High-Fidelity Drug-Induced Liver Injury Screen Using Human Pluripotent Stem Cell-Derived Organoids.

BACKGROUND & AIMS: Preclinical identification of compounds at risk of causing drug induced liver injury (DILI) remains a significant challenge in drug development, highlighting a need for a predictive human system to study complicated DILI mechanism and susceptibility to individual drug. Here, we established a human liver organoid (HLO)-based screening model for analyzing DILI pathology at organoid resolution. METHODS: We first developed a reproducible method to generate HLO from storable foregut progenitors from pluripotent stem cell (PSC) lines with reproducible bile transport function. The qRT-PCR and single cell RNA-seq determined hepatocyte transcriptomic state in cells of HLO relative to primary hepatocytes. Histological and ultrastructural analyses were performed to evaluate micro-anatomical architecture. HLO based drug-induced liver injury assays were transformed into a 384 well based high-speed live imaging platform. RESULTS: HLO, generated from 10 different pluripotent stem cell lines, contain polarized immature hepatocytes with bile canaliculi-like architecture, establishing the unidirectional bile acid transport pathway. Single cell RNA-seq profiling identified diverse and zonal hepatocytic populations that in part emulate primary adult hepatocytes. The accumulation of fluorescent bile acid into organoid was impaired by CRISPR-Cas9-based gene editing and transporter inhibitor treatment with BSEP. Furthermore, we successfully developed an organoid based assay with multiplexed readouts measuring viability, cholestatic and/or mitochondrial toxicity with high predictive values for 238 marketed drugs at 4 different concentrations (Sensitivity: 88.7%, Specificity: 88.9%). LoT positively predicts genomic predisposition (CYP2C9∗2) for Bosentan-induced cholestasis. CONCLUSIONS: Liver organoid-based Toxicity screen (LoT) is a potential assay system for liver toxicology studies, facilitating compound optimization, mechanistic study, and precision medicine as well as drug screening applications.

Analysis of 612 Benign Papillomas Diagnosed at Core Biopsy: Rate of Upgrade to Malignancy, Factors Associated With Upgrade, and a Proposal for Selective Surgical Excision.

BACKGROUND. Despite numerous published studies, management of benign papillomas without atypia remains controversial. OBJECTIVE. The purpose of this study was to determine the malignancy upgrade rate of benign papillomas, identify risk factors for upgrade, and formulate criteria for selective surgery. METHODS. This retrospective study included benign papillomas without atypia diagnosed on percutaneous biopsy between December 1, 2000, and December 31, 2019. Papillomas that did not undergo surgical excision or at least 2 years of imaging and/or clinical follow-up were excluded. Clinical, imaging, and histopathologic features were extracted from the electronic medical record. Features associated with upgrade to malignancy were identified. Multivariable logistic regression was performed. RESULTS. The study included 612 benign papillomas in 543 women (mean age, 54.5 ± 12.1 [SD] years); 466 papillomas were excised, and 146 underwent imaging or clinical surveillance. The upgrade rate to malignancy was 2.3% (14/612). Upgrade rate was associated (p < .05) with radiology-pathology correlation (50.0% if discordant vs 2.1% if concordant), patient age (5.6% for 60 years and older vs 0.7% for younger than 60 years), presenting symptoms (6.7% if palpable mass or pathologic nipple discharge vs 1.3% if no symptoms), and lesion size (7.3% if ≥ 10 mm vs 0.6% if < 10 mm). Three of 14 upgraded papillomas were associated with four or more metachronous or concurrent peripheral papillomas. No incidental papilloma or papilloma reported as completely excised on core biopsy histopathologic analysis was upgraded. A predictive model combining radiology-pathology discordance, symptoms (palpable mass or nipple discharge), age 60 years old and older, size 10 mm or larger, and presence of four or more metachronous or concurrent peripheral papillomas achieved an AUC of 0.91, sensitivity of 79%, and spec-ificity of 89% for upgrade. Selective surgery based on presence of any of these five factors, although excluding from surgery incidental papillomas and papillomas reported as completely excised on histopathology, would have spared 294 of 612 lesions from routine excision and identified all 14 upgraded lesions. CONCLUSION. Benign nonatypical papillomas have a low malignancy upgrade rate; routine surgical excision may not be necessary. Selective excision is recommended for lesions satisfying any of the five criteria. Incidental papillomas or papillomas completely excised on histopathology may undergo imaging follow-up. CLINICAL IMPACT. The proposed criteria for selective surgery of benign papillomas on core biopsy would reduce surgeries without delaying diagnosis of malignancy.

p66ShcA functions as a contextual promoter of breast cancer metastasis.

BACKGROUND: The p66ShcA redox protein is the longest isoform of the Shc1 gene and is variably expressed in breast cancers. In response to a variety of stress stimuli, p66ShcA becomes phosphorylated on serine 36, which allows it to translocate from the cytoplasm to the mitochondria where it stimulates the formation of reactive oxygen species (ROS). Conflicting studies suggest both pro- and anti-tumorigenic functions for p66ShcA, which prompted us to examine the contribution of tumor cell-intrinsic functions of p66ShcA during breast cancer metastasis. METHODS: We tested whether p66ShcA impacts the lung-metastatic ability of breast cancer cells. Breast cancer cells characteristic of the ErbB2+/luminal (NIC) or basal (4T1) subtypes were engineered to overexpress p66ShcA. In addition, lung-metastatic 4T1 variants (4T1-537) were engineered to lack endogenous p66ShcA via Crispr/Cas9 genomic editing. p66ShcA null cells were then reconstituted with wild-type p66ShcA or a mutant (S36A) that cannot translocate to the mitochondria, thereby lacking the ability to stimulate mitochondrial-dependent ROS production. These cells were tested for their ability to form spontaneous metastases from the primary site or seed and colonize the lung in experimental (tail vein) metastasis assays. These cells were further characterized with respect to their migration rates, focal adhesion dynamics, and resistance to anoikis in vitro. Finally, their ability to survive in circulation and seed the lungs of mice was assessed in vivo. RESULTS: We show that p66ShcA increases the lung-metastatic potential of breast cancer cells by augmenting their ability to navigate each stage of the metastatic cascade. A non-phosphorylatable p66ShcA-S36A mutant, which cannot translocate to the mitochondria, still potentiated breast cancer cell migration, lung colonization, and growth of secondary lung metastases. However, breast cancer cell survival in the circulation uniquely required an intact p66ShcA S36 phosphorylation site. CONCLUSION: This study provides the first evidence that both mitochondrial and non-mitochondrial p66ShcA pools collaborate in breast cancer cells to promote their maximal metastatic fitness.

Changes in peripapillary blood vessel density in Graves' orbitopathy after orbital decompression surgery as measured by optical coherence tomography angiography.

The purpose is to evaluate the utility of optical coherence tomography (OCT) angiography in the evaluation of Graves' orbitopathy (GO) and response to orbital decompression in patients with and without dysthyroid optic neuropathy (DON). This was a single-center, prospective case series in a cohort of 12 patients (24 orbits) with GO and ±DON, (6 orbits) who underwent bilateral orbital decompression. All patients underwent pre- and postoperative OCT angiography of the peripapillary area. Vessel density indices were calculated in a 4.5 mm × 4.5 mm ellipsoid centered on the optic disk using split-spectrum amplitude decorrelation angiography algorithm, producing the vessel density measurements. Mean change in vessel density indices was compared between pre- and postoperative sessions and between patients with and without DON. Patient 1, a 34-year-old male with GO and unilateral DON OD, showed a significant reduction in blood vessel density indices oculus dexter (OD) (DON eye) after decompression while a more modest reduction was found oculus sinister (OS) with the greatest change noted intrapapillary. Patient 2, a 50-year-old male with DON OU, showed worsening neuropathy following decompression OD that was confirmed by angiographic density indices. Patient 3, a 55-year-female with DON, showed a reduction in blood vessel density OD and increased density OS. Patients without DON showed overall less impressive changes in indices as compared to those with DON. Using OCT angiography, response to surgical treatment in GO orbits, more so in orbits with DON, can be demonstrated and quantified using vessel density indices with reproducibility.

Complete inferior rectus muscle transection secondary to orbital blowout fracture.

Complete extraocular muscle transection is uncommon in the setting of blunt trauma. We report a case of a 53-year-old male that developed diplopia after hitting his face directly on a concrete slab after a fall. On examination, he had a right hypertropia with a complete infraduction deficit. A CT scan of the face showed an orbital floor blowout fracture with complete inferior rectus transection. On surgical exploration, the distal and proximal ends of the muscle were identified and sutured together, and the floor fracture was repaired. At his post-operative visits, the patient had a persistent infraduction deficit, but subjectively had improved diplopia.

FXR-Gankyrin axis is involved in development of pediatric liver cancer.

The development of hepatoblastoma (HBL) is associated with failure of hepatic stem cells (HSC) to differentiate into hepatocytes. Despite intensive investigations, mechanisms of the failure of HSC to differentiate are not known. We found that oncogene Gankyrin (Gank) is involved in the inhibition of differentiation of HSC via triggering degradation of tumor suppressor proteins (TSPs) Rb, p53, C/EBPα and HNF4α. Our data show that the activation of a repressor of Gank, farnesoid X receptor, FXR, after initiation of liver cancer by Diethylnitrosamine (DEN) prevents the development of liver cancer by inhibiting Gank and rescuing tumor suppressor proteins. We next analyzed FXR-Gank-Tumor suppressor pathways in a large cohort of HBL patients which include 6 controls and 53 HBL samples. Systemic analysis of these samples and RNA-Seq approach revealed that the FXR-Gank axis is activated; markers of hepatic stem cells are dramatically elevated and hepatocyte markers are reduced in HBL samples. In the course of these studies, we found that RNA binding protein CUGBP1 is a new tumor suppressor protein which is reduced in all HBL samples. Therefore, we generated CUGBP1 KO mice and examined HBL signatures in the liver of these mice. Micro-array studies revealed that the HBL-specific molecular signature is developed in livers of CUGBP1 KO mice at very early ages. Thus, we conclude that FXR-Gank-TSPs-Stem cells pathway is a key determinant of liver cancer in animal models and in pediatric liver cancer. Our data provide a strong basis for development of FXR-Gank-based therapy for treatment of patients with hepatoblastoma.

Syntheses of Families of Enantiopure and Diastereopure Cobalt Catalysts Derived from Trications of the Formula [Co(NH2CHArCHArNH2)3]3.

Aerobic reactions of CoX2 (X = OAc, Cl) or Co(ClO4)2 with (S,S)-1,2-diphenylethylenediamine [(S,S)-dpen] in CH3OH, followed by HCl or HClO4 additions, give the diastereomeric lipophobic salts Λ-[Co((S,S)-dpen)3]3+3Cl- [Λ-(S,S)-13+3Cl-] or Δ-(S,S)-13+3ClO4- (60-65%) with high degrees of selectivity. Anion metatheses (room temperature) and equilibrations (charcoal, CH3OH, 70 °C) show that the former is more stable than Δ-(S,S)-13+3Cl-, and the latter is more stable than Λ-(S,S)-13+3ClO4-. Additional anion metatheses lead to large families of lipophilic salts Λ- and Δ-(S,S)-13+2X-X'- [X/X' = Cl/BArf [BArf = B(3,5-C6H3(CF3)2)4], PF6/BArf, BF4/BArf, PhBF3/BArf, Cl/BArf20 [BArf20 = B(C6F5)4], BArf/BArf, BArf20/BArf20, BF4/BF4, PF6/PF6]. Mixed salts of the formula Λ- and Δ-[Co((S,S)-NH2CHArCHArNH2)3]3+2Cl-BArf- are similarly prepared (Ar = 4-C6H4n-Bu, 4-C6H4Cl, 4-C6H4CF3, 4-C6H4OCH3, α-naphthyl, ß-naphthyl, 2-C6H4OBn). The diastereotopic NHH' protons exhibit different 1H NMR signals; one shifts far downfield when X/X' = Cl/BArf (δ ca. 8.0 vs 4.0 ppm). This is believed to arise from hydrogen bonding between the two Cl- anions and the two C3 faces of the D3-symmetric trication, each of which feature three synperiplanar NH groups. When all of the anions are poor hydrogen-bond acceptors (e.g., BArf-, BF4-, ClO4-), equilibria favor Δ diastereomers.

Octahedral Werner complexes with substituted ethylenediamine ligands: a stereochemical primer for a historic series of compounds now emerging as a modern family of catalysts.

As reported by Alfred Werner in 1911-1912, salts of the formally D3 symmetric [Co(en)3]3+ (en = ethylenediamine) trication were among the first chiral inorganic compounds to be resolved into enantiomers, the absolute configurations of which are denoted Λ (left handed helix) or Δ (right handed helix). After a >100 year dormant period during which few useful reactions of these substitution inert complexes were described, carbon substituted derivatives have recently been found to be potent catalysts for enantioselective organic synthesis. This review systematically outlines the fascinating range of stereoisomers that can arise, such as conformers associated with the five membered chelate rings (λ/δ), alignment modes of the C-C bonds with the C3 symmetry axis (lel/ob), geometric isomers (fac/mer), and configurational diastereomers (R/S) arising from carbon stereocenters. These analyses demonstrate a profound stereochemical diversity that can be applied in catalyst optimization. Efforts are made to bridge the often orthogonal nomenclature systems inorganic and organic chemists employ to describe these phenomena.

Cooperation of C/EBP family proteins and chromatin remodeling proteins is essential for termination of liver regeneration.

UNLABELLED: Liver cancer is the fifth most common cancer. A highly invasive surgical resection of the liver tumor is the main approach used to eliminate the tumor. Mechanisms that terminate liver regeneration when the liver reaches the original size are not known. The aims of this work were to generate an animal model that fails to stop liver regeneration after surgical resections and elucidate mechanisms that are involved in termination of liver regeneration. Because epigenetic control of liver function has been previously implicated in the regulation of liver proliferation, we generated C/EBPα-S193A knockin mice, which have alterations in formation of complexes of C/EBP family proteins with chromatin remodeling proteins. The C/EBPα-S193A mice have altered liver morphology and altered liver function leading to changes of glucose metabolism and blood parameters. Examination of the proliferative capacity of C/EBPα-S193A livers showed that livers of S193A mice have a higher rate of proliferation after birth, but stop proliferation at the age of 2 months. These animals have increased liver proliferation in response to liver surgery as well as carbon tetrachloride (CCl4 )-mediated injury. Importantly, livers of C/EBPα-S193A mice fail to stop liver regeneration after surgery when livers reach the original, preresection, size. The failure of S193A livers to stop regeneration correlates with the epigenetic repression of key regulators of liver proliferation C/EBPα, p53, FXR, SIRT1, PGC1α, and TERT by C/EBPß-HDAC1 complexes. The C/EBPß-HDAC1 complexes also repress promoters of enzymes of glucose synthesis PEPCK and G6Pase. CONCLUSION: Proper cooperation of C/EBP and chromatin remodeling proteins is essential for the termination of liver regeneration after surgery and for maintenance of liver functions.

Spontaneous Decompression Fracture in Thyroid Eye Disease.

This is a case of a 44-year-old female with a history of Graves' orbitopathy presented to the emergency department after waking from a nap with sudden onset of left facial and periorbital swelling, ecchymosis, and subconjunctival hemorrhage. A CT scan obtained in the emergency department revealed a left blowout fracture and enlarged extraocular muscles. The patient lives with her mother and both adamantly denied any trauma. The patient had sustained a spontaneous orbital fracture; a process reported but few times in the medical literature.

Intracranial Extension of an Orbital Epidermoid Cyst.

Epidermoid and dermoid cysts represent the most common cystic lesions of the orbit and commonly arise from bony sutures or the intradiplpoic space of orbital bones. Massive intracranial extension of an epidermoid cyst arising from the intradiploic space of an orbital bone is very rarely seen. We present a case of a 55-year-old male who was incidentally found to have massive intracranial extension of an intradiploic epidermoid cyst of the superolateral orbital bone with minimal symptoms. The cyst was completely excised via a pterional craniotomy and lateral orbitotomy by neurosurgery and oculoplastic surgery teams. The patient suffered no complications and is doing very well.

Age-associated change of C/EBP family proteins causes severe liver injury and acceleration of liver proliferation after CCl4 treatments.

The aged liver is more sensitive to the drug treatments and has a high probability of developing liver disorders such as fibrosis, cirrhosis, and cancer. Here we present mechanisms underlying age-associated severe liver injury and acceleration of liver proliferation after CCl4 treatments. We have examined liver response to CCl4 treatments using old WT mice and young C/EBPα-S193D knockin mice, which express an aged-like isoform of C/EBPα. Both animal models have altered chromatin structure as well as increased liver injury and proliferation after acute CCl4 treatments. We found that these age-related changes are associated with the repression of key regulators of liver biology: C/EBPα, Farnesoid X Receptor (FXR) and telomere reverse transcriptase (TERT). In quiescent livers of old WT and young S193D mice, the inhibition of TERT is mediated by HDAC1-C/EBPα complexes. After CCl4 treatments, TERT, C/EBPα and FXR are repressed by different mechanisms. These mechanisms include the increase of a dominant negative isoform, C/EBPß-LIP, and subsequent repression of C/EBPα, FXR, and TERT promoters. C/EBPß-LIP also disrupts Rb-E2F1 complexes in C/EBPα-S193D mice after CCl4 treatments. To examine if these alterations are involved in drug-mediated liver diseases, we performed chronic treatments of mice with CCl4. We found that C/EBPα-S193D mice developed fibrosis much more rapidly than WT mice. Thus, our data show that the age-associated alterations of C/EBP proteins create favorable conditions for the increased liver proliferation after CCl4 treatments and for development of drug-mediated liver diseases.

Transcriptional and translational regulation of C/EBPß-HDAC1 protein complexes controls different levels of p53, SIRT1, and PGC1α proteins at the early and late stages of liver cancer.

Cancer changes biological processes in the liver by altering gene expression at the levels of transcription, translation, and protein modification. The RNA binding protein CUGBP1 is a key regulator of translation of CCAAT enhancer binding protein ß and histone deacetylase 1 (HDAC1). These proteins form complexes that are involved in the regulation of liver biology. Here we show a critical role of the translational activation of CCAAT/enhancer binding protein ß-HDAC1 complexes in the development of liver cancer mediated by diethylnitrosamine. We found that diethylnitrosamine increases the levels of CUGBP1 and activates CUGBP1 by phosphorylation, leading to the formation of the CUGBP1-eIF2 complex, which is an activator of translation of CUGBP1-dependent mRNAs. The elevation of the CUGBP1-eIF2 complex increases translation of C/EBPß and HDAC1, resulting in an increase of C/EBPß-HDAC1 complexes at later stages of liver cancer. We found that C/EBPß-HDAC1 complexes repress promoters of three key regulators of liver functions: p53, SIRT1, and PGC1α. As the result of this suppression, the p53-, SIRT1-, and PGC1α-dependent downstream pathways are reduced, leading to increased liver proliferation. We also found that the proper regulation of C/EBPß-HDAC1 complexes is required for the maintenance of biological levels of p53, SIRT1, and PGC1α in quiescent livers and at early stages of liver cancer. Taken together, these studies showed that the development of liver cancer includes a tight regulation of levels of C/EBPß-HDAC1 complexes on the levels of transcription, translation, and posttranslational modifications.

A statewide assessment of smoke-free policy in multiunit housing settings.

INTRODUCTION: The prevalence of smoke-free policies in multiunit housing (MUH) in South Dakota was examined. Owner beliefs about smoke-free policies were identified. METHODS: Stratified random sampling included 27 South Dakota counties classified as frontier, large rural, or urban. Data collection with MUH owners in selected counties employed a telephone survey with mailed backup. RESULTS: The owner response rate was 41.5% (324/780). A written smoke-free policy was reported by 175 (54.0%) owners, and 31 (10%) reported a verbal smoke-free policy. Owners in large rural counties (57.4%) had more written smoke-free policies than owners in urban (52.2%) and frontier (53.5%) counties. Only 8.5% of properties had policies covering both buildings and grounds. Owners without policies were more than twice as likely to manage U.S. Department of Housing and Urban Development subsidized units and were three times as likely to be current smokers. Owners without a smoke-free policy anticipated that a policy would decrease maintenance costs but increase turnover and vacancy rates. Nearly one-half (47.9%) of owners with no smoke-free policy had previously considered implementing a policy. Owners self-reported beliefs about smoke-free policies identified perceived benefits such as decreased maintenance and costs, improved tenant safety and health, and conscientious tenants. Perceived drawbacks included increased outdoor maintenance, enforcement problems, concerns about long-term tenants who smoke, and freedom/rights of smokers. CONCLUSIONS: This study provides a baseline assessment of smoke-free polices in MUH settings. Perceptions of owners without smoke-free policies focused on economic concerns that were inconsistent with reports from those owners with smoke-free policies.

p66ShcA promotes malignant breast cancer phenotypes by alleviating energetic and oxidative stress.

Significant efforts have focused on identifying targetable genetic drivers that support the growth of solid tumors and/or increase metastatic ability. During tumor development and progression to metastatic disease, physiological and pharmacological selective pressures influence parallel adaptive strategies within cancer cell sub-populations. Such adaptations allow cancer cells to withstand these stressful microenvironments. This Darwinian model of stress adaptation often prevents durable clinical responses and influences the emergence of aggressive cancers with increased metastatic fitness. However, the mechanisms contributing to such adaptive stress responses are poorly understood. We now demonstrate that the p66ShcA redox protein, itself a ROS inducer, is essential for survival in response to physiological stressors, including anchorage independence and nutrient deprivation, in the context of poor outcome breast cancers. Mechanistically, we show that p66ShcA promotes both glucose and glutamine metabolic reprogramming in breast cancer cells, to increase their capacity to engage catabolic metabolism and support glutathione synthesis. In doing so, chronic p66ShcA exposure contributes to adaptive stress responses, providing breast cancer cells with sufficient ATP and redox balance needed to withstand such transient stressed states. Our studies demonstrate that p66ShcA functionally contributes to the maintenance of aggressive phenotypes and the emergence of metastatic disease by forcing breast tumors to adapt to chronic and moderately elevated levels of oxidative stress.

The PDZ1 and PDZ3 domains of MAGI-1 regulate the eight-exon isoform of the coxsackievirus and adenovirus receptor.

Epithelial integrity is essential for homeostasis and poses a formidable barrier to pathogen entry. Major factors for viral entry into epithelial cells are the localization and abundance of the primary receptor. The coxsackievirus and adenovirus receptor (CAR) is a primary receptor for these two pathogenic groups of viruses. In polarized epithelia, a low-abundance, alternatively spliced eight-exon isoform of CAR, CAR(Ex8), is localized apically where it can support viral infection from the air-exposed surface. Using biochemical, cell biology, genetic, and spectroscopic approaches, we show that the levels of apical CAR(Ex8) are negatively regulated by the PDZ domain-containing protein MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1) and that two MAGI-1 PDZ domains, PDZ1 and PDZ3, regulate CAR(Ex8) levels in opposing ways. Similar to full-length MAGI-1, expression of the isolated PDZ3 domain significantly reduces cell surface CAR(Ex8) abundance and adenovirus infection. In contrast, the PDZ1 domain is able to rescue CAR(Ex8) and adenovirus infection from MAGI-1-mediated suppression. These data suggest a novel cell-based strategy to either suppress viral infection or augment adenovirus-based gene therapy.