ChIP-qPCR for Polycomb Group Proteins During Neuronal Differentiation of Human Pluripotent Stem Cells.

Neuronal differentiation is an intricate and a complex process which involves crosstalk among various signaling pathways, growth factors, transcription factors, and epigenetic modifiers. During different stages of neuronal development, there are various histone modifiers which drive the expression of lineage-specific genes. Polycomb group proteins are one of the histone modifiers that control transcriptional repression of specific genes in development, differentiation, and functionality of various tissues. Chromatin immunoprecipitation (ChIP) is a widely used technique to investigate the interaction of proteins and DNA; ChIP combined with quantitative real-time PCR (qPCR) gives a quantitative data about the occupancy of specific protein on a particular stretch of DNA, and this can help us investigate how a protein regulates expression of a specific gene. In this chapter, we describe a protocol for ChIP coupled to qPCR during early neuronal differentiation to identify the specific genomic targets regulated by components of Polycomb repressive complex 1.

Osilodrostat: A Novel Steroidogenesis Inhibitor to Treat Cushing's Disease.

OBJECTIVE: To review data on efficacy and safety of osilodrostat (Isturisa), a novel oral steroidogenesis inhibitor for treatment of Cushing's disease (CD), a life-threatening endocrine disorder. DATA SOURCES: A PubMed/CINAHL search from inception to September 25, 2020, was performed using the following keywords: osilodrostat, 11-beta hydroxylase, pituitary, ACTH hypersecretion, and Cushing's disease. STUDY SELECTION AND DATA EXTRACTION: Phase 2 and 3 clinical trials and supplementary documents investigating osilodrostat were obtained from a primary literature search, the manufacturer's website, and the Food and Drug Administration website. These articles evaluated the clinical pharmacology, efficacy, safety, adverse events, warnings, and precautions for osilodrostat. DATA SYNTHESIS: Osilodrostat was efficacious and safe in the treatment of CD in mostly middle-aged Caucasian women. A pivotal phase 3 study revealed a significant difference in 24-hour mean urinary free cortisol (primary end point) between osilodrostat and placebo (86% vs 29%; P < 0.001). RELEVANCE TO PATIENT CARE AND CLINICAL PRACTICE: Osilodrostat provides a potent and consistent effect in reducing life-threatening supraphysiological levels of cortisol in patients with CD. Hypocortisolism adverse effects can be mitigated by slowly increasing osilodrostat's dose at ≥2-week intervals. QT interval prolongation was noted; therefore, the QT interval must be monitored by the electrocardiogram. Increased levels of cortisol precursors during treatment with osilodrostat may increase the risk of hypokalemia, edema, and hypertension. CONCLUSIONS: Osilodrostat was efficacious in decreasing cortisol levels and safe in treating patients who have failed or are ineligible for pituitary surgery. Although risks exist, a pivotal clinical trial revealed efficacy in 86% of participants.

Fallot type of absent pulmonary valve syndrome - A case report.

Absent pulmonary valve syndrome (APVS) is a rare congenital cardiac malformation characterized by absent, dysplastic, or rudimentary pulmonary valve leaflets in association with other cardiac anomalies. It has an incidence of 3-6% in cases of tetralogy of Fallot (TOF) and 0.2-0.4% of live-born infants with congenital heart disease (CHD). Absent pulmonary valve leads to dilated main pulmonary artery; presenting as a pulsatile, paracardiac cystic lesion on antenatal ultrasound (USG). We report a case of this rare anomaly in association with ventricular septal defect (VSD), TOF, and left axis deviation of heart detected at 23 weeks of gestation.

PRC1 catalytic unit RING1B regulates early neural differentiation of human pluripotent stem cells.

BACKGROUND: Polycomb group (PcG) proteins are histone modifiers which control gene expression by assembling into large repressive complexes termed - Polycomb repressive complex (PRC); RING1B, core catalytic subunit of PRC1 that performs H2AK119 monoubiquitination leading to gene repression. The role of PRC1 complex during early neural specification in humans is unclear; we have tried to uncover the role of PRC1 in neuronal differentiation using human pluripotent stem cells as an in vitro model. RESULTS: We differentiated both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) towards neural progenitor stage evident from the expression of NESTIN, TUJ1, NCAD, and PAX6. When we checked the total expression of RING1B and BMI1, we saw that they were significantly upregulated in differentiated neural progenitors compared to undifferentiated cells. Further, we used Chromatin Immunoprecipitation coupled with qPCR to determine the localization of RING1B, and the repressive histone modification H2AK119ub1 at the promoters of neuronal specific genes. We observed that RING1B localized to and catalyzed H2AK119ub1 modification at promoters of TUJ1, NCAM, and NESTIN during early differentiation and later RING1B was lost from its promoter leading their expression; while functional RING1B persisted significantly on mature neuronal genes such as IRX3, GSX2, SOX1, NEUROD1 and FOXG1 in neural progenitors. CONCLUSION: The results of our study show that PRC1 catalytic component RING1B occupies neuronal gene promoters in human pluripotent stem cells and may prevent their precocious expression. However, when neuronal inductive signals are given, RING1B is not only removed from neuronal gene promoters, but the inhibitory H2AK119ub1 modification is also lost.

Inhibition of RING1B alters lineage specificity in human embryonic stem cells.

Polycomb group (PcG) proteins are histone modifiers which are known to perform transcriptional repression and have been shown to be critical during murine embryonic development. PcGs are broadly characterized into polycomb repressive complex 1 (PRC1) and 2 and (PRC2). RING1B, core catalytic unit of PRC1 performs H2AK119 monoubiquitination leading to transcriptional repression. We used human embryonic stem cell (hESC) line to study the fate of pluripotent stem cells (PSCs) under inhibition of RING1B, as its role in human development is still to be completely explored. Embryoid bodies (EBs) were generated to differentiate hESCs using hanging drop method. PRT4165 (synthetic RING1B catalytic activity inhibitor) was added to undifferentiated and differentiated cells for 24 h. When we inhibited RING1B in undifferentiated cells, OCT4 levels remained unchanged indicating RING1B does not regulate pluripotency. The drug when added to differentiated cells led to decrease in the levels of RING1B, BMI1, and H2AK119ub1. Interestingly, we also report that the differentiated cells show an increased expression of neuroectodermal markers: SOX1 and PAX6 as well as expression of other neuroectodermal markers such as TUJ1, FOXG1, and NCAM. However, there was reduction in expression of endodermal (SOX17 and FOXA2) mesodermal marker BRACHYURY and TBX5 in treated EBs compared with control EBs. In summary, alteration of RING1B catalytic activity in hESCs during differentiation promotes neuroectodermal differentiation thus, we demonstrate critical role of RING1B in regulating neural differentiation. The strategy of inhibiting RING1B could be used to direct PSCs towards early neuronal fate.

Polycomb repressive complex 1: Regulators of neurogenesis from embryonic to adult stage.

Development of vertebrate nervous system is a complex process which involves differential gene expression and disruptions in this process or in the mature brain, may lead to neurological disorders and diseases. Extensive work that spanned several decades using rodent models and recent work on stem cells have helped uncover the intricate process of neuronal differentiation and maturation. There are various morphological changes, genetic and epigenetic modifications which occur during normal mammalian neural development, one of the chromatin modifications that controls vital gene expression are the posttranslational modifications on histone proteins, that controls accessibility of translational machinery. Among the histone modifiers, polycomb group proteins (PcGs), such as Ezh2, Eed and Suz12 form large protein complexes-polycomb repressive complex 2 (PRC2); while Ring1b and Bmi1 proteins form core of PRC1 along with accessory proteins such as Cbx, Hph, Rybp and Pcgfs catalyse histone modifications such as H3K27me3 and H2AK119ub1. PRC1 proteins are known to play critical role in X chromosome inactivation in females but they also repress the expression of key developmental genes and tightly regulate the mammalian neuronal development. In this review we have discussed the signalling pathways, morphogens and nuclear factors that initiate, regulate and maintain cells of the nervous system. Further, we have extensively reviewed the recent literature on the role of Ring1b and Bmi1 in mammalian neuronal development and differentiation; as well as highlighted questions that are still unanswered.

Localization Methods for Excisional Biopsy in Women With Nonpalpable Mammographic Abnormalities.

INTRODUCTION: With the advent and proliferation of breast cancer screening programs, more women are being diagnosed with mammographic abnormalities that require tissue diagnosis. If imaged-guided biopsy is not possible or previous image-guided biopsies reveal pathologies that require more extensive surgery, guided excisional biopsy/lumpectomy may be necessary. METHODS: Fifteen women were enrolled in the study of the feasibility of off-site or day-before wire-localization excisional biopsy of the breast with mammographic abnormalities. Five patients had their localization wire placed the day before, whereas 10 patients had their localization the same day with surgery in a distant procedure room under straight local anesthesia. RESULTS: Two of the 15 patients had an eventual cancer diagnosis from their wire-localized excisional breast biopsy. All patients had their mammographic abnormality removed with the previously placed core biopsy clip, and there was 100% radiologic/clinical correlation. All patients' wounds healed primarily without any surgical site infections. CONCLUSION: The protocol answers 2 questions concerning the wire-localized excisional breast biopsy technique. The series shows that the wire-localization technique can be performed the night before or in a location away from the procedure room that would allow better synchronization with surgical schedules or allow the procedure to take place in low-cost settings away from the expense of the hospital operating room.