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Abstract Context Preclinical studies show seliciclib (R-roscovitine) suppresses neoplastic corticotroph proliferation and pituitary adrenocorticotrophic hormone (ACTH) production. Objective To evaluate seliciclib as an effective pituitary-targeting treatment for patients with Cushing disease (CD). Methods Two prospective, open-label, phase 2 trials, conducted at a tertiary referral pituitary center, included adult patients with de novo, persistent, or recurrent CD who received oral seliciclib 400 mg twice daily for 4 consecutive days each week for 4 weeks. The primary endpoint in the proof-of-concept single-center study was normalization of 24-hour urinary free cortisol (UFC; ≤ 50 µg/24 hours) at study end; in the pilot multicenter study, primary endpoint was UFC normalization or ≥ 50% reduction in UFC from baseline to study end. Results Sixteen patients were consented and 9 were treated. Mean UFC decreased by 42%, from 226.4 ± 140.3 µg/24 hours at baseline to 131.3 ± 114.3 µg/24 hours by study end. Longitudinal model showed significant UFC reductions from baseline to each treatment week. Three patients achieved ≥ 50% UFC reduction (range, 55%-75%), and 2 patients exhibited 48% reduction; none achieved UFC normalization. Plasma ACTH decreased by 19% (P = 0.01) in patients who achieved ≥ 48% UFC reduction. Three patients developed grade ≤ 2 elevated liver enzymes, anemia, and/or elevated creatinine, which resolved with dose interruption/reduction. Two patients developed grade 4 liver-related serious adverse events that resolved within 4 weeks of seliciclib discontinuation. Conclusion Seliciclib may directly target pituitary corticotrophs in CD and reverse hypercortisolism. Potential liver toxicity of seliciclib resolves with treatment withdrawal. The lowest effective dose requires further determination.

Abstract Context Postoperative hyponatremia leads to prolonged hospital length of stay and readmission within 30 days. Objective To assess 3 strategies for reducing rates of postoperative hyponatremia and analyze risk factors for hyponatremia. Design Two retrospective analyses and 1 prospective study. Setting Tertiary referral hospital. Patients Patients undergoing transsphenoidal surgery for pituitary adenomas and other sellar and parasellar pathologies. Intervention(s) Phase 1: no intervention. Phase 2: postoperative day (POD) 7 sodium testing and patient education. Phase 3: fluid restriction to 1 L/day on discharge in addition to phase 2 interventions. Main outcome measures Rates of early and delayed hyponatremia and readmissions. Secondary outcomes were risk factors for hyponatremia and readmission costs. Results In phase 1, 296 patients underwent transsphenoidal surgery. Twenty percent developed early and 28% delayed hyponatremia. Thirty-eight percent underwent POD 7 sodium testing. Readmission rates were 15% overall and 4.3% for hyponatremia. In phase 2 (n = 316), 22% developed early and 25% delayed hyponatremia. Eighty-nine percent complied with POD 7 sodium testing. Readmissions were unchanged although severity of hyponatremia was reduced by 60%. In phase 3 (n = 110), delayed hyponatremia was reduced 2-fold [12.7%, relative risk (RR) = 0.52] and readmissions 3-fold [4.6%, RR = 0.30 (0.12–0.73)]; readmissions for hyponatremia were markedly reduced. Hyponatremia readmission increased costs by 30%. Conclusions Restricting fluid to 1 L/day on discharge decreases rates of delayed hyponatremia and readmissions by 50%. Standardized patient education and POD 7 sodium testing decreases severity of hyponatremia but does not impact readmission rates. These protocols should be considered standard practice for patients undergoing transsphenoidal surgery.

Cushing disease caused by adrenocorticotropin (ACTH)-secreting pituitary adenomas leads to hypercortisolemia predisposing to diabetes, hypertension, osteoporosis, central obesity, cardiovascular morbidity, and increased mortality. There is no effective pituitary targeted pharmacotherapy for Cushing disease. Here, we generated germline transgenic zebrafish with overexpression of pituitary tumor transforming gene (PTTG/securin) targeted to the adenohypophyseal proopiomelanocortin (POMC) lineage, which recapitulated early features pathognomonic of corticotroph adenomas, including corticotroph expansion and partial glucocorticoid resistance. Adult Tg:Pomc-Pttg fish develop neoplastic coticotrophs and pituitary cyclin E up-regulation, as well as metabolic disturbances mimicking hypercortisolism caused by Cushing disease. Early development of corticotroph pathologies in Tg:Pomc-Pttg embryos facilitated drug testing in vivo. We identified a pharmacologic CDK2/cyclin E inhibitor, R-roscovitine (seliciclib; CYC202), which specifically reversed corticotroph expansion in live Tg:Pomc-Pttg embryos. We further validated that orally administered R-roscovitine suppresses ACTH and corticosterone levels, and also restrained tumor growth in a mouse model of ACTH-secreting pituitary adenomas. Molecular analyses in vitro and in vivo showed that R-roscovitine suppresses ACTH expression, induces corticotroph tumor cell senescence and cell cycle exit by up-regulating p27, p21 and p57, and downregulates cyclin E expression. The results suggest that use of selective CDK inhibitors could effectively target corticotroph tumor growth and hormone secretion.

Helicobacter pylori infection is associated with several gastric diseases, including gastritis, peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma. Due to the prevalence and severeness of H. pylori infection, a thorough understanding of this pathogen is necessary. Lipopolysaccharide, one of the major virulence factors of H. pylori, can exert immunomodulating and immunostimulating functions on the host. In this study, the HP0044 and HP1275 genes were under investigation. These two genes potentially encode GDP-D-mannose dehydratase (GMD) and phosphomannomutase (PMM)/phosphoglucomutase (PGM), respectively, and are involved in the biosynthesis of fucose. HP0044 and HP1275 knockout mutants were generated; both mutants displayed a truncated LPS, suggesting that the encoded enzymes are not only involved in fucose production but are also important for LPS construction. In addition, these two gene knockout mutants exhibited retarded growth, increased surface hydrophobicity and autoaggregation as well as being more sensitive to the detergent SDS and the antibiotic novobiocin. Furthermore, the LPS-defective mutants also had significantly reduced bacterial infection, adhesion and internalization in the in vitro cell line model. Moreover, disruptions of the HP0044 and HP1275 genes in H. pylori altered protein sorting into outer membrane vesicles. The critical roles of HP0044 and HP1275 in LPS biosynthesis, bacterial fitness and pathogenesis make them attractive candidates for drug inventions against H. pylori infection.

Loss of retinoblastoma (Rb) tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39) mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

Both endocrine and exocrine pancreatic cells arise from pancreatic-duodenal homeobox 1 (pdx1)-positive progenitors. The molecular mechanisms controlling cell fate determination and subsequent proliferation, however, are poorly understood. Unlike endocrine cells, less is known about exocrine cell specification. We report here the identification and characterization of a novel exocrine cell determinant gene, exocrine differentiation and proliferation factor (exdpf), which is highly expressed in the exocrine cell progenitors and differentiated cells of the developing pancreas in zebrafish. Knockdown of exdpf by antisense morpholino caused loss or significant reduction of exocrine cells due to lineage-specific cell cycle arrest but not apoptosis, whereas the endocrine cell mass appeared normal. Real-time PCR results demonstrated that the cell cycle arrest is mediated by up-regulation of cell cycle inhibitor genes p21(Cip), p27(Kip), and cyclin G1 in the exdpf morphants. Conversely, overexpression of exdpf resulted in an overgrowth of the exocrine pancreas and a severe reduction of the endocrine cell mass, suggesting an inhibitory role for exdpf in endocrine cell progenitors. We show that exdpf is a direct target gene of pancreas-specific transcription factor 1a (Ptf1a), a transcription factor critical for exocrine formation. Three consensus Ptf1a binding sites have been identified in the exdpf promoter region. Luciferase assay demonstrated that Ptf1a promotes transcription of the exdpf promoter. Furthermore, exdpf expression in the exocrine pancreas was lost in ptf1a morphants, and overexpression of exdpf successfully rescued exocrine formation in ptf1a-deficient embryos. Genetic evidence places expdf downstream of retinoic acid (RA), an instructive signal for pancreas development. Knocking down exdpf by morpholino abolished ectopic carboxypeptidase A (cpa) expression induced by RA. On the other hand, exdpf mRNA injection rescued endogenous cpa expression in embryos treated with diethylaminobenzaldehyde, an inhibitor of RA signaling. Moreover, exogenous RA treatment induced anterior ectopic expression of exdpf and trypsin in a similar pattern. Our study provides a new understanding of the molecular mechanisms controlling exocrine cell specification and proliferation by a novel gene, exdpf. Highly conserved in mammals, the expression level of exdpf appears elevated in several human tumors, suggesting a possible role in tumor pathogenesis.

Rationale: Hyponatremia occurs in approximately 20% of patients after transsphenoidal pituitary surgery (TSS). Symptomatic delayed hyponatremia from SIADH is a major cause of morbidity and hospital readmission. Prophylactic fluid restriction (FR) protocols starting 2-5 days after surgery may potentially reduce rates of symptomatic hyponatremia and readmissions. Methods Patients who underwent TSS for sellar and parasellar masses at a tertiary referral center were discharged with a one-liter fluid restriction protocol. Serum sodium levels were measured on post-operative day (POD) 7 with telephone follow-up care. Rates of hyponatremia and readmissions were compared to a cohort of 316 post-operative patients treated prior to the protocol. Numerical variables were summarized as mean ± SD and significance testing calculated by Chi-square and t-test. Results 105 patients were enrolled on the FR protocol upon discharge. Twelve patients were excluded as they had diabetes insipidus or prolonged hospitalization beyond POD 7. Ninety-three patients were included in the analysis, of whom 74.3% had pituitary adenomas, 5.4% Rathke's Cleft cysts, 8.6% meningiomas, 5.4% apoplexy, 2.2% craniopharyngiomas, and 4.3% other masses. Hyponatremia occurred in 13/93 (14%) patients. Mean POD 7 sodium was 137.9 mmol/L across the entire cohort. Two patients (2.1%) were readmitted for hyponatremia, while 3 were re-admitted for other causes. 88/93 (94.6%) of patients complied with the FR protocol. Of the 5 non-compliant patients, 2 developed hyponatremias (40%) compared to 3/88 (3.4%) in those who complied (p=0.008). Mean POD7 sodium was 138 (+/-4.5) mmol/L in those that complied and 135 (+/- 4.7) mmol/L in those who did not. Overall readmission rate for those with postoperative hyponatremia was 23% compared to 2.5% for those with normal sodium levels (p=0.0023). 15.4% of patients with hyponatremia were re-admitted for hyponatremia treatment, compared to 0/80 (0%) of those who did not develop post-operative hyponatremia (p=0.0004). Age and BMI did not impact hyponatremia rates. In 316 patients treated in 2012-2018 prior to the FR protocol, 78 (24.7%) developed delayed hyponatremia with 6% readmitted for hyponatremia. Thirty nine percent of patients with delayed hyponatremia were re-admitted, compared to 7.6% for those without hyponatremia. Compared to patients not on FR, patients on the FR protocol had 50% reduced risk of hyponatremia (OR=0.49 (95% CI 0.26-0.94, p=0.03), and a 3-fold reduced risk of overall readmissions (OR=0.31; 95% CI 0.12-0.81, p=0.0157) and readmission for hyponatremia (OR= 0.34;95% CI 0.08-1.5, p=0.16). A post-operative CSF leak or use of a lumbar drain were associated with an increased readmission rate in the historical cohort. Conclusion Instituting a one-liter daily FR protocol in patients after TSS results in significantly reduced rates of hyponatremia, overall readmissions, and readmission for symptomatic hyponatremia. A FR protocol should become routine practice in the post-operative care of patients undergoing pituitary surgery. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

This study aimed to investigate the expression of Twist in gastric cancer tissues and its correlation between Twist and the epithelial-mesenchymal transition (EMT). By means of RT-PCR and Western blot, the mRNA and protein expressions of Twist, E-cadherin, and Vimentin in 61 gastric cancer tissues and adjacent normal tissues were detected. The positive rates of Twist, E-cadherin, and Vimentin mRNA expression in gastric cancer tissues were 73.9. 40.6, and 60.9 %, respectively; compared to the expression of these genes in adjacent normal tissues (2.9, 75.4, and 27.5 %), the differences were significant ( p  < 0.05). The E-cadherin protein expression level in gastric cancer tissues was significantly lower than that in the adjacent normal tissues ( p  < 0.05). After the transfection of Twist siRNA into the MKN45 cells, the protein expression of Twist was significantly reduced ( p  < 0.05), the protein expression of E-cadherin was significantly increased, and the number of cells that passed through the Transwell chamber was significantly lower than that in the non-transfected control group as well as the transfected control group ( p  < 0.05). Twist may be associated with the epithelial-mesenchymal transition in gastric cancer and the tumorigenesis, invasion, and metastasis of gastric cancer.

Cereal endosperms, which consist of an outer aleurone layer and an inner starchy endosperm, provide over 70% staple food for human [www.fao.org]. Rice endosperm development occurs in the caryopsis of a grain. During the grain filling stage, sucrose produced in photosynthetic leaves is transported to grains via the phloem [ 1, 2]. It has been proposed that sucrose, upon arrival in the caryopsis through the phloem located at the dorsal vascular bundle, is partially hydrolyzed by cell wall invertases （CINs） into glu- cose and fructose [3, 4]. Since endosperm is symplastically isolated from maternal tissues, it is assumed that these sug- ars are delivered to the endosperm actively through an ap- oplastic pathway by sugar transporters located in aleurone [5, 6]. However, the exact role of aleurone in regulating sugar transport remains unknown.

Loss of retinoblastoma (Rb) tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39) mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130), and cdkn1a (p21) expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.