Synergistic Cytotoxicity of Extracts of Chaga Mushroom and Microalgae against Mammalian Cancer Cells In Vitro.

Chaga mushroom (Inonotus obliquus) contains bioactive metabolites and has been used to treat various ailments, including cancer. Similarly, marine microalgae are considered a sustainable food supplement with anticancer and antioxidant properties. This study investigated the cytotoxicity of different extracts prepared from I. obliquus and microalgae using cultured human and canine cancer cell lines (MCF-7, HepG2, HOS, D-17, and DH-82). MTS cell viability assay was used to study the cytotoxicity of I. obliquus and microalgae extracts, and a synergy matrix effect was used to study the combined effect of the extracts. Isobologram analysis and the highest single agent synergy model were applied to study and validate the synergy between the extracts from I. obliquus and microalgae. Ethanol-based extraction and supercritical water extract significantly inhibited the growth of various mammalian cancer cells compared to aqueous extracts. Osteosarcoma cells were more susceptible to the supercritical extracts of I. obliquus and chlorophyll-free and sugar-free ethanol extracts of microalgae. A combination of ethanol-based I. obliquus extract and chlorophyll-free microalgae extract resulted in a synergistic interaction with various tested cancer cells. This study provides experimental evidence supporting the potential therapeutic application of I. obliquus and microalgae extracts with a synergistic effect to inhibit the growth of various mammalian cancer cells. Additional in vivo studies are required to fully explore possible therapeutic applications of these unique mixtures to be used in treating cancers.

Factors and Barriers to Goals-of-Care Conversations for Patients With Cancer and Inpatient Mortality.

PURPOSE: Conversations about personal values and goals of care (GOC) at the end of life are essential in caring for patients with advanced cancer. However, GOC conversations may be influenced by patient and oncologist factors during transitions of care. METHODS: We electronically administered surveys to medical oncologists of inpatients who died from May 1, 2020, to May 31, 2021. Primary outcomes included oncologists' knowledge of inpatient death, anticipation of patient death, and recollection of GOC discussions. Secondary outcomes, including GOC documentation and advance directives (ADs), were collected retrospectively from electronic health records. Outcomes were analyzed for association with patient, oncologist, and patient-oncologist relationship factors. RESULTS: For 75 patients who died, 104/158 (66%) surveys were completed by 40 inpatient and 64 outpatient oncologists. Eighty-one oncologists (77.9%) were aware of patients' deaths, 68 (65.4%) anticipated patients' deaths within 6 months, and 67 (64.4%) recalled having GOC discussions before or during the terminal hospitalization. Outpatient oncologists were more likely to report knowledge of patient death (P < .001), as were those with longer therapeutic relationships (P < .001). Inpatient oncologists were more likely to correctly anticipate patient death (P = .014). Secondary outcomes revealed 21.3% of patients had documented GOC discussions before admission and 33.3% had ADs; patients with a longer duration of cancer diagnosis were more likely to have ADs (P = .003). Oncologist-reported barriers to GOC included unrealistic expectations from patients or family (25%) and decreased patient participation because of clinical conditions (15%). CONCLUSION: Most oncologists recalled having GOC discussions for patients with inpatient mortality, yet documentation of serious illness conversations remained suboptimal. Further studies are needed to examine barriers to GOC conversations and documentation during transitions of care and across health care settings.

Results of Leveraging Pharmaceutical Patient Assistance Programs to Expand Access to High Cost Medications in a Student-Run Free Clinic.

High costs make many medications inaccessible to patients in the United States. Uninsured and underinsured patients are disproportionately affected. Pharmaceutical companies offer patient assistance programs (PAPs) to lower the cost-sharing burden of expensive prescription medications for uninsured patients. PAPs are used by various clinics, particularly oncology clinics and those caring for underserved communities, to expand patients' access to medications. Prior studies describing the implementation of PAPs in student-run free clinics have demonstrated cost-savings during the first few years of using PAPs. However, there is a lack of data regarding the efficacy and cost savings of longitudinal use of PAPs across several years. This study describes the growth of PAP use at a student-run free clinic in Nashville, Tennessee over ten years, demonstrating that PAPs can be used reliably and sustainably to expand patients' access to expensive medications. From 2012 to 2021, we increased the number of medications available through PAPs from 8 to 59 and the number of patient enrollments from 20 to 232. In 2021, our PAP enrollments demonstrated potential cost savings of over $1.2 million. Strategies, limitations, and future directions of PAP use are also discussed, highlighting that PAPs can be a powerful tool for free clinics in serving underserved communities.

A single-cell, time-resolved profiling of Xenopus mucociliary epithelium reveals nonhierarchical model of development.

The specialized cell types of the mucociliary epithelium (MCE) lining the respiratory tract enable continuous airway clearing, with its defects leading to chronic respiratory diseases. The molecular mechanisms driving cell fate acquisition and temporal specialization during mucociliary epithelial development remain largely unknown. Here, we profile the developing Xenopus MCE from pluripotent to mature stages by single-cell transcriptomics, identifying multipotent early epithelial progenitors that execute multilineage cues before specializing into late-stage ionocytes and goblet and basal cells. Combining in silico lineage inference, in situ hybridization, and single-cell multiplexed RNA imaging, we capture the initial bifurcation into early epithelial and multiciliated progenitors and chart cell type emergence and fate progression into specialized cell types. Comparative analysis of nine airway atlases reveals an evolutionary conserved transcriptional module in ciliated cells, whereas secretory and basal types execute distinct function-specific programs across vertebrates. We uncover a continuous nonhierarchical model of MCE development alongside a data resource for understanding respiratory biology.

Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors.

Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRASG12C shows increased antitumor activity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.

Sodium-Glucose Cotransporter-2 Inhibitor-associated Euglycemic Diabetic Ketoacidosis: Lessons From a Case Series of 4 Patients Undergoing Coronary Artery Bypass Grafting Surgery.

BACKGROUND: Euglycemic diabetic ketoacidosis (DKA) is a potentially life-threatening adverse condition associated with use of sodium-glucose cotransporter-2 inhibitors (SGLT2i). This risk is further pronounced in the perioperative period. There is no consensus for when SGLT2i should be held preoperatively, and recommendations from various organizations have evolved from 1 day to 3 to 4 days in the latest American Diabetes Association guidelines. Further study of patients with perioperative euglycemic DKA is required to help clarify the optimal timing of preoperative discontinuation of SGLT2i agents. METHODS: In this retrospective, single-centre case series we examined 4 patients who developed postoperative euglycemic DKA after coronary artery bypass grafting, 3 of whom underwent semiurgent surgery. We characterized their clinical course, predisposing factors and treatment characteristics. RESULTS: The SGLT2i were held for 1 to 5 days preoperatively, with times since last dose before surgery being 54, 79, 80 and 151 hours. Surgery was semiurgent for 3 patients, and elective for 1 patient. Three patients were diagnosed with euglycemic DKA within 24 hours after surgery. The fourth patient developed euglycemic DKA on postoperative day 3 in the context of significant hypovolemia and exhibited potential signs of protracted SGLT2i action at 7 days since the last dose. CONCLUSIONS: The duration of SGLT2i action and risk for DKA is variable and complex. Providers should hold SGLT2i at least 3 days before elective major surgery, with potentially longer times in high-risk patients. Careful vigilance should be used for perioperative DKA development in all patients recently exposed to SGLT2i.

Single-cell transcriptome and cell type-specific molecular pathways of human non-alcoholic steatohepatitis.

The aim of this study is to characterize cell type-specific transcriptional signatures in non-alcoholic steatohepatitis (NASH) to improve our understanding of the disease. We performed single-cell RNA sequencing on liver biopsies from 10 patients with NASH. We applied weighted gene co-expression network analysis and validated our findings using a publicly available RNA sequencing data set derived from 160 patients with non-alcoholic fatty liver disease (NAFLD) and 24 controls with normal liver histology. Our study provides a comprehensive single-cell analysis of NASH pathology in humans, describing 19,627 single-cell transcriptomes from biopsy-proven NASH patients. Our data suggest that the previous notion of "NASH-associated macrophages" can be explained by an up-regulation of normally existing subpopulations of liver macrophages. Similarly, we describe two distinct populations of activated hepatic stellate cells, associated with the level of fibrosis. Finally, we find that the expression of several circulating markers of NAFLD are co-regulated in hepatocytes together with predicted effector genes from NAFLD genome-wide association studies (GWAS), coupled to abnormalities in the complement system. In sum, our single-cell transcriptomic data set provides insights into novel cell type-specific and general biological processes associated with inflammation and fibrosis, emphasizing the importance of studying cell type-specific biological processes in human NASH.

Nasal interferon responses to community rhinovirus infections are similar in controls and children with asthma.

BACKGROUND: Rhinovirus (RV) is the main cause of asthma exacerbations in children. Some studies reported that persons with asthma have attenuated interferon (IFN) responses to experimental RV infection compared with healthy individuals. However, responses to community-acquired RV infections in controls and children with asthma have not been compared. OBJECTIVE: To evaluate nasal cytokine responses after natural RV infections in people with asthma and healthy children. METHODS: We compared nasal cytokine expression among controls and children with asthma during healthy, virus-negative surveillance weeks and self-reported RV-positive sick weeks. A total of 14 controls and 21 patients with asthma were studied. Asthma disease severity was based on symptoms and medication use. Viral genome was detected by multiplex polymerase chain reaction. Nasal cytokine protein levels were determined by multiplex assays. RESULTS: Two out of 47 surveillance weeks tested positive for RV, illustrating an asymptomatic infection rate of 5%. A total of 38 of 47 sick weeks (81%) tested positive for the respiratory virus. Of these, 33 (87%) were positive for RV. During well weeks, nasal interleukin 8 (IL-8), IL-12, and IL-1ß levels were higher in children with asthma than controls. Compared with healthy virus-negative surveillance weeks, IL-8, IL-13, and interferon beta increased during colds only in patients with asthma. In both controls and children with asthma, the nasal levels of interferon gamma, interferon lambda-1, IL-1ß, IL-8, and IL-10 increased during RV-positive sick weeks. During RV infection, IL-8, IL-1ß, and tumor necrosis factor-α levels were strongly correlated. CONCLUSION: In both controls and patients with asthma, natural RV infection results in robust type II and III IFN responses.

MK-8719, a Novel and Selective O-GlcNAcase Inhibitor That Reduces the Formation of Pathological Tau and Ameliorates Neurodegeneration in a Mouse Model of Tauopathy.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT: MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

Biophysical and Immunological Characterization and In Vivo Pharmacokinetics and Toxicology in Nonhuman Primates of the Anti-PD-1 Antibody Pembrolizumab.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.

IL-1ß prevents ILC2 expansion, type 2 cytokine secretion, and mucus metaplasia in response to early-life rhinovirus infection in mice.

BACKGROUND: Early-life wheezing-associated respiratory infection with human rhinovirus (RV) is associated with asthma development. RV infection of 6-day-old immature mice causes mucous metaplasia and airway hyperresponsiveness which is associated with the expansion of IL-13-producing type 2 innate lymphoid cells (ILC2s) and dependent on IL-25 and IL-33. We examined regulation of this asthma-like phenotype by IL-1ß. METHODS: Six-day-old wild-type or NRLP3-/- mice were inoculated with sham or RV-A1B. Selected mice were treated with IL-1 receptor antagonist (IL-1RA), anti-IL-1ß, or recombinant IL-1ß. RESULTS: Rhinovirus infection induced Il25, Il33, Il4, Il5, Il13, muc5ac, and gob5 mRNA expression, ILC2 expansion, mucus metaplasia, and airway hyperresponsiveness. RV also induced lung mRNA and protein expression of pro-IL-1ß and NLRP3 as well as cleavage of caspase-1 and pro-IL-1ß, indicating inflammasome priming and activation. Lung macrophages were a major source of IL-1ß. Inhibition of IL-1ß signaling with IL-1RA, anti-IL-1ß, or NLRP3 KO increased RV-induced type 2 cytokine immune responses, ILC2 number, and mucus metaplasia, while decreasing IL-17 mRNA expression. Treatment with IL-1ß had the opposite effect, decreasing IL-25, IL-33, and mucous metaplasia while increasing IL-17 expression. IL-1ß and IL-17 each suppressed Il25, Il33, and muc5ac mRNA expression in cultured airway epithelial cells. Finally, RV-infected 6-day-old mice showed reduced IL-1ß mRNA and protein expression compared to mature mice. CONCLUSION: Macrophage IL-1ß limits type 2 inflammation and mucous metaplasia following RV infection by suppressing epithelial cell innate cytokine expression. Reduced IL-1ß production in immature animals provides a mechanism permitting asthma development after early-life viral infection.

Early Quantification of Systemic Inflammatory Proteins Predicts Long-Term Treatment Response to Tofacitinib and Etanercept.

The application of machine learning to longitudinal gene-expression profiles has demonstrated potential to decrease the assessment gap, between biochemical determination and clinical manifestation, of a patient's response to treatment. Although psoriasis is a proven testing ground for treatment-response prediction using transcriptomic data from clinically accessible skin biopsies, these biopsies are expensive, invasive, and challenging to obtain from certain body areas. Response prediction from blood biochemical measurements could be a cheaper, less invasive predictive platform. Longitudinal profiles for 92 inflammatory and 65 cardiovascular disease proteins were measured from the blood of psoriasis patients at baseline, and 4-weeks, following tofacitinib (janus kinase-signal transducer and activator of transcription-inhibitor) or etanercept (tumor necrosis factor-inhibitor) treatment, and predictive models were developed by applying machine-learning techniques such as bagging and ensembles. This data driven approach developed predictive models able to accurately predict the 12-week clinical endpoint for psoriasis following tofacitinib (area under the receiver operating characteristic curve [auROC] = 78%), or etanercept (auROC = 71%) treatment in a validation dataset, revealing a robust predictive protein signature including well-established psoriasis markers such as IL-17A and IL-17C, highlighting potential for biologically meaningful and clinically useful response predictions using blood protein data. Although most blood classifiers were outperformed by simple models trained using Psoriasis Area Severity Index scores, performance might be enhanced in future studies by measuring a wider variety of proteins.

Multiplex proteomics identifies novel CSF and plasma biomarkers of early Alzheimer's disease.

To date, the development of disease-modifying therapies for Alzheimer's disease (AD) has largely focused on the removal of amyloid beta Aß fragments from the CNS. Proteomic profiling of patient fluids may help identify novel therapeutic targets and biomarkers associated with AD pathology. Here, we applied the Olink™ ProSeek immunoassay to measure 270 CSF and plasma proteins across 415 Aß- negative cognitively normal individuals (Aß- CN), 142 Aß-positive CN (Aß+ CN), 50 Aß- mild cognitive impairment (MCI) patients, 75 Aß+ MCI patients, and 161 Aß+ AD patients from the Swedish BioFINDER study. A validation cohort included 59 Aß- CN, 23 Aß- + CN, 44 Aß- MCI and 53 Aß+ MCI. To compare protein concentrations in patients versus controls, we applied multiple linear regressions adjusting for age, gender, medications, smoking and mean subject-level protein concentration, and corrected findings for false discovery rate (FDR, q < 0.05). We identified, and replicated, altered levels of ten CSF proteins in Aß+ individuals, including CHIT1, SMOC2, MMP-10, LDLR, CD200, EIF4EBP1, ALCAM, RGMB, tPA and STAMBP (- 0.14 < d < 1.16; q < 0.05). We also identified and replicated alterations of six plasma proteins in Aß+ individuals OSM, MMP-9, HAGH, CD200, AXIN1, and uPA (- 0.77 < d < 1.28; q < 0.05). Multiple analytes associated with cognitive performance and cortical thickness (q < 0.05). Plasma biomarkers could distinguish AD dementia (AUC = 0.94, 95% CI = 0.87-0.98) and prodromal AD (AUC = 0.78, 95% CI = 0.68-0.87) from CN. These findings reemphasize the contributions of immune markers, phospholipids, angiogenic proteins and other biomarkers downstream of, and potentially orthogonal to, Aß- and tau in AD, and identify candidate biomarkers for earlier detection of neurodegeneration.

Molecular Profiling of Ulcerative Colitis Subjects from the TURANDOT Trial Reveals Novel Pharmacodynamic/Efficacy Biomarkers.

BACKGROUND AND AIMS: To define pharmacodynamic and efficacy biomarkers in ulcerative colitis [UC] patients treated with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody, in the TURANDOT study. METHODS: Transcriptome, proteome and immunohistochemistry data were generated in peripheral blood and intestinal biopsies from 357 subjects in the TURANDOT study. RESULTS: In peripheral blood, C-C motif chemokine receptor 9 [CCR9] gene expression demonstrated a dose-dependent increase relative to placebo, but in inflamed intestinal biopsies CCR9 gene expression decreased with increasing PF-00547659 dose. Statistical models incorporating the full RNA transcriptome in inflamed intestinal biopsies showed significant ability to assess response and remission status. Oncostatin M [OSM] gene expression in inflamed intestinal biopsies demonstrated significant associations with, and good accuracy for, efficacy, and this observation was confirmed in independent published studies in which UC patients were treated with infliximab or vedolizumab. Compared with the placebo group, intestinal T-regulatory cells demonstrated a significant increase in the intermediate 22.5-mg dose cohort, but not in the 225-mg cohort. CONCLUSIONS: CCR9 and OSM are implicated as novel pharmacodynamic and efficacy biomarkers. These findings occur amid coordinated transcriptional changes that enable the definition of surrogate efficacy biomarkers based on inflamed biopsy or blood transcriptomics data.ClinicalTrials.gov identifierNCT01620255.

Reduction of Inflammatory and Cardiovascular Proteins in the Blood of Patients with Psoriasis: Differential Responses between Tofacitinib and Etanercept after 4 Weeks of Treatment.

Patients with psoriasis have an increased risk of myocardial infarction, and psoriasis is now recognized as an independent risk factor for coronary heart disease and cardiovascular mortality. To understand the effects of psoriasis medications on systemic inflammation associated with cardiovascular risks, we studied blood proteins related to inflammation and cardiovascular disease archived from a phase 3 clinical trial of tofacitinib and etanercept in adults with moderate-to-severe psoriasis. A total of 157 blood proteins were quantified by a proximity extension assay from 266 patients at baseline and week 4. Protein changes in the blood after 1 month of treatment were compared between tofacitinib (10 mg two times a day) and etanercept (50 mg biweekly), and by response status at week 12. Tofacitinib and etanercept commonly reduced IL-6, CCL20, and CXCL10, but IL-17A was significantly reduced only in responders of either treatment. Compared with etanercept, tofacitinib showed a wider spectrum of cardiovascular blood protein reduction, but the protein reduction effects of tofacitinib were strictly confined to treatment responders. Tumor necrosis factor receptor 1, E-selectin, hK11, tumor necrosis factor-related activation-induced cytokine, CHI3L1, IL-16, and matrix metalloproteinase-12 were cardiovascular proteins significantly reduced only in tofacitinib responders. Our data suggest that a short-term systemic psoriasis treatment can cause reductions in circulating inflammatory and other proteins associated with cardiovascular risks.

Cutaneous Findings and Systemic Associations in Women With Polycystic Ovary Syndrome.

IMPORTANCE: Understanding of the associations among cutaneous findings, systemic abnormalities, and fulfillment of the diagnostic criteria in women suspected of having polycystic ovary syndrome (PCOS) is incomplete. OBJECTIVE: To identify cutaneous and systemic features of PCOS that help distinguish women who do and do not meet the diagnostic criteria. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cross-sectional study of a racially diverse referred sample of women seen at the University of California, San Francisco, Polycystic Ovary Syndrome Multidisciplinary Clinic over a 6-year period between May 18, 2006, and October 25, 2012. Participants were 401 women referred for suspected PCOS. In total, 68.8% (276 of 401) met the Rotterdam PCOS diagnostic criteria, while 12.0% (48 of 401) did not. Overall, 11.5% (46 of 401) had insufficient data to render a diagnosis, 1.7% (7 of 401) were excluded from the study, and 6.0% (24 of 401) refused to participate in the study. EXPOSURE: Comprehensive skin examination and transvaginal ultrasonography. All patients were tested for levels of total testosterone, free testosterone, dehydroepiandrosterone (DHEAS), androstenedione, luteinizing hormone, and follicle-stimulating hormone. Levels of serum cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides were obtained, in addition to 0-hour and 2-hour oral glucose tolerance test (OGTT) results, with measurement of glucose and insulin levels. MAIN OUTCOMES AND MEASURES: Findings from comprehensive skin examination, laboratory testing, and transvaginal ultrasonography. RESULTS: In total, 401 women with suspected PCOS were included in the study. The median patient age was 28 years. Compared with women who did not meet the diagnostic criteria for PCOS, women who met the criteria had higher rates of hirsutism (53.3% [144 of 270] vs 31.2% [15 of 48], P = .005) (with higher mean modified Ferriman-Gallwey scores of 8.6 vs 5.6, P = .001), acne (61.2% [164 of 268] vs 40.4% [19 of 47], P = .004), and acanthosis nigricans (AN) (36.9% [89 of 241] vs 20.0% [9 of 45], P = .03). Cutaneous distributions also varied. Women who met the PCOS criteria demonstrated more severe truncal hirsutism and higher rates of axillary AN. Women who met the PCOS criteria had elevated total testosterone levels (40.7% [105 of 258] vs 4.3% [2 of 47], P < .001). Among women with PCOS, the presence of hirsutism (43.9% [54 of 123] vs 30.9% [34 of 110], P = .04) or AN (53.3% [40 of 75] vs 27.0% [40 of 148], P < .001) was associated with higher rates of elevated free testosterone levels as well as several metabolic abnormalities, including insulin resistance, dyslipidemia, and increased body mass index. Although the prevalence of acne was increased among women with PCOS, there were minimal differences in acne types and distribution between the women meeting vs not meeting the PCOS criteria. CONCLUSIONS AND RELEVANCE: Hirsutism and AN are the most reliable cutaneous markers of PCOS and require a comprehensive skin examination to diagnose. When present, hirsutism and AN should raise clinical concern that warrants further diagnostic evaluation for metabolic comorbidities that may lead to long-term complications. Acne and androgenic alopecia are prevalent but unreliable markers of biochemical hyperandrogenism among this population.

Imidazotriazines: Spleen Tyrosine Kinase (Syk) Inhibitors Identified by Free-Energy Perturbation (FEP).

There has been significant interest in spleen tyrosine kinase (Syk) owing to its role in a number of disease states, including autoimmunity, inflammation, and cancer. Ongoing therapeutic programs have resulted in several compounds that are now in clinical use. Herein we report our optimization of the imidazopyrazine core scaffold of Syk inhibitors through the use of empirical and computational approaches. Free-energy perturbation (FEP) methods with MCPRO+ were undertaken to calculate the relative binding free energies for several alternate scaffolds. FEP was first applied retrospectively to determine if there is any predictive value; this resulted in 12 of 13 transformations being predicted in a directionally correct manner. FEP was then applied in a prospective manner to evaluate 17 potential targets, resulting in the realization of imidazotriazine 17 (3-(4-(3,4-dimethoxyphenylamino)imidazo[1,2-f][1,2,4]triazin-2-yl)benzamide), which shows a tenfold improvement in activity relative to the parent compound and no increase in atom count. Optimization of 17 led to compounds with nanomolar cellular activity.

ERG promotes the maintenance of hematopoietic stem cells by restricting their differentiation.

The balance between self-renewal and differentiation is crucial for the maintenance of hematopoietic stem cells (HSCs). Whereas numerous gene regulatory factors have been shown to control HSC self-renewal or drive their differentiation, we have relatively few insights into transcription factors that serve to restrict HSC differentiation. In the present work, we identify ETS (E-twenty-six)-related gene (ERG) as a critical factor protecting HSCs from differentiation. Specifically, loss of Erg accelerates HSC differentiation by >20-fold, thus leading to rapid depletion of immunophenotypic and functional HSCs. Molecularly, we could demonstrate that ERG, in addition to promoting the expression of HSC self-renewal genes, also represses a group of MYC targets, thereby explaining why Erg loss closely mimics Myc overexpression. Consistently, the BET domain inhibitor CPI-203, known to repress Myc expression, confers a partial phenotypic rescue. In summary, ERG plays a critical role in coordinating the balance between self-renewal and differentiation of HSCs.