add-on of AM833 after one week of treatment with semaglutide. Body weight and food intake were measured daily. Body composition was assessed by magnetic resonance scan pre- and post-treatment. In the DIO rat, both concurrent (-13.1% ± 0.7%) and add-on (-12.8% ± 1.2%) treatment modes induced equivalent weight loss that was greater than each monotherapy (-6.3% ± 0.7%, 2 nmol/kg semaglutide and -5.8% ± 0.9%, 2 nmol/kg AM833) relative to initial body weights. Both combination groups achieved normalization of body weight to that of lean age-matched control rats. Reductions in cumulative food intake corresponded with the extent of weight loss. In the DIO mouse, weight loss in the combination groups was not significantly different (-9.6% ± 1.5%, concurrent vs. -11.5% ± 1.2%, add-on) but was greater than that observed in each monotherapy group (-1.9% ±1.2%, 1 nmol/kg semaglutide and +1.5% ± 2.2%, 10 nmol/kg AM833). In the DIO mouse, body weight did not normalize to match that of lean controls with combination treatment. In both rodent models, combination therapy at submaximal doses of AM833 and semaglutide achieved significantly greater weight loss compared to the monotherapy groups highlighting the potential of this combination for further clinical development.
Corticosterone Enhances Formation of Neutral but Not Fear Memory During Infectious Illness
Authors: Hill Alice , Johnston Colin , Spencer-Segal Joanna L
Published: May 2021

Survivors of critical illness often report traumatic memories of their illness period, and these memories are thought to contribute to development of neuropsychiatric disorders, such as PTSD. Many patients are treated with high doses of glucocorticoids for their vasoactive and anti-inflammatory properties, and glucocorticoids have also been shown to prevent the development of PTSD after trauma. Due to their activity in the hippocampus and amygdala, the putative protective effect of glucocorticoids may occur via memory formation during illness. To examine the effect of glucocorticoids on memory formation during acute infectious illness, male and female C57BL/6 mice (N=80, 40 male/40 female) underwent cecal ligation and puncture and were treated with either corticosterone (16 mg/kg) or vehicle in the early afternoon daily for five days beginning on the day of surgery. All mice were habituated to a neutral object in their home cage for five days and underwent one 30-minute footshock/no shock training session during the illness period. After physiologic recovery (2 weeks), the mice underwent behavioral testing including open field exploration, object recognition testing in which they were presented with both the familiar (habituated) object and a novel object, and testing in the shock context. The results showed that drug treatment had no effect on behavior in the open field, including time spent in the center (VEH: 20.19±10.81 vs CORT: 22.32±12.87 sec; P=0.476). Drug treatment increased overall object exploration (12.28±10.79 vs 19.17±15.88 sec; P=0.049). Corticosterone-treated mice showed a preference for the familiar object (60.9±23.0% of total exploration time with familiar object; P=0.015), while vehicle-treated mice did not (54.1±23.3%; P=0.378). The increase in overall object exploration seen in corticosterone-treated mice could be accounted for by an increase in exploration of the familiar object. History of footshock increased freezing in the training context (3.96±2.54% vs 36.08±15.42%; P<0.0001) and corticosterone treatment had no effect (18.06±17.65% vs 22.16±21.19%; P=0.557). In conclusion, administration of corticosterone during infectious illness facilitated memory of a neutral object from the illness period, and recovered mice exhibited a preference for this object over a novel one. Corticosterone treatment had no impact on fear memory formed during illness. This is consistent with human literature suggesting that hydrocortisone decreases PTSD symptoms without impacting traumatic memories. These findings suggest that glucocorticoids selectively enhance the formation, consolidation, and/or recall of neutral but not fear memories during illness, which may rely on hippocampal circuitry. We further suggest that accurate memories of the illness period may influence patients’ perception of this experience and alter their risk for psychiatric sequelae.
Epigenetic Regulation of Gonadotropin Hormone Beta Subunit Gene Expression
Authors: Ruggiero Rebecca E , Coss Djurdjica
Published: May 2021

Gonadotropin releasing hormone (GnRH) from the hypothalamus regulates the synthesis and secretion of gonadotropin hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH regulates steroidogenesis in both sexes and ovulation in females, while FSH stimulates folliculogenesis in females and spermatogenesis in males. LH and FSH are heterodimers of a common alpha subunit and a unique beta subunit, which provides biological specificity and is the rate limiting factor in hormone synthesis. Immediate early genes, early growth response 1 (Egr1) and fos proto-oncogene (cFos) are critical in the induction of LH-beta and FSH-beta subunits by GnRH, respectfully. However, gaps exist in our understanding of developmental initiation and hormonal regulation of gonadotropin gene expression. Specifically, epigenetic mechanisms that may play a role in beta subunit transcriptional regulation are unknown. The aim of this work was to identify transcriptional cofactors that are recruited to gonadotropin beta-subunit promoters with or without GnRH. Transcription factors interact with cofactors that recruit chromatin remodeling enzymes in order to regulate transcription. Identification of cofactors may explain tight regulation of gonadotropin hormone levels in reproductive physiology. Previous studies identified regions on the beta-subunit promoters that are necessary for GnRH responsiveness. These regions were used to pull down interacting proteins that bind to these response elements using nuclear extracts from the immortalized mature gonadotrope cell line, LβT2. Using a discovery proteomics approach, we identified different transcriptional cofactors that are recruited to beta subunit promoters with or without GnRH. Approximately 1500-2000 proteins were identified per pulldown. As expected, proteins known to interact with beta subunit promoters, such as Egr1, cFos and cJun were identified in the DNA pulldown experiments as positive controls. We identified 63 proteins unique for LH-beta promoter under control conditions and 60 unique for FSH-beta promoter, of which 7 proteins for LH-beta and 8 proteins for FSH-beta may play a role as corepressors. We further identified 97 proteins that were pulled down with the LH-beta promoter following GnRH treatment, of which 9 proteins were also pulled down with Egr1 as potential coactivator candidates. We also identified 72 proteins that were pulled down with the FSH-beta promoter following GnRH treatment, of which 6 proteins were pulled down with cFos as potential coactivator candidates. Functional studies to identify roles of these cofactors in gonadotropin hormone expression are in progress. The identification of epigenetic regulators will allow for better understanding of the transcriptional regulation of gonadotropin beta-subunit gene expression, which is critical for reproductive function.
Expression of Kisspeptin, Neurokinin B, and Dynorphin During Pubertal Development in Female Sheep
Authors: Aerts Eliana G , Harlow KaLynn E , Griesgraber Max J , Bowdridge Elizabeth C , Hardy Steven L , Nestor Casey C , Hileman Stanley M
Published: May 2021

Puberty onset depends upon an increase in pulsatile GnRH/LH secretion, which in sheep is the result of reduced sensitivity to estrogen negative feedback. Neurons within the arcuate nucleus of the hypothalamus (ARC) expressing kisspeptin, neurokinin B (NKB), and dynorphin (i.e. KNDy neurons) express estrogen receptors and are believed to play a key role in mediating the effects of estrogen on GnRH/LH secretion. Therefore, the purpose of this study was to assess changes in kisspeptin, NKB, and dynorphin within the ARC across pubertal development in female sheep. Blood samples were collected at 12-minute intervals for 4 hours and assessed for LH secretion in five age groups of ewes: 5 months (n=6), 6 months (n=6), 7 months (n=5), 8 months (n=5), and 10 months (n=6) of age. Following each bleed, ewes were sacrificed, hypothalamic tissue containing the ARC was collected, and then processed for use in dual immunofluorescence and RNAscope. Mean LH and LH pulse frequencies followed the expected patterns: concentrations and frequencies were low during the prepubertal ages (5-7 months of age), intermediate during the peripubertal age (8 months of age), and elevated in the postpubertal age group (10 months of age). Using immunofluorescence, kisspeptin and NKB immuno-positive cell numbers did not change significantly (P > 0.50) over time with cell numbers averaging 235.3±16.8 and 231.3±16.8, respectively. Colocalization of kisspeptin and NKB was greater than 90% for all age groups. Using RNAscope, the total number of cells expressing mRNA for kisspeptin and dynorphin did not change significantly (P > 0.05) over time with cell numbers averaging 46.7±12.0 and 28.3±10.0 cells/hemisection, respectively. Taken together, our data suggest that the increase in LH secretion that drives puberty onset is not limited by changes in kisspeptin, NKB, or dynorphin expression, but may instead depend on other factors such as changes in receptor expression or changes in KNDy neuron activity via a reduction in inhibitory and/or an increase in stimulatory afferent inputs.
GH Is a Component of SASP in Aging Tissue
Authors: Chesnokova Vera M , Zonis Svetlana M , Estrada Hannah , Barrett Robert , Melmed Shlomo
Published: May 2021

Deficient GH signaling results in lifespan extension in murine and human models, while patients with uncontrolled acromegaly and transgenic mice overexpressing GH have a shorter lifespan. Colon polyp development increases with age, and also with GH excess in acromegaly. Aging is characterized by senescent cell accumulation with p53/p21 or p16 upregulation as well as cell cycle arrest and expression of a senescence-associated secretory phenotype (SASP). Senescence is reinforced by the SASP, which comprises pro-inflammatory cytokines, chemokines, growth modulators, angiogenic factors, and matrix metalloproteinases. SASP contributes to pro-aging phenotypes, and depletion or removal of senescent cells increases lifespan by partially protecting from age-related pathologies. Senescence, triggered by genotoxic insult with DNA damage, can be reversed by p53 inactivation; senescent cells with low p53 and unrepaired DNA damage can then re-enter the cell cycle (Beausejour et al, EMBO 2003), potentially resulting in chromosomal instability. We showed GH induction in non-pituitary senescent cells leading to suppressed p53/p21 (Chesnokova PNAS 2013). We now show that, in senescent human colon cells (hNCC) and in 3-dimensional human intestinal organoids, non-pituitary GH (npGH) is a component of SASP. In response to DNA damage, npGH is expressed and secreted locally as measured by RT-PCR, WB, and ELISA. High autocrine/paracrine GH further exacerbate DNA damage and reverses senescent in colon cells, enabling them to re-enter the cell cycle, as evidenced by p53 downregulation and increased Ki67 expression. Senescent colon cells expressing high intracellular GH form colonies in soft agar, indicative of cell transformation and proliferation, while GH deletion by shRNA results in Ki67 downregulation and decreased colony formation and size. The SASP protein CXCL1, a chemo-attractant that also functions to eliminate senescent cells, dose-dependently activates GH in hNCC and in intestinal organoids. GH, in turn, suppresses CXCL1 expression. Consistent with these findings, colon CXCL1 was induced in GHRKO mice, and also in hNCC with abrogated GH signaling by shRNA. Taken together with our finding that GH accumulates in aging normal human colon tissue and co-localizes in cells expressing senescence-associated β-galactosidase, these results suggest that GH, as a SASP component, initiates senescent cell proliferation and transformation. By inhibiting CXCL1, GH also functions to attenuate immune-mediated senescent cell elimination that protects aging tissue from deleterious effects of SASP. These mechanisms may underly an initial step in age-associated epithelial polyp development.
Mice With Targeted Deletion of ARC Kisspeptin Exhibit Immature Gametogenesis and Impaired Fertility
Authors: Nandankar Nimisha , Negron Ariel L , Wolfe Andrew M , Levine Jon E , Radovick Sally
Published: May 2021

Hypothalamic kisspeptin is primarily synthesized in two discrete nuclei - the anteroventral periventricular (AVPV) and the arcuate (ARC) nuclei. We have previously developed a selective, conditional ARC kisspeptin knock-out (KO) mouse line, namely the Pdyn-Cre/Kissfl/fl KO mice, that exhibited normal puberty onset in both sexes, but impaired estrous cyclicity and LH pulsatility in Pdyn-Cre/Kissfl/fl KO females. To examine the end-organ effect of the lack of ARC kisspeptin, we examined gametogenesis, gonad morphology, and fertility. Hematoxylin and eosin (H&E) staining of serial-sectioned whole ovaries demonstrated that Pdyn-Cre/Kissfl/fl KO female mice lacked corpora lutea - their ovarian folliculogenesis did not progress beyond antral follicle development, suggesting an ovulatory defect in Pdyn-Cre/Kissfl/fl KO females. 75% of the Pdyn-Cre/Kissfl/fl KO male mice had testes exhibiting a striking decrease in mature sperm in the seminiferous tubules. The remaining 25% showed evidence of mature sperm. Further evidence of a hypogonadal phenotype of the Pdyn-Cre/Kissfl/fl KO mice included the significantly low weight and small size of the ovaries, uteri, and testes when compared to control littermates. In a controlled, continuous mating paradigm with proven WT males, 2-4-month-old Pdyn-Cre/Kissfl/fl KO female mice failed to become pregnant or produce any pups, whereas age-matched WT females exhibited normal pregnancies to term. Thus, Pdyn-Cre/Kissfl/fl KO females have complete infertility. Ongoing studies of male fertility data suggest that Pdyn-Cre/Kissfl/fl KO males are subfertile, in accordance with their variable spermatogenesis phenotype - some KO males sired pups when paired with proven, WT females, whereas other KO males are infertile. Future experiments include assessing the capability of Pdyn-Cre/Kissfl/fl KO mice to respond to chronic, exogenous kisspeptin and GnRH administration to rescue abnormal LH pulsatility and estrous cyclicity in females, as well as the impaired fertility in both sexes.
Mutation of the GnRHR Proximal Promoter AP-1 Element in Mice Results in Suboptimal GnRH Induction of LH and an Abnormal Reproductive Phenotype
Authors: Hausken Krist N , Noel Sekoni D , Kim Han Kyeol , Carroll Rona Stephanie , Kaiser Ursula B
Published: May 2021

Reproduction is regulated by the gonadotropins, LH and FSH, which are synthesized and secreted by pituitary gonadotrophs in response to hypothalamic GnRH in a pulse frequency dependent manner. The gonadotroph decodes GnRH pulsatility via the GnRH receptor (GnRHR), which increases in expression and cell surface density before estrus and is responsible for downstream signaling cascades that differentially favor gonadotropin expression. The gonadotroph Gnrhr promoter contains a tripartite enhancer, including an AP-1 element that is necessary for full GnRH induction of Gnrhr expression in vitro. We previously generated an AP-1 knock-in (KI) mouse model with a single point mutation (C-269T) in the Gnrhr promoter AP-1 binding motif that resulted in an abnormal reproductive phenotype in female mice. Compared to wildtype (WT) littermates, female KI mice had a significant delay in first estrus, disrupted estrous cyclicity, fewer corpora lutea, and smaller litters. Males had no apparent reproductive phenotype. Basal serum gonadotropin levels were similar between WT and KI mice, but gonadectomy induced a significantly lower rise in serum LH levels of KI mice relative to WT mice, concomitant with significantly lower pituitary Gnrhr, Lhb, and Fshb mRNA levels in both sexes. We have now extended the characterization of these mice by measuring LH pulsatility and assessing GnRH induction of LH in vivo and in vitro. The frequency and amplitude of LH pulses over three hours were similar in ovariectomized WT and KI mice; however, KI mice had significantly reduced LH secretion, as measured by area under the curve. Similarly, GnRH treatment induced a diminished LH response in intact KI compared to WT males. In vitro cultures of hemi-pituitaries from gonadectomized WT and KI males were exposed to 0.01 nM GnRH and LH secretion into culture media was measured by ELISA at 0, 0.5, 1, 2, and 4 hours. There was no difference in basal LH secretion between WT and KI pituitaries but GnRH induction of LH was significantly lower in cultures from AP-1 mutant mice, indicating a direct impairment of GnRH action at the level of the pituitary. Taken together, these data indicate that the gonadotroph Gnrhr AP-1 promoter motif is critical for normal reproductive function. Prevention of AP-1 binding to the Gnrhr proximal promoter element decreases GnRH-induced Gnrhr, Lhb, and Fshb levels, impairs GnRH-stimulated LH secretion, and disrupts pubertal development and reproductive cyclicity in female mice.