AI-driven designed protein epigenomics.

The biological revolutions of computationally designed proteins, induced pluripotent stem cells (iPSCs), and the CRISPR-Cas9 system finally enables modifications that can spur deep understanding of spatial requirement of epigenetic information. This commentary describes the utility of a computationally designed protein, EED Binder (EB), when fused to dCas9 (EBdCas9) for identifying critical sites of PRC2 dependent histone H3K27me3 marks in the chromatin. By using EBdCas9 and gRNA, PRC2 function can be inhibited at specific loci, resulting in precise reduction of EZH2 and H3K27me3 marks, and in some (but not all) locations, activation of the gene and functional outcomes (such as regulation of cell cycle or trophoblast transdifferentiation). Interestingly, a functional TATA box located more than 500bp upstream of a TBX18 TSS was found to be repressed by PRC2, supporting the theory that epigenetic regulators control the repression of transcriptional elements on the promoter region. The EBdCas9 technology may provide a useful tool for controlling gene regulation through epigenomic control.

dCas9 fusion to computer-designed PRC2 inhibitor reveals functional TATA box in distal promoter region.

Bifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.

Prolactinoma: Medical and Surgical Considerations.

Prolactinomas are the most common secretory tumor of the pituitary gland. Clinical symptoms may be due to prolactin oversecretion, localized mass effect, or a combination of both. Although the mainstay of prolactinoma management is medical therapy with dopamine agonists, endoscopic endonasal or transcranial surgery, radiation therapy, or a combination of these is an important treatment option in select cases. This article discusses prolactinoma phenotypes, clinical presentations, and clinically pertinent medical and surgical considerations when managing these tumors.

UTILITY OF AFIRMA GENE EXPRESSION CLASSIFIER FOR EVALUATION OF INDETERMINATE THYROID NODULES AND CORRELATION WITH ULTRASOUND RISK ASSESSMENT: SINGLE INSTITUTIONAL EXPERIENCE.

Objective: We assessed our experience with Afirma gene expression classifier (GEC) combined with sono-graphic risk assessment, using both the American Thyroid Association (ATA) and the Thyroid Imaging Reporting and Data System (TI-RADS) in evaluating indeterminate thyroid nodules. Methods: We identified 98 patients with 101 nodules who had a second fine needle aspiration biopsy (FNA) between January 1, 2014, and September 30, 2017, and sent to Veracyte for cytopathology and subsequent Afirma GEC testing. A second FNA biopsy was performed if the initial cytopathology was either Bethesda III or IV (n = 94) or nondiagnostic (n = 7). We correlated cytopathology, histopathology, and Afirma GEC results with sonographic risk assessment using both the ATA system and TI-RADS. Results: The mean age of the cohort was 57.4 ± 12.3 years; 84% women and 60% white. Repeat FNA was benign in 51 of 101 nodules, and of the remaining 50 nodules, 18 (36%) were GEC-benign and 32 (64%) GEC-suspicious. Eighteen of the 32 GEC-suspicious nodules underwent surgery with the following results: 7 benign (39%), 1 follicular thyroid carcinoma (6%), 6 follicular variant of papillary thyroid cancer (33%), and 4 noninvasive follicular tumor with papillary-like nuclear features (22%). The malignancy rate among the surgical cohort was 39% (without noninvasive follicular tumor with papillary-like nuclear features [NIFTP]) and 61% (with NIFTP) and about 50% and 20% of this group scored in the high suspicion category by ATA and TR5 by TI-RADS, respectively. Conclusion: Afirma GEC was useful in avoiding surgery in one-third of indeterminate nodules and performed similarly to ATA and TI-RADS. However, the use of echogenicity in scoring may underestimate the risk of malignancy in patients with indeterminate nodules. Abbreviations: ATA = American Thyroid Association; AUS = Atypia of Undetermined Significance; FLUS = Follicular Lesion of Undetermined Significance; FN = follicular neoplasm; FNA = fine needle aspiration; FTC = follicular thyroid cancer; FVPTC = follicular variant of papillary thyroid cancer; GEC = Gene Expression Classifier; ND = nondiagnostic; NIFTP = noninvasive follicular tumor with papillary-like nuclear features; TI-RADS = Thyroid Imaging Reporting and Data System; TR = TI-RADS.

Pituitary Dysfunction Among Men Presenting with Hypogonadism.

PURPOSE OF REVIEW: Hypogonadism is a common endocrine dysfunction. This review focuses on the most up-to-date guideline for evaluation of pituitary function among men presenting with signs and symptoms of hypogonadism. RECENT FINDINGS: The clinician must differentiate between primary (testicular) and secondary (pituitary-hypothalamic or central) hypogonadisms and be aware of adult-onset hypogonadism. If gonadotropins are low or inappropriately normal, the clinician must consider potential reversible causes in the hypothalamus-pituitary axis. Also, it is critical to understand the pitfalls of testosterone testing. When clinically indicated, evaluation of other pituitary hormone functions as well as pituitary magnetic resonance imaging may be necessary. Furthermore, it is essential to recognize that pituitary incidentalomas are common. Patients with microprolactinoma are more likely to present with symptoms of sexual dysfunction while those with macroprolactinoma are more likely to present with symptoms of mass effect. Some functional pituitary tumors respond to drug therapy while other nonfunctional tumors require surgical intervention. It is important for the clinician to understand the proper work-up of the hypogonadal patient with pituitary dysfunction and when necessary to refer to an endocrinologist or a neurosurgeon.

TFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes.

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX.  Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.

Single-Cell Transcriptomic Analysis of Cardiac Differentiation from Human PSCs Reveals HOPX-Dependent Cardiomyocyte Maturation.

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.

First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor.

The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein-protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.

ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling.

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.

ZnT-1 protects HL-1 cells from simulated ischemia-reperfusion through activation of Ras-ERK signaling.

Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.

Distinct activities of several RNase J proteins in methanogenic archaea.

RNA degradation plays an important role in the control of gene expression in all domains of life, including Archaea. While analyzing RNA degradation in different archaea, we faced an interesting situation. The members of a group of methanogenic archaea, including Methanocaldococcus jannaschii, contain neither the archaeal exosome nor RNase II/R homologs. However, looking for potential ribonucleases revealed proteins related to the recently discovered ribonuclease RNase J. RNase J is unique among known ribonucleases because it may combine endo- and 5'→3' exo-ribonucleolytic activities in a single polypeptide. Here, we report the characterization of the ribonuclease activities of three RNase J homologs encoded in the genome of the methanogenic archaeon Methanocaldococcus jannaschii. The analysis of the recombinant archaeal proteins purified from E. coli revealed an optimal activity at 60°C. Whereas mjRNase J1 and -J3 displayed exclusively 5'→3' exonucleolytic activity, mjRNase J2 is an endonuclease with no apparent exonuclease activity. The exonucleolytic activity of both mjRNase J1 and -J3 is enhanced in molecules harboring monophosphate at the 5' end. mjRNase J3, and to some extent mjRNase J2, degrade ssDNA. Together, these results reveal that in archaea lacking the exosome and RNase II/R, RNA and perhaps also DNA are possibly degraded by the coordinated activities of several RNase J proteins. Unlike bacteria, in archaea RNase J proteins provide separately the exo- and endonucleolytic activities that are probably essential for RNA degradation.

Unusual mid-shaft fractures during long-term bisphosphonate therapy.

BACKGROUND: Bisphosphonates are the most commonly prescribed medications for the treatment of osteoporosis. Although existing evidence supports a good safety profile, there is concern that chronic administration of these agents could result in severe suppression of bone turnover with increased risk of nonvertebral fractures. OBJECTIVE: The objective of this study was to report the clinical presentation, selected bone histomorphometry and X-ray images of patients who developed mid-shaft long bone fractures during bisphosphonate therapy, six of whom had bone biopsy for histomorphometery. RESULTS: Of the 13 patients who sustained atraumatic mid-shaft fractures, 10 were on alendronate and three were on risedronate therapy before the fractures. In addition to bisphosphonates, three patients were on oestrogen and two on tamoxifen concomitantly. Four patients with glucocorticoid-induced osteoporosis were on alendronate for 3-11 years along with glucocorticoid therapy. Bone histomorphometry showed severe suppression of bone turnover in five patients and low bone turnover in one patient. CONCLUSION: Long-term bisphosphonate therapy may increase the risk of unusual long bone mid-shaft fractures. This is probably due to prolonged suppression of bone turnover, which could lead to accumulation of microdamage and development of hypermineralized bone. At present, the scope of this complication in the larger context of patients receiving bisphosphonate therapy remains unknown, but appears to be small.

Prevalence of vitamin D depletion among morbidly obese patients seeking gastric bypass surgery.

BACKGROUND: Abnormalities in calcium and vitamin D metabolism have been reported after bariatric surgery. The purpose of this study was to evaluate vitamin D nutritional status among morbidly obese patients before gastric bypass surgery. METHODS: We prospectively studied 279 morbidly obese patients seeking gastric bypass surgery for vitamin D nutritional status as assessed by serum 25-hydroxyvitamin D level. In addition, serum samples were analyzed for calcium, alkaline phosphatase (AP), intact parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D. RESULTS: Mean patient age was 43 +/- 9 years; 87% of the study patients were women, and 72% were white. Serum calcium and AP levels were normal in 88% and 89% of the patients, respectively. Vitamin D depletion, defined as serum 25-hydroxyvitamin D level