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Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of non-enveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoV) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent-RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA., Teaser: Polymerase of a positive-sense RNA virus forms LLPS to regulate replication as an elegant solution for an enigmatic question.

We recently discovered that steroid receptor coactivators (SRCs) SRCs-1, 2 and 3, are abundantly expressed in cardiac fibroblasts (CFs) and their activation with the SRC small molecule stimulator MCB-613 improves cardiac function and dramatically lowers pro-fibrotic signaling in CFs post-myocardial infarction. These findings suggest that CF-derived SRC activation could be beneficial in the mitigation of chronic heart failure after ischemic insult. However, the cardioprotective mechanisms by which CFs contribute to cardiac pathological remodeling are unclear. Here we present studies designed to identify the molecular and cellular circuitry that governs the anti-fibrotic effects of an MCB-613 derivative, MCB-613-10-1, in CFs. We performed cytokine profiling and whole transcriptome and proteome analyses of CF-derived signals in response to MCB-613-10-1. We identified the NRF2 pathway as a direct MCB-613-10-1 therapeutic target for promoting resistance to oxidative stress in CFs. We show that MCB-613-10-1 promotes cell survival of anti-fibrotic CFs exposed to oxidative stress by suppressing apoptosis. We demonstrate that an increase in HMOX1 expression contributes to CF resistance to oxidative stress-mediated apoptosis via a mechanism involving SRC co-activation of NRF2, hence reducing inflammation and fibrosis. We provide evidence that MCB-613-10-1 acts as a protectant against oxidative stress-induced mitochondrial damage. Our data reveal that SRC stimulation of the NRF2 transcriptional network promotes resistance to oxidative stress and highlights a mechanistic approach toward addressing pathologic cardiac remodeling., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We then validated differences in key pathways through functional studies and determined whether these cultures recapitulate known features of the infant intestinal epithelium. RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell, and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex vivo model to advance studies of infant-specific diseases and drug discovery for this population., Importance: Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begin during infancy, tools that appropriately reflect intestinal development and diseases are critical. Infant enteroids exhibit key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases, and responses to pathogens., Competing Interests: The authors declare no conflict of interest.

The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3β, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.

The initial step in estrogen-regulated transcription is the binding of a ligand to its cognate receptors, named estrogen receptors (ERα and ERβ). Phytochemicals present in foods and environment can compete with endogenous hormones to alter physiological responses. We screened 224 flavonoids in our engineered biosensor ERα and ERβ PRL-array cell lines to characterize their activity on several steps of the estrogen signaling pathway. We identified 83 and 96 flavonoids that can activate ERα or ERβ, respectively. While most act on both receptors, many appear to be subtype-selective, including potent flavonoids that activate ER at sub-micromolar concentrations. We employed an orthogonal assay using a transgenic zebrafish in vivo model that validated the estrogenic potential of these compounds. To our knowledge, this is the largest study thus far on flavonoids and the ER pathway, facilitating the identification of a new set of potential endocrine disruptors acting on both ERα and ERβ., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

In this study we present an inducible biosensor model for the Estrogen Receptor Beta (ERβ), GFP-ERβ:PRL-HeLa, a single-cell-based high throughput (HT) in vitro assay that allows direct visualization and measurement of GFP-tagged ERβ binding to ER-specific DNA response elements (EREs), ERβ-induced chromatin remodeling, and monitor transcriptional alterations via mRNA fluorescence in situ hybridization for a prolactin (PRL)-dsRED2 reporter gene. The model was used to accurately (Z' = 0.58-0.8) differentiate ERβ-selective ligands from ERα ligands when treated with a panel of selective agonists and antagonists. Next, we tested an Environmental Protection Agency (EPA)-provided set of 45 estrogenic reference chemicals with known ERα in vivo activity and identified several that activated ERβ as well, with varying sensitivity, including a subset that is completely novel. We then used an orthogonal ERE-containing transgenic zebrafish (ZF) model to cross validate ERβ and ERα selective activities at the organism level. Using this environmentally relevant ZF assay, some compounds were confirmed to have ERβ activity, validating the GFP-ERβ:PRL-HeLa assay as a screening tool for potential ERβ active endocrine disruptors (EDCs). These data demonstrate the value of sensitive multiplex mechanistic data gathered by the GFP-ERβ:PRL-HeLa assay coupled with an orthogonal zebrafish model to rapidly identify environmentally relevant ERβ EDCs and improve upon currently available screening tools for this understudied nuclear receptor., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

In this short review we discuss the current view of how the estrogen receptor (ER), a pivotal member of the nuclear receptor superfamily of transcription factors, regulates gene transcription at the single cell and allele level, focusing on in vitro cell line models. We discuss central topics and new trends in molecular biology including phenotypic heterogeneity, single cell sequencing, nuclear phase separated condensates, single cell imaging, and image analysis methods, with particular focus on the methodologies and results that have been reported in the last few years using microscopy-based techniques. These observations augment the results from biochemical assays that lead to a much more complex and dynamic view of how ER, and arguably most transcription factors, act to regulate gene transcription., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Humans are continuously exposed to a variety of toxicants and chemicals which is exacerbated during and after environmental catastrophes such as floods, earthquakes, and hurricanes. The hazardous chemical mixtures generated during these events threaten the health and safety of humans and other living organisms. This necessitates the development of rapid decision-making tools to facilitate mitigating the adverse effects of exposure on the key modulators of the endocrine system, such as the estrogen receptor alpha (ERα), for example. The mechanistic stages of the estrogenic transcriptional activity can be measured with high content/high throughput microscopy-based biosensor assays at the single-cell level, which generates millions of object-based minable data points. By combining computational modeling and experimental analysis, we built a highly accurate data-driven classification framework to assess the endocrine disrupting potential of environmental compounds. The effects of these compounds on the ERα pathway are predicted as being receptor agonists or antagonists using the principal component analysis (PCA) projections of high throughput, high content image analysis descriptors. The framework also combines rigorous preprocessing steps and nonlinear machine learning algorithms, such as the Support Vector Machines and Random Forest classifiers, to develop highly accurate mathematical representations of the separation between ERα agonists and antagonists. The results show that Support Vector Machines classify the unseen chemicals correctly with more than 96% accuracy using the proposed framework, where the preprocessing and the PCA steps play a key role in suppressing experimental noise and unraveling hidden patterns in the dataset.

Objective: To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing in vitro fertilization., Design: Basic science research with a preclinical allosteric FSHR agonist., Setting: University hospital., Patient(s): Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS., Intervention(s): Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle., Main Outcome Measure(s): Estradiol (E 2 ) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells., Result(s): TOP5300 consistently stimulated E 2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS., Conclusion(s): TOP5300 appears to stimulate E 2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E 2 production., Competing Interests: Declaration of interests J.Z.G. reports funding from NIH R01 HD032067 and Baylor College of Medicine Department of Obstetrics and Gynecology for the submitted work. D.M. has nothing to disclose. H.Y. is the Chief executive officer and reports stock options from CanWell Pharma. F.S.S. has nothing to disclose. H.L.J. has nothing to disclose. W.E.G. has nothing to disclose. M.M.M. has nothing to disclose. S.P. has nothing to disclose., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Measuring single cell responses to the universe of chemicals (drugs, natural products, environmental toxicants etc.) is of paramount importance to human health as phenotypic variability in sensing stimuli is a hallmark of biology that is considered during high throughput screening. One of the ways to approach this problem is via high throughput, microscopy-based assays coupled with multi-dimensional single cell analysis methods. Here, we will summarize some of the efforts in this vast and growing field, focusing on phenotypic screens (e.g., Cell Painting), single cell analytics and quality control, with particular attention to environmental toxicology and drug screening. We will discuss advantages and limitations of high throughput assays with various end points and levels of complexity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Microscopy-generated images contain quantitative information that can be extracted using a variety of analysis methods and automated pipelines. In this protocol, basic aspects of image analysis are outlined, including software installation, data import, image processing functions, and analytical tools that can be used to extract information from microscopy data using the open-source software ImageJ/Fiji. Step-by-step protocols for analyzing objects (i.e., nuclei) in a fluorescence image are included, focusing on segmentation routines. © 2023 Wiley Periodicals LLC., (© 2023 Wiley Periodicals LLC.)

Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3α (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Despite the knowledge that protein translation and various metabolic reactions that create and sustain cellular life occur in the cytoplasm, the structural organization within the cytoplasm remains unclear. Recent models indicate that cytoplasm contains viscous fluid and elastic solid phases. We separated these viscous fluid and solid elastic compartments, which we call the cytosol and cytomatrix, respectively. The distinctive composition of the cytomatrix included structural proteins, ribosomes, and metabolome enzymes. High-throughput analysis revealed unique biosynthetic pathways within the cytomatrix. Enrichment of biosynthetic pathways in the cytomatrix indicated the presence of immobilized biocatalysis. Enzymatic immobilization and segregation can surmount spatial impediments, and the local pathway segregation may form cytoplasmic organelles. Protein translation was reprogrammed within the cytomatrix under the restriction of protein synthesis by drug treatment. The cytosol and cytomatrix are an elaborately interconnected network that promotes operational flexibility in healthy cells and the survival of malignant cells., Competing Interests: The authors declare no competing interests. Patent No: US 11,428,693 B2; Data of Patent: Aug. 30, 2022 “Method of high throughput screening of chemical compounds suppressing nucleolar hypertrophy” Applicant: Baylor College of Medicine, Houston, TX (US) Inventor: Tattym E. Shaiken, Houston, TX (US) Patent materials were incorporated, in part, in the Result section “Assessment of protein synthesis in the cytosol and the cytomatrix”, (© 2023 The Author(s).)

We develop a machine learning framework that integrates high content/high throughput image analysis and artificial neural networks (ANNs) to model the separation between chemical compounds based on their estrogenic receptor activity. Natural and man-made chemicals have the potential to disrupt the endocrine system by interfering with hormone actions in people and wildlife. Although numerous studies have revealed new knowledge on the mechanism through which these compounds interfere with various hormone receptors, it is still a very challenging task to comprehensively evaluate the endocrine disrupting potential of all existing chemicals and their mixtures by pure in vitro or in vivo approaches. Machine learning offers a unique advantage in the rapid evaluation of chemical toxicity through learning the underlying patterns in the experimental biological activity data. Motivated by this, we train and test ANN classifiers for modeling the activity of estrogen receptor-α agonists and antagonists at the single-cell level by using high throughput/high content microscopy descriptors. Our framework preprocesses the experimental data by cleaning, scaling, and feature engineering where only the middle 50% of the values from each sample with detectable receptor-DNA binding is considered in the dataset. Principal component analysis is also used to minimize the effects of experimental noise in modeling where these projected features are used in classification model building. The results show that our ANN-based nonlinear data-driven framework classifies the benchmark agonist and antagonist chemicals with 98.41% accuracy.

Proteasome inhibitors have become the standard of care for multiple myeloma (MM). Blocking protein degradation particularly perturbs the homeostasis of short-lived polypeptides such as transcription factors and epigenetic regulators. To determine how proteasome inhibitors directly impact gene regulation, we performed an integrative genomics study in MM cells. We discovered that proteasome inhibitors reduce the turnover of DNA-associated proteins and repress genes necessary for proliferation through epigenetic silencing. Specifically, proteasome inhibition results in the localized accumulation of histone deacetylase 3 (HDAC3) at defined genomic sites, which reduces H3K27 acetylation and increases chromatin condensation. The loss of active chromatin at super-enhancers critical for MM, including the super-enhancer controlling the proto-oncogene c-MYC, reduces metabolic activity and cancer cell growth. Epigenetic silencing is attenuated by HDAC3 depletion, suggesting a tumor-suppressive element of this deacetylase in the context of proteasome inhibition. In the absence of treatment, HDAC3 is continuously removed from DNA by the ubiquitin ligase SIAH2. Overexpression of SIAH2 increases H3K27 acetylation at c-MYC-controlled genes, increases metabolic output, and accelerates cancer cell proliferation. Our studies indicate a novel therapeutic function of proteasome inhibitors in MM by reshaping the epigenetic landscape in an HDAC3-dependent manner. As a result, blocking the proteasome effectively antagonizes c-MYC and the genes controlled by this proto-oncogene., Competing Interests: Authors’ Disclosures: P.L. is an advisory board member of Karyopharm Therapeutics. All other authors declare they have no competing interests.

To investigate the role of adrenergic signalling (AS) in the host immune response and Porphyromonas gingivalis virulence, we compared norepinephrine (NE) and isoproterenol (ISO) responses in Galleria mellonella . P. gingivalis infection was evaluated by survival; humoral immune responses (i.e. melanization and cecropin and gloverin mRNA expression); cellular immune responses (i.e. haemocyte count, nodulation by histology); and P. gingivalis recovery (CFU/mL). P. gingivalis was cultivated in the presence of ISO (PgISO) or NE and injected into the larvae for survival evaluation. Finally, we co-injected ISO and PgISO to evaluate the concomitant effects on the immune response and bacterial virulence. None of the ligands were toxic to the larvae; ISO increased haemocyte number, even after P. gingivalis infection, by mobilizing sessile haemocytes in a β-adrenergic-specific manner, while NE showed the opposite effect. ISO treatment reduced larval mortality and the number of recovered bacteria, while NE increased mortality and showed no effect on bacterial recovery. ISO and NE had similar effects on melanization and decreased the expression of cecropin. Although co-cultivation with NE and ISO increased the gene expression of bacterial virulence factors in vitro , only the injection of PgISO increased larval death, which was partially reversed by circulating ISO. Therefore, α- and β-adrenergic signalling had opposite effects after P. gingivalis infection. Ultimately, the catecholamine influence on the immune response overcame the effect of more virulent strains. The effect of AS directly on the pathogen found in vitro did not translate to the in vivo setting.

The United States Environmental Protection Agency (EPA) has been pursuing new high throughput in vitro assays to characterize endocrine disrupting chemicals (EDCs) that interact with estrogen receptor signaling. We characterize two new PRL-HeLa cell models expressing either inducible C-terminal (iGFP-ER) or N-terminal (iER-GFP) tagged estrogen receptor-α (ERα) that allows direct visualization of chromatin binding. These models are an order of magnitude more sensitive, detecting 87 - 93% of very weak estrogens tested compared to only 27% by a previous PRL-HeLa variant and compares favorably to the 73% detected by an EPA-developed computational model using in vitro data. Importantly, the chromatin binding assays distinguished agonist- and antagonist-like phenotypes without activity specific assays. Finally, analysis of complex environmentally relevant chemical mixtures demonstrated how chromatin binding data can be used in risk assessment models to predict activity. These new assays should be a useful in vitro tool to screen for estrogenic activity., Competing Interests: The authors declare no competing interests., (© 2022.)

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here, we reported a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with one of four strains of RSV representing both major subgroups as well as historic and more contemporary genotypes (RSV/A/Tracy [GA1], RSV/A/Ontario [ON], RSV/B/18537 [GB1], and RSV/B/Buenos Aires [BA]) via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response, including transcriptional changes and levels of secreted cytokines and growth factors. IMPORTANCE Infection with the respiratory syncytial virus (RSV) early in life is essentially guaranteed and can lead to severe disease. Most RSV studies have involved either of two historic RSV/A strains infecting one of two cell lines, HEp-2 or A549 cells. However, RSV contains ample variation within two evolving subgroups (A and B), and HEp-2 and A549 cell lines are genetically distinct. Here, we measured viral action and host response in both HEp-2 and A549 cells infected with four RSV strains from both subgroups and representing both historic and more contemporary strains. We discovered a subgroup-dependent difference in viral gene expression and found A549 cells were more potently antiviral and more sensitive, albeit subtly, to viral variation. Our findings revealed important differences between RSV subgroups and two widely used cell lines and provided baseline data for experiments with model systems better representative of natural RSV infection.

The ubiquitin-proteasome pathway precisely controls the turnover of transcription factors in the nucleus, playing an important role in maintaining appropriate quantities of these regulatory proteins. The transcription factor c-MYC is essential for normal development and is a critical cancer driver. Despite being highly expressed in several tissues and malignancies, the c-MYC protein is also continuously targeted by the ubiquitin-proteasome pathway, which can either facilitate or inhibit c-MYC degradation. Deubiquitinating proteases can remove ubiquitin chains from target proteins and rescue them from proteasomal digestion. This study sought to determine novel elements of the ubiquitin-proteasome pathway that regulate c-MYC levels. We performed an overexpression screen with 41 human proteases to identify which deubiquitinases stabilize c-MYC. We discovered that the highly expressed Otubain-1 (OTUB1) protease increases c-MYC protein levels. Confirming its role in enhancing c-MYC activity, we found that elevated OTUB1 correlates with inferior clinical outcomes in the c-MYC-dependent cancer multiple myeloma, and overexpression of OTUB1 accelerates the growth of myeloma cells. In summary, our study identifies OTUB1 as a novel amplifier of the proto-oncogene c-MYC.

Background: Diverse toxicants and mixtures that affect hormone responsive cells [endocrine disrupting chemicals (EDCs)] are highly pervasive in the environment and are directly linked to human disease. They often target the nuclear receptor family of transcription factors modulating their levels and activity. Many high-throughput assays have been developed to query such toxicants; however, single-cell analysis of EDC effects on endogenous receptors has been missing, in part due to the lack of quality control metrics to reproducibly measure cell-to-cell variability in responses., Objective: We began by developing single-cell imaging and informatic workflows to query whether the single cell distribution of the estrogen receptor- α (ER), used as a model system, can be used to measure effects of EDCs in a sensitive and reproducible manner., Methods: We used high-throughput microscopy, coupled with image analytics to measure changes in single cell ER nuclear levels on treatment with ∼ 100 toxicants, over a large number of biological and technical replicates., Results: We developed a two-tiered quality control pipeline for single cell analysis and tested it against a large set of biological replicates, and toxicants from the EPA and Agency for Toxic Substances and Disease Registry lists. We also identified a subset of potentially novel EDCs that were active only on the endogenous ER level and activity as measured by single molecule RNA fluorescence in situ hybridization (RNA FISH)., Discussion: We demonstrated that the distribution of ER levels per cell, and the changes upon chemical challenges were remarkably stable features; and importantly, these features could be used for quality control and identification of endocrine disruptor toxicants with high sensitivity. When coupled with orthogonal assays, ER single cell distribution is a valuable resource for high-throughput screening of environmental toxicants. https://doi.org/10.1289/EHP9297.

Transcription is a highly regulated sequence of stochastic processes utilizing many regulators, including nuclear receptors (NR) that respond to stimuli. Endocrine disrupting chemicals (EDCs) in the environment can compete with natural ligands for nuclear receptors to alter transcription. As nuclear dynamics can be tightly linked to transcription, it is important to determine how EDCs affect NR mobility. We use an EPA-assembled set of 45 estrogen receptor-α (ERα) ligands and EDCs in our engineered PRL-Array model to characterize their effect upon transcription using fluorescence in situ hybridization and fluorescence recovery after photobleaching (FRAP). We identified 36 compounds that target ERα-GFP to a transcriptionally active, visible locus. Using a novel method for multi-region FRAP analysis we find a strong negative correlation between ERα mobility and inverse agonists. Our findings indicate that ERα mobility is not solely tied to transcription but affected highly by the chemical class binding the receptor., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

The epithelial-mesenchymal transition (EMT) has been implicated in conferring stem cell properties and therapeutic resistance to cancer cells. Therefore, identification of drugs that can reprogram EMT may provide new therapeutic strategies. Here, we report that cells derived from claudin-low mammary tumors, a mesenchymal subtype of triple-negative breast cancer, exhibit a distinctive organoid structure with extended "spikes" in 3D matrices. Upon a miR-200 induced mesenchymal-epithelial transition (MET), the organoids switch to a smoother round morphology. Based on these observations, we developed a morphological screening method with accompanying analytical pipelines that leverage deep neural networks and nearest neighborhood classification to screen for EMT-reversing drugs. Through screening of a targeted epigenetic drug library, we identified multiple class I HDAC inhibitors and Bromodomain inhibitors that reverse EMT. These data support the use of morphological screening of mesenchymal mammary tumor organoids as a platform to identify drugs that reverse EMT., (© 2021. The Author(s).)

Xia-Gibbs syndrome (XGS) is a rare Mendelian disease typically caused by de novo stop-gain or frameshift mutations in the AT-hook DNA binding motif containing 1 (AHDC1) gene. Patients usually present in early infancy with hypotonia and developmental delay and later exhibit intellectual disability (ID). The overall presentation is variable, however, and the emerging clinical picture is still evolving. A detailed phenotypic analysis of 34 XGS individuals revealed five core phenotypes (delayed motor milestones, speech delay, low muscle tone, ID, and hypotonia) in more than 80% of individuals and an additional 12 features that occurred more variably. Seizures and scoliosis were more frequently associated with truncations that arise before the midpoint of the protein although the occurrence of most features could not be predicted by the mutation position. Transient expression of wild type and different patient truncated AHDC1 protein forms in human cell lines revealed abnormal patterns of nuclear localization including a diffuse distribution of a short truncated form and nucleolar aggregation in mid-protein truncated forms. Overall, both the occurrence of variable phenotypes and the different distribution of the expressed protein reflect the heterogeneity of this syndrome., (© 2021 Wiley Periodicals LLC.)

Although current antiretroviral therapies (ART) are successful in controlling HIV-1 infection, a stable viral reservoir reactivates when ART is discontinued. Consequently, there is a major research effort to develop approaches to disrupt the latent viral reservoir and enhance the immune system's ability to clear HIV-1. A number of small molecules, termed latency reversal agents (LRAs), have been identified which can reactivate latent HIV-1 in cell lines and patients' cells ex vivo. However, clinical trials have suggested that combinations of LRAs will be required to efficiently reactivate HIV-1 in vivo, especially LRAs that act synergistically by functioning through distinct pathways. To identify novel LRAs, we used an image-based assay to screen a natural compound library for the ability to induce a low level of aggregation of resting primary CD4+ T cells from healthy donors. We identified celastrol as a novel LRA. Celastrol functions synergistically with other classes of LRA to reactivate latent HIV-1 in a Jurkat cell line, suggesting a novel mechanism in its LRA activity. Additionally, celastrol does not appear to activate resting CD4+ T cells at levels at which it can reactivate latent HIV-1. Celastrol appears to represent a novel class of LRAs and it therefore can serve as a lead compound for LRA development., Competing Interests: The authors have declared that no competing interests exist.

There is an unmet need for preclinical models to understand the pathogenesis of human respiratory viruses and predict responsiveness to immunotherapies. Airway organoids can serve as an ex vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a noninvasive technique to generate human nose organoids (HNOs) as an alternative to biopsy-derived organoids. We made air-liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hypersecretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation), while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration-dependent manner. Thus, the HNO-ALI model can serve as an alternative to lung organoids to study respiratory viruses and test therapeutics. IMPORTANCE Preclinical models that recapitulate aspects of human airway disease are essential for the advancement of novel therapeutics and vaccines. Here, we report a versatile airway organoid model, the human nose organoid (HNO), that recapitulates the complex interactions between the host and virus. HNOs are obtained using noninvasive procedures and show divergent responses to SARS-CoV-2 and RSV infection. SARS-CoV-2 induces severe damage to cilia and the epithelium, no interferon-λ response, and minimal mucus secretion. In striking contrast, RSV induces hypersecretion of mucus and a profound interferon-λ response with ciliary damage. We also demonstrated the usefulness of our ex vivo HNO model of RSV infection to test the efficacy of palivizumab, an FDA-approved monoclonal antibody to prevent severe RSV disease in high-risk infants. Our study reports a breakthrough in both the development of a novel nose organoid model and in our understanding of the host cellular response to RSV and SARS-CoV-2 infection.

Many oncogenic insults deregulate RNA splicing, often leading to hypersensitivity of tumors to spliceosome-targeted therapies (STTs). However, the mechanisms by which STTs selectively kill cancers remain largely unknown. Herein, we discover that mis-spliced RNA itself is a molecular trigger for tumor killing through viral mimicry. In MYC-driven triple-negative breast cancer, STTs cause widespread cytoplasmic accumulation of mis-spliced mRNAs, many of which form double-stranded structures. Double-stranded RNA (dsRNA)-binding proteins recognize these endogenous dsRNAs, triggering antiviral signaling and extrinsic apoptosis. In immune-competent models of breast cancer, STTs cause tumor cell-intrinsic antiviral signaling, downstream adaptive immune signaling, and tumor cell death. Furthermore, RNA mis-splicing in human breast cancers correlates with innate and adaptive immune signatures, especially in MYC-amplified tumors that are typically immune cold. These findings indicate that dsRNA-sensing pathways respond to global aberrations of RNA splicing in cancer and provoke the hypothesis that STTs may provide unexplored strategies to activate anti-tumor immune pathways., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chordomas are rare spinal tumors addicted to expression of the developmental transcription factor brachyury. In chordomas, brachyury is super-enhancer associated and preferentially downregulated by pharmacologic transcriptional CDK inhibition, leading to cell death. To understand the underlying basis of this sensitivity, we dissect the brachyury transcription regulatory network and compare the consequences of brachyury degradation with transcriptional CDK inhibition. Brachyury defines the chordoma super-enhancer landscape and autoregulates through binding its super-enhancer, and its locus forms a transcriptional condensate. Transcriptional CDK inhibition and brachyury degradation disrupt brachyury autoregulation, leading to loss of its transcriptional condensate and transcriptional program. Compared with transcriptional CDK inhibition, which globally downregulates transcription, leading to cell death, brachyury degradation is much more selective, inducing senescence and sensitizing cells to anti-apoptotic inhibition. These data suggest that brachyury downregulation is a core tenet of transcriptional CDK inhibition and motivates developing strategies to target brachyury and its autoregulatory feedback loop., Competing Interests: C.Y.L. is an inventor of intellectual property licensed to Syros Pharmaceuticals, and is the Vice President of Biology and a shareholder of Kronos Bio, Inc. S.L.S. is a shareholder and serves on the Board of Directors of Jnana Therapeutics; is a shareholder of Forma Therapeutics and Decibel Therapeutics; is a shareholder and advises Kojin Therapeutics, Kisbee Therapeutics, Exo Therapeutics, and Eikonizo Therapeutics; serves on the Scientific Advisory Boards of Eisai Co., Ltd., Ono Pharma Foundation, Biogen, Inc., and F-Prime Capital Partners and the Board of Advisers of the Genomics Institute of the Novartis Research Foundation; and is a Novartis Faculty Scholar. M.J.W. is an employee and equity holder of Kojin Therapeutics. P.A.C. is an advisor to Pfizer, Inc. and nference, Inc. R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. P.W. is a current employee of The Institute of Cancer Research, which has a Rewards to Inventors scheme and has a commercial interest in development of inhibitors of the WNT pathway, CDK8/19, and other CDKs, with intellectual property licensed to Merck and Cyclacel Pharmaceuticals. P.W. is also a consultant for Astex Pharmaceuticals, CV6 Therapeutics, Nextechinvest, Nuevolution, Black Diamond, and STORM Therapeutics and holds equity in Chroma Therapeutics, Nextech, Black Diamond, and STORM. B.N. is an inventor on patent applications related to the dTAG system (WO/2017/024318, WO/2017/024319, WO/2018/148443, and WO/2018/148440). N.S.G. is a Scientific Founder, member of the Scientific Advisory Board (SAB), and equity holder in C4 Therapeutics, Syros, Soltego (board member), B2S, Allorion, and Inception. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voroni, Arbella, Deerfield, and Sanofi., (© 2020 The Authors.)

A comprehensive evaluation of toxic chemicals and understanding their potential harm to human physiology is vital in mitigating their adverse effects following exposure from environmental emergencies. In this work, we develop data-driven classification models to facilitate rapid decision making in such catastrophic events and predict the estrogenic activity of environmental toxicants as estrogen receptor-α (ERα) agonists or antagonists. By combining high-content analysis, big-data analytics, and machine learning algorithms, we demonstrate that highly accurate classifiers can be constructed for evaluating the estrogenic potential of many chemicals. We follow a rigorous, high throughput microscopy-based high-content analysis pipeline to measure the single cell-level response of benchmark compounds with known in vivo effects on the ERα pathway. The resulting high-dimensional dataset is then pre-processed by fitting a non-central gamma probability distribution function to each feature, compound, and concentration. The characteristic parameters of the distribution, which represent the mean and the shape of the distribution, are used as features for the classification analysis via Random Forest (RF) and Support Vector Machine (SVM) algorithms. The results show that the SVM classifier can predict the estrogenic potential of benchmark chemicals with higher accuracy than the RF algorithm, which misclassifies two antagonist compounds.

Advanced Bladder Cancer (BLCA) remains a clinical challenge that lacks effective therapeutic measures. Here, we show that distinct, stage-wise metabolic alterations in BLCA are associated with the loss of function of aldehyde oxidase (AOX1). AOX1 associated metabolites have a high predictive value for advanced BLCA and our findings demonstrate that AOX1 is epigenetically silenced during BLCA progression by the methyltransferase activity of EZH2. Knockdown (KD) of AOX1 in normal bladder epithelial cells re-wires the tryptophan-kynurenine pathway resulting in elevated NADP levels which may increase metabolic flux through the pentose phosphate (PPP) pathway, enabling increased nucleotide synthesis, and promoting cell invasion. Inhibition of NADP synthesis rescues the metabolic effects of AOX1 KD. Ectopic AOX1 expression decreases NADP production, PPP flux and nucleotide synthesis, while decreasing invasion in cell line models and suppressing growth in tumor xenografts. Further gain and loss of AOX1 confirm the EZH2-dependent activation, metabolic deregulation, and tumor growth in BLCA. Our findings highlight the therapeutic potential of AOX1 and provide a basis for the development of prognostic markers for advanced BLCA.