Asymmetric allostery in estrogen receptor-α homodimers drives responses to the ensemble of estrogens in the hormonal milieu.

The estrogen receptor-α (ER) is thought to function only as a homodimer but responds to a variety of environmental, metazoan, and therapeutic estrogens at subsaturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations-receptor conformational heterodimers-mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining the binding of the same ligand in crystal structures of ER in the agonist vs. antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist vs. antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from the ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric vs. dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing different modes for ligand-dependent regulation of ER activity.

Asymmetric Allostery in Estrogen Receptor-α Homodimers Drives Responses to the Ensemble of Estrogens in the Hormonal Milieu.

The estrogen receptor-α (ER) is thought to function only as a homodimer, but responds to a variety of environmental, metazoan, and therapeutic estrogens at sub-saturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations -receptor conformational heterodimers-mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining binding of the same ligand in crystal structures of ER in the agonist versus antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist versus antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric versus dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing new modes for ligand-dependent regulation of ER activity. Significance: The estrogen receptor-α (ER) regulates transcription in response to a hormonal milieu that includes low levels of estradiol, a variety of environmental estrogens, as well as ER antagonists such as breast cancer anti-hormonal therapies. While ER has been studied as a homodimer, the variety of ligand and receptor concentrations in different tissues means that the receptor can be occupied with two different ligands, with only one ligand in the dimer, or as a monomer. Here, we use X-ray crystallography and molecular dynamics simulations to reveal a new mode for ligand regulation of ER activity whereby sequence-identical homodimers can act as functional or conformational heterodimers having unique signaling characteristics, with ligand-selective allostery operating across the dimer interface integrating two different signaling outcomes.

Estimating migration timing and abundance in partial migratory systems by integrating continuous antenna detections with physical captures.

Many populations migrate between two different habitats (e.g. wintering/foraging to breeding area, mainstem-tributary, river-lake, river-ocean, river-side channel) as part of their life history. Detection technologies, such as passive integrated transponder (PIT) antennas or sonic receivers, can be placed at boundaries between habitats (e.g. near the confluence of rivers) to detect migratory movements of marked animals. Often, these detection systems have high detection probabilities and detect many individuals but are limited in their ability to make inferences about abundance because only marked individuals can be detected. Here, we introduce a mark-recapture modelling approach that uses detections from a double-array PIT antenna system to imply movement directionality from arrays and estimate migration timing. Additionally, when combined with physical captures, the model can be used to estimate abundances for both migratory and non-migratory groups and help quantify partial migration. We first test our approach using simulation, and results indicate our approach displayed negligible bias for total abundance (less than ±1%) and slight biases for state-specific abundance estimates (±1%-6%). We fit our model to array detections and physical captures of three native fishes (humpback chub [Gila cypha], flannelmouth sucker [Catostomus latipinnis] and bluehead sucker [Catostomus discobolus]) in the Little Colorado River (LCR) in Grand Canyon, AZ, a system that exhibits partial migration (i.e. includes residents and migrants). Abundance estimates from our model confirm that, for all three species, migratory individuals are much more numerous than residents. There was little difference in movement timing between 2021 (a year without preceding winter/spring floods) and 2022 (a year with a small flood occurring in early April). In both years, flannelmouth sucker arrived in mid-March whereas humpback chub and bluehead sucker arrivals occurred early- to mid-April. With humpback chub and flannelmouth sucker, movement timing was influenced by body size so that large individuals were more likely to arrive early compared to smaller individuals. With more years of data, this model framework could be used to evaluate ecological questions pertaining to flow cues and movement timing or intensity, relative trends in migrants versus residents and ecological drivers of skipped spawning.

Investigation of an mpox outbreak affecting many vaccinated persons in Chicago, IL-March 2023-June 2023.

BACKGROUND: After months of few mpox cases, an increased number of cases were reported in Chicago during May 2023; predominantly among fully vaccinated patients. We investigated the outbreak scope, differences between vaccinated and unvaccinated patients, and hypotheses for monkeypox virus (MPXV) infection after vaccination. METHODS: We interviewed patients and reviewed medical records to assess demographic, behavioral, and clinical characteristics, mpox vaccine status, and vaccine administration routes. We evaluated serum antibody levels after infection and compared patient viral genomes with MPXV sequences in available databases. We discussed potential vaccine compromise with partners who manufactured, handled, and administered vaccine associated with breakthrough infections. RESULTS: During March 18-June 27, 2023, we identified 49 mpox cases; 57% of these mpox patients were fully vaccinated (FV). FV patients received both JYNNEOS doses subcutaneously (57%), intradermally (7%), or via heterologous administration (36%). FV patients had more median sex partners (3, IQR=1-4) versus not fully vaccinated (NFV) patients (1, IQR=1-2). Thirty-six of 37 sequenced specimens belonged to lineage B.1.20 of clade IIb MPXV, which did not demonstrate any amino acid changes relative to B.1, the predominant lineage from May 2022. Vaccinated patients demonstrated expected humoral antibody responses; none were hospitalized. No vaccine storage excursions were identified. Approximately 63% of people at risk for mpox in Chicago were FV during this period. CONCLUSIONS: Our investigation indicated cases were likely due to frequent behaviors associated with mpox transmission, even with relatively high vaccine effectiveness and vaccine coverage. Cases after vaccination might occur in similar populations.

Skeletal Muscle Endogenous Estrogen Production Ameliorates the Metabolic Consequences of a High-Fat Diet in Male Mice.

AIMS: The role of skeletal muscle estrogen and its ability to mitigate the negative impact of a high-fat diet (HFD) on obesity-associated metabolic impairments is unknown. To address this, we developed a novel mouse model to determine the role of endogenous 17ß-estradiol (E2) production in males in skeletal muscle via inducible, skeletal muscle-specific aromatase overexpression (SkM-Arom↑). METHODS: Male SkM-Arom↑ mice and littermate controls were fed a HFD for 14 weeks prior to induction of SkM-Arom↑ for a period of 6.5 weeks. Glucose tolerance, insulin action, adipose tissue inflammation, and body composition were assessed. Indirect calorimetry and behavioral phenotyping experiments were performed using metabolic cages. Liquid chromatography mass spectrometry was used to determine circulating and tissue (skeletal muscle, hepatic, and adipose) E2 and testosterone concentrations. RESULTS: SkM-Arom↑ significantly increased E2 in skeletal muscle, circulation, the liver, and adipose tissue. SkM-Arom↑ ameliorated HFD-induced hyperglycemia, hyperinsulinemia, impaired glucose tolerance, adipose tissue inflammation, and reduced hepatic lipid accumulation while eliciting skeletal muscle hypertrophy. CONCLUSION: Enhanced skeletal muscle aromatase activity in male mice induces weight loss, improves metabolic and inflammatory outcomes and mitigates the negative effects of a HFD. Additionally, our data demonstrate for the first time skeletal muscle E2 has anabolic effects on the musculoskeletal system.

Prevalence and Dose Dependency Analysis of Pentosan Polysulfate Sodium Maculopathy.

PURPOSE: We describe a retrospective cohort study investigating the prevalence of pentosan polysulfate sodium (PPS) maculopathy in patients with PPS exposure, as well as the relationship between cumulative PPS exposure and the presence of PPS-maculopathy. METHODS: Patients were identified through review of the electronic medical record system. Available diagnostic imaging was reviewed for signs of PPS-maculopathy. Patients were also contacted to determine cumulative exposure. RESULTS: Of the 335 identified eligible patient records, 84 had sufficient diagnostic imaging. Sixteen patients had definitive signs of PPS-maculopathy, 6 had likely signs of PPS-maculopathy, and 62 had no signs. The mean cumulative PPS exposure and standard error of the mean (SEM) for patients with any signs of PPS-maculopathy was 1946.0 g (396.0 g), significantly higher than the mean cumulative PPS exposure for patients without such signs of 782.3 g (105.3 g). No significant difference in BCVA was noted. The odds ratio (OR, 95% confidence interval (95% CI)) of PPS-maculopathy was significantly elevated in patients with cumulative PPS exposures of 1500-2000 g [OR 4.72 (0.856-26.02 95% CI)] and greater than 2000 g [OR 28.33 (2.388-336.1, 95% CI)]. Logistic regression analysis confirmed a positive dose response relationship. CONCLUSIONS: We describe the concerning incidence of PPS-maculopathy in a multispecialty ophthalmology practice's patient population and investigate the dose-dependency of PPS-maculopathy. Patients with PPS-maculopathy were shown to have a higher average exposure to PPS than those without the maculopathy. Patients with cumulative PPS exposures greater than 1500 g were shown to have an increased risk of PPS-maculopathy.

Design, synthesis, biological evaluation and crystal structure determination of dual modulators of carbonic anhydrases and estrogen receptors.

Multi-target compounds have become increasingly important for the development of safer and more effective drug candidates. In this work, we devised a combined ligand-based and structure-based multi-target repurposing strategy and applied it to a series of hexahydrocyclopenta[c]quinoline compounds synthesized previously. The in silico analyses identified human Carbonic Anhydrases (hCA) and Estrogen Receptors (ER) as top scoring candidates for dual modulation. hCA isoforms IX and XII, and ER subtypes ER⍺ and/or ERß are co-expressed in various cancer cell types, including breast and prostate cancer cells. ER⍺ is the primary target of anti-estrogen therapy in breast cancer, and the hCA IX isoform is a therapeutic target in triple-negative breast cancer. ER⍺-mediated transcriptional programs and hCA activity in cancer cells promote favorable microenvironments for cell proliferation. Interestingly, several lines of evidence indicate that the combined modulation of these two targets may provide significant therapeutic benefits. Moving from these first results, two additional hexahydrocyclopenta[c]quinoline derivatives bearing a sulfonamide zinc binding group (hCA) and a phenolic hydroxyl (ER) pharmacophoric group placed at the appropriate locations were designed and synthesized. Interestingly, these compounds were able to directly modulate the activities of both hCA and ER targets. In cell-based assays, they inhibited proliferation of breast and prostate cancer cells with micromolar potency and cell type-selective efficacy. The compounds inhibited hCA activity with nanomolar potency and isoform-selectivity. In transactivation assays, they reduced estrogen-driven ER activity with micro-molar potency. Finally, crystal structures of the synthesized ligands in complex with the two targets revealed that the compounds bind directly to the hCA active site, as well as to the ER ligand-binding domain, providing structural explanation to the observed activity and a rationale for optimization of their dual activity. To the best of our knowledge, this work describes the design, synthesis and biological characterization of the first dual modulators of hCA and ER, laying the ground for the structure-based optimization of their multi-target activity.

Langerhans' cell histiocytosis of the retropharynx: first reported case.

Langerhans' cell histiocytosis (LCH) is a rare condition characterised by histiocyte proliferation leading to destructive granulomatous lesions. It may occur anywhere in the body but extraosseous manifestations affecting the head and neck are particularly uncommon. Here, we present the first reported case of a mass arising in the retropharyngeal space caused by LCH. The patient was a 33-year-old man with various symptoms which are presented. Although rare, LCH can affect a variety of tissues in the head and neck. Clinicians need to be cognisant of its inclusion in the differential diagnoses for similar cases in their practice, in particular because of potential difficulties in diagnosis.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor α (ERα). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2-infected hamsters localized lung pathology with increased ERα lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ERα and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ERα interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology.

Hydroxychloroquine blocks SARS-CoV-2 entry into the endocytic pathway in mammalian cell culture.

Hydroxychloroquine (HCQ), a drug used to treat lupus and malaria, was proposed as a treatment for SARS-coronavirus-2 (SARS-CoV-2) infection, albeit with controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ's mechanism of actions in vitro is needed. Recently, anesthetics were shown to disrupt ordered clusters of monosialotetrahexosylganglioside1 (GM1) lipid. These same lipid clusters recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to endocytic lipids, away from phosphatidylinositol 4,5 bisphosphate (PIP2) clusters. Here we employed super-resolution imaging of cultured mammalian cells (VeroE6, A549, H1793, and HEK293T) to show HCQ directly perturbs clustering of ACE2 receptor with both endocytic lipids and PIP2 clusters. In elevated (high) cholesterol, HCQ moves ACE2 nanoscopic distances away from endocytic lipids. In cells with resting (low) cholesterol, ACE2 primarily associates with PIP2 clusters, and HCQ moves ACE2 away from PIP2 clusters-erythromycin has a similar effect. We conclude HCQ inhibits viral entry through two distinct mechanisms in high and low tissue cholesterol and does so prior to inhibiting cathepsin-L. HCQ clinical trials and animal studies will need to account for tissue cholesterol levels when evaluating dosing and efficacy.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ERα). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ERα binding mode. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [ 18 F]fluoroestradiol (FES) localized lung pathology with increased ERα lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ERα expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ERα interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ER). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ER binding mode. In cultured cells, S DNA transfection increased ER cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [18F]fluoroestradiol (FES) localized lung pathology with increased ER lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ER expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ER interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.

Detection of SARS-CoV-2 infection in asymptomatic populations using the DiaSorin molecular Simplexa and Roche Cobas EUA assays.

Identification of asymptomatic patients is necessary to control the COVID-19 pandemic and testing is one of the measures to detect this population. We evaluated the clinical correlation of the DiaSorin Molecular Simplexa COVID-19 Direct (DiaSorin Molecular) and Roche Cobas 6800 SARS-CoV-2 (Roche) assays using 253 oropharyngeal (OP) swab specimens collected from asymptomatic patients. Agreement between DiaSorin Molecular and Roche was 97% (95% CI, 0.94 to 0.99), with a κ statistic of 0.90 (95% CI, 0.83 to 0.97) and a PPA of 89% (95% CI, 0.76 to 0.96) and NPA of 99% (95% CI, 0.97 to 0.99). Simple regression analysis of Ct values revealed a regression line of y = 1.065*X - 5.537 with a Pearson's r of 0.8542, indicating a good correlation between both platforms. The DiaSorin Molecular assay demonstrates clinical performance comparable to that of Roche in this population.

Activation of Crtc2/Creb1 in skeletal muscle enhances weight loss during intermittent fasting.

The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, maintenance of higher energy expenditure during the fast and reduced binge-eating during the feeding period. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.

Dual-mechanism estrogen receptor inhibitors.

Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.

Chemical systems biology reveals mechanisms of glucocorticoid receptor signaling.

Glucocorticoids display remarkable anti-inflammatory activity, but their use is limited by on-target adverse effects including insulin resistance and skeletal muscle atrophy. We used a chemical systems biology approach, ligand class analysis, to examine ligands designed to modulate glucocorticoid receptor activity through distinct structural mechanisms. These ligands displayed diverse activity profiles, providing the variance required to identify target genes and coregulator interactions that were highly predictive of their effects on myocyte glucose disposal and protein balance. Their anti-inflammatory effects were linked to glucose disposal but not muscle atrophy. This approach also predicted selective modulation in vivo, identifying compounds that were muscle-sparing or anabolic for protein balance and mitochondrial potential. Ligand class analysis defined the mechanistic links between the ligand-receptor interface and ligand-driven physiological outcomes, a general approach that can be applied to any ligand-regulated allosteric signaling system.

A data-centric approach to generative modelling for 3D-printed steel.

The emergence of additive manufacture (AM) for metallic material enables components of near arbitrary complexity to be produced. This has potential to disrupt traditional engineering approaches. However, metallic AM components exhibit greater levels of variation in their geometric and mechanical properties compared to standard components, which is not yet well understood. This uncertainty poses a fundamental barrier to potential users of the material, since extensive post-manufacture testing is currently required to ensure safety standards are met. Taking an interdisciplinary approach that combines probabilistic mechanics and uncertainty quantification, we demonstrate that intrinsic variation in AM steel can be well described by a generative statistical model that enables the quality of a design to be predicted before manufacture. Specifically, the geometric variation in the material can be described by an anisotropic spatial random field with oscillatory covariance structure, and the mechanical behaviour by a stochastic anisotropic elasto-plastic material model. The fitted generative model is validated on a held-out experimental dataset and our results underscore the need to combine both statistical and physics-based modelling in the characterization of new AM steel products.