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The coronavirus SARS-CoV-2 is the causative agent of the ongoing severe acute respiratory disease pandemic COVID-19. Tissue and cellular tropism is one key to understanding the pathogenesis of SARS-CoV-2. We investigate the expression and subcellular localization of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), within the upper (nasal) and lower (pulmonary) respiratory tracts of human donors using a diverse panel of banked tissues. Here, we report our discovery that the ACE2 receptor protein robustly localizes within the motile cilia of airway epithelial cells, which likely represents the initial or early subcellular site of SARS-CoV-2 viral entry during host respiratory transmission. We further determine whether ciliary ACE2 expression in the upper airway is influenced by patient demographics, clinical characteristics, comorbidities, or medication use, and show the first mechanistic evidence that the use of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARBs) does not increase susceptibility to SARS-CoV-2 infection through enhancing the expression of ciliary ACE2 receptor. These findings are crucial to our understanding of the transmission of SARS-CoV-2 for prevention and control of this virulent pathogen.

Cyclic-3',5'-adenosine monophosphate (cAMP) is an ancient second messenger but organizing signaling selectivity on the nanoscale is poorly understood. Examining transport of a new fluorescent cAMP probe, Bock and coworkers observe "buffered diffusion" and establish phosphodiesterase activity can organize cAMP nanodomains, while Zhao and coworkers find that protein kinase A regulatory subunits assemble liquid droplets to further localize cAMP signaling., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC., Competing Interests: Declaration of Interests M.O. acknowledges Partnership for New York City for a BioAccelerate Award, funding large-scale synthesis of SMAP-1154. Mt. Sinai has filed patents on behalf of M.O. on SMAP-1154 and SMAP-DT-061. CWRU has filed a patent on behalf of M.O. and G.N. describing combinations of '1154 and '061 with kinase inhibitors. G.N. has an ownership interest in RAPPTA Therapeutics LLC. D.K.N. is a co-founder, shareholder, and scientific adviser for Artris Therapeutics and Frontier Medicines. J. Sage receives research funding from Stemcentrx/Abbvie, Pfizer, and Revolution Medicines and owns stock in Forty Seven Inc., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We investigated the expression and subcellular localization of the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), within the upper (nasal) and lower (pulmonary) respiratory tracts of healthy human donors. We detected ACE2 protein expression within the cilia organelle of ciliated airway epithelial cells, which likely represents the initial or early subcellular site of SARS-CoV-2 viral entry during respiratory transmission. We further determined whether ACE2 expression in the cilia of upper respiratory cells was influenced by patient demographics, clinical characteristics, co-morbidities, or medication use, and found no evidence that the use of angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARBs) increases ACE2 protein expression.

Cancer genomics studies have identified thousands of putative cancer driver genes 1 . Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif 2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

Primary cilia in many cell types contain a periaxonemal subcompartment called the inversin compartment. Four proteins have been found to assemble within the inversin compartment: INVS, ANKS6, NEK8, and NPHP3. The function of the inversin compartment is unknown, but it appears to be critical for normal development, including left-right asymmetry and renal tissue homeostasis. Here we combine superresolution imaging of human RPE1 cells, a classic model for studying primary cilia in vitro, with a genetic dissection of the protein-protein binding relationships that organize compartment assembly to develop a new structural model. We observe that INVS is the core structural determinant of a compartment composed of novel fibril-like substructures, which we identify here by three-dimensional single-molecule superresolution imaging. We find that NEK8 and ANKS6 depend on INVS for localization to these fibrillar assemblies and that ANKS6-NEK8 density within the compartment is regulated by NEK8. Together, NEK8 and ANKS6 are required downstream of INVS to localize and concentrate NPHP3 within the compartment. In the absence of these upstream components, NPHP3 is redistributed within cilia. These results provide a more detailed structure for the inversin compartment and introduce a new example of a membraneless compartment organized by protein-protein interactions.

Adult mesenchymal stem cells, including preadipocytes, possess a cellular sensory organelle called the primary cilium. Ciliated preadipocytes abundantly populate perivascular compartments in fat and are activated by a high-fat diet. Here, we sought to understand whether preadipocytes use their cilia to sense and respond to external cues to remodel white adipose tissue. Abolishing preadipocyte cilia in mice severely impairs white adipose tissue expansion. We discover that TULP3-dependent ciliary localization of the omega-3 fatty acid receptor FFAR4/GPR120 promotes adipogenesis. FFAR4 agonists and ω-3 fatty acids, but not saturated fatty acids, trigger mitosis and adipogenesis by rapidly activating cAMP production inside cilia. Ciliary cAMP activates EPAC signaling, CTCF-dependent chromatin remodeling, and transcriptional activation of PPARγ and CEBPα to initiate adipogenesis. We propose that dietary ω-3 fatty acids selectively drive expansion of adipocyte numbers to produce new fat cells and store saturated fatty acids, enabling homeostasis of healthy fat tissue., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The purpose of this study was to characterize implicit gender bias among residents in US Emergency Medicine and OB/GYN residencies., Methods: We conducted a survey of all allopathic Emergency Medicine and OB/GYN residency programs including questions about leadership as well as an implicit association test (IAT) for unconscious gender bias. We used descriptive statistics to analyze the Likert-type survey responses and used standard IAT analysis methods. We conducted univariate and multivariate analyses to identify factors that were associated with implicit bias. We conducted a subgroup analysis of study sites involved in a multi-site intervention study to determine if responses were different in this group., Results: Overall, 74% of the programs had at least one respondent. Out of 14,234 eligible, 1634 respondents completed the survey (11.5%). Of the five sites enrolled in the intervention study, 244 of 359 eligible residents completed the survey (68%). Male residents had a mean IAT score of 0.31 (SD 0.23) and females 0.14 (SD 0.24), both favoring males in leadership roles and the difference was statistically significant (p < 0.01). IAT scores did not differ by postgraduate year (PGY). Multivariable analysis of IAT score and participant demographics confirmed a significant association between female gender and lower IAT score. Explicit bias favoring males in leadership roles was associated with increased implicit bias favoring males in leadership roles (r = 0.1 p < 0.001)., Conclusions: We found that gender bias is present among US residents favoring men in leadership positions, this bias differs between male and female residents, and is associated with discipline. Implicit bias did not differ across training years, and is associated with explicit bias.

E2F transcription factors are central regulators of cell division and cell fate decisions. E2F4 often represents the predominant E2F activity in cells. E2F4 is a transcriptional repressor implicated in cell cycle arrest and whose repressive activity depends on its interaction with members of the RB family. Here we show that E2F4 is important for the proliferation and the survival of mouse embryonic stem cells. In these cells, E2F4 acts in part as a transcriptional activator that promotes the expression of cell cycle genes. This role for E2F4 is independent of the RB family. Furthermore, E2F4 functionally interacts with chromatin regulators associated with gene activation and we observed decreased histone acetylation at the promoters of cell cycle genes and E2F targets upon loss of E2F4 in RB family-mutant cells. Taken together, our findings uncover a non-canonical role for E2F4 that provide insights into the biology of rapidly dividing cells.

The PBX1 homeodomain transcription factor is converted by t(1;19) chromosomal translocations in acute leukemia into the chimeric E2A-PBX1 oncoprotein. Fusion with E2A confers potent transcriptional activation and constitutive nuclear localization, bypassing the need for dimerization with protein partners that normally stabilize and regulate import of PBX1 into the nucleus, but the mechanisms underlying its oncogenic activation are incompletely defined. We demonstrate here that E2A-PBX1 self-associates through the PBX1 PBC-B domain of the chimeric protein to form higher-order oligomers in t(1;19) human leukemia cells, and that this property is required for oncogenic activity. Structural and functional studies indicate that self-association facilitates the binding of E2A-PBX1 to DNA. Mutants unable to self-associate are transformation defective, however their oncogenic activity is rescued by the synthetic oligomerization domain of FKBP, which confers conditional transformation properties on E2A-PBX1. In contrast to self-association, PBX1 protein domains that mediate interactions with HOX DNA-binding partners are dispensable. These studies suggest that oligomeric self-association may compensate for the inability of monomeric E2A-PBX1 to stably bind DNA and circumvents protein interactions that otherwise modulate PBX1 stability, nuclear localization, DNA binding, and transcriptional activity. The unique dependence on self-association for E2A-PBX1 oncogenic activity suggests potential approaches for mechanism-based targeted therapies.

Opioid analgesics are powerful pain relievers; however, over time, pain control diminishes as analgesic tolerance develops. The molecular mechanisms initiating tolerance have remained unresolved to date. We have previously shown that desensitization of the μ-opioid receptor and interaction with β-arrestins is controlled by carboxyl-terminal phosphorylation. Here we created knockin mice with a series of serine- and threonine-to-alanine mutations that render the receptor increasingly unable to recruit β-arrestins. Desensitization is inhibited in locus coeruleus neurons of mutant mice. Opioid-induced analgesia is strongly enhanced and analgesic tolerance is greatly diminished. Surprisingly, respiratory depression, constipation, and opioid withdrawal signs are unchanged or exacerbated, indicating that β-arrestin recruitment does not contribute to the severity of opioid side effects and, hence, predicting that G-protein-biased µ-agonists are still likely to elicit severe adverse effects. In conclusion, our findings identify carboxyl-terminal multisite phosphorylation as key step that drives acute μ-opioid receptor desensitization and long-term tolerance.

EZH2 is frequently amplified in human melanomas. In this issue of Cancer Cell, Zingg et al. find that EZH2 overexpression silences genes for the primary cilium, causing deciliation, Wnt pathway activation, and progression of Braf V600E - or Nras Q61N -driven melanomas, thus defining a tumor-suppressor role for cilia in cancer., (Copyright © 2018 Elsevier Inc. All rights reserved.)

GTPases are molecular switches that cycle between the inactive GDP-bound state and the active GTP-bound state. GTPases exchange nucleotides either by its intrinsic nucleotide exchange or by interaction with guanine nucleotide exchange factors (GEFs). Monitoring the nucleotide exchange in vitro , together with reconstitution of direct interactions with regulatory proteins, provides key insights into how a GTPase is activated. In this protocol, we describe core methods to monitor nucleotide exchange using fluorescent N-Methylanthraniloyl (MANT)-guanine nucleotide.

The lateral ventricle subventricular zone (SVZ) is a frequent and consequential site of pediatric and adult glioma spread, but the cellular and molecular mechanisms mediating this are poorly understood. We demonstrate that neural precursor cell (NPC):glioma cell communication underpins this propensity of glioma to colonize the SVZ through secretion of chemoattractant signals toward which glioma cells home. Biochemical, proteomic, and functional analyses of SVZ NPC-secreted factors revealed the neurite outgrowth-promoting factor pleiotrophin, along with required binding partners SPARC/SPARCL1 and HSP90B, as key mediators of this chemoattractant effect. Pleiotrophin expression is strongly enriched in the SVZ, and pleiotrophin knock down starkly reduced glioma invasion of the SVZ in the murine brain. Pleiotrophin, in complex with the binding partners, activated glioma Rho/ROCK signaling, and ROCK inhibition decreased invasion toward SVZ NPC-secreted factors. These findings demonstrate a pathogenic role for NPC:glioma interactions and potential therapeutic targets to limit glioma invasion. PAPERCLIP., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Highly conserved intraflagellar transport (IFT) protein complexes direct both the assembly of primary cilia and the trafficking of signaling molecules. IFT complexes initially accumulate at the base of the cilium and periodically enter the cilium, suggesting an as-yet-unidentified mechanism that triggers ciliary entry of IFT complexes. Using affinity-purification and mass spectrometry of interactors of the centrosomal and ciliopathy protein, CEP19, we identify CEP350, FOP, and the RABL2B GTPase as proteins organizing the first known mechanism directing ciliary entry of IFT complexes. We discover that CEP19 is recruited to the ciliary base by the centriolar CEP350/FOP complex and then specifically captures GTP-bound RABL2B, which is activated via its intrinsic nucleotide exchange. Activated RABL2B then captures and releases its single effector, the intraflagellar transport B holocomplex, from the large pool of pre-docked IFT-B complexes, and thus initiates ciliary entry of IFT., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dendritic release of dopamine activates dopamine D2 autoreceptors, which are inhibitory G protein-coupled receptors (GPCRs), to decrease the excitability of dopamine neurons. This study used tagged D2 receptors to identify the localization and distribution of these receptors in living midbrain dopamine neurons. GFP-tagged D2 receptors were found to be unevenly clustered on the soma and dendrites of dopamine neurons within the substantia nigra pars compacta (SNc). Physiological signaling and desensitization of the tagged receptors were not different from wild type receptors. Unexpectedly, upon desensitization the tagged D2 receptors were not internalized. When tagged D2 receptors were expressed in locus coeruleus neurons, a desensitizing protocol induced significant internalization. Likewise, when tagged µ-opioid receptors were expressed in dopamine neurons they too were internalized. The distribution and lack of agonist-induced internalization of D2 receptors on dopamine neurons indicate a purposefully regulated localization of these receptors.

Despite the wide administration of several effective vaccines, rotavirus (RV) remains the single most important etiological agent of severe diarrhea in infants and young children worldwide, with an annual mortality of over 200,000 people. RV attachment and internalization into target cells is mediated by its outer capsid protein VP4. To better understand the molecular details of RV entry, we performed tandem affinity purification coupled with high-resolution mass spectrometry to map the host proteins that interact with VP4. We identified an actin-binding protein, drebrin (DBN1), that coprecipitates and colocalizes with VP4 during RV infection. Importantly, blocking DBN1 function by siRNA silencing, CRISPR knockout (KO), or chemical inhibition significantly increased host cell susceptibility to RV infection. Dbn1 KO mice exhibited higher incidence of diarrhea and more viral antigen shedding in their stool samples compared with the wild-type littermates. In addition, we found that uptake of other dynamin-dependent cargos, including transferrin, cholera toxin, and multiple viruses, was also enhanced in DBN1-deficient cells. Inhibition of cortactin or dynamin-2 abrogated the increased virus entry observed in DBN1-deficient cells, suggesting that DBN1 suppresses dynamin-mediated endocytosis via interaction with cortactin. Our study unveiled an unexpected role of DBN1 in restricting the entry of RV and other viruses into host cells and more broadly to function as a crucial negative regulator of diverse dynamin-dependent endocytic pathways., Competing Interests: The authors declare no conflict of interest.

Introduction: Sponge cytology is a novel screening tool for esophageal cancer but has been unable to be validated for widespread use. Our aim was to apply fluorescent in situ hybridization to sponge cytology samples in order to evaluate the safety and efficacy of this modality in screening for esophageal cancer., Materials and Methods: At a single, multidisciplinary, NCI-designated cancer center, patients completed sponge cytology sampling prior to upper endoscopy. Samples were analyzed by p53 fluorescent in situ hybridization, and results were compared to the endoscopic diagnosis., Results: Fifty patients were enrolled (96 % Caucasian, 68 % male, median age of 67). All patients successfully swallowed the capsule. No complications (string breakage, bleeding, mucosal injury) occurred. Endoscopy revealed that 38 % had normal esophageal mucosa and 62 % had an esophageal mucosal abnormality. In total, six samples demonstrated p53 loss (94 % specificity for any abnormality). The sensitivity of the p53 fluorescent in situ hybridization probe was13.3 % for any abnormality, 10 % for intestinal metaplasia, and 0 % for dysplasia or esophageal cancer., Discussion: Esophageal sponge cytology is a promising, safe, and tolerable method for collecting esophageal cell samples. However, our data suggest that p53 fluorescent in situ hybridization does not improve the sensitivity for detecting cancer in these samples.

Rotaviruses (RVs) are the leading cause of severe gastroenteritis in young children, accounting for half a million deaths annually worldwide. RV encodes non-structural protein 1 (NSP1), a well-characterized interferon (IFN) antagonist, which facilitates virus replication by mediating the degradation of host antiviral factors including IRF3 and β-TrCP. Here, we utilized six human and animal RV NSP1s as baits and performed tandem-affinity purification coupled with high-resolution mass spectrometry to comprehensively characterize NSP1-host protein interaction network. Multiple Cullin-RING ubiquitin ligase (CRL) complexes were identified. Importantly, inhibition of cullin-3 (Cul3) or RING-box protein 1 (Rbx1), by siRNA silencing or chemical perturbation, significantly impairs strain-specific NSP1-mediated β-TrCP degradation. Mechanistically, we demonstrate that NSP1 localizes to the Golgi with the host Cul3-Rbx1 CRL complex, which targets β-TrCP and NSP1 for co-destruction at the proteasome. Our study uncovers a novel mechanism that RV employs to promote β-TrCP turnover and provides molecular insights into virus-mediated innate immunity inhibition., Competing Interests: The authors have declared that no competing interests exist.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Emerging evidence indicates that methamphetamine (MA) abuse can impact cardiovascular disease. In humans, MA abuse is associated with an increased risk of stroke as well as an earlier age at which the stroke occurs. However, little is known about how chronic daily MA exposure can impact ischemic outcome in either humans or animal models. In the present study, mice were injected with MA (10 mg/kg, i.p.) or saline once daily for 10 consecutive days. Twenty-four hours after the final injection, mice were subjected to transient middle cerebral artery occlusion (tMCAO) for one hour followed by reperfusion. Mice were tested for novel object memory at 96 h post-reperfusion, just prior to removal of brains for quantification of infarct volume using 2,3,5-Triphenyltetrazolium Chloride (TTC) staining. Mice treated with MA prior to tMCAO showed decreased object memory recognition and increased infarct volume compared to saline-treated mice. These findings indicate that chronic MA exposure can worsen both cognitive and morphological outcomes following cerebral ischemia.

Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. Ciliopathies are a spectrum of human diseases resulting from defects in cilia structure or function. The mechanisms regulating the assembly of ciliary multiprotein complexes and the transport of these complexes to the base of cilia remain largely unknown. Combining proteomics, in vivo imaging and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy and Wdpcp), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector), and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy-associated protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with ciliopathies in human patients.

Background: Although tumor length has received little attention for staging of esophageal cancer, it may be a valid prognostic feature for node positivity and survival., Methods: Through retrospective review of a prospective institutional database, esophageal cancer patients who completed esophagectomy without neoadjuvant chemoradiation were analyzed. Pathologic tumor lengths were compared with node positivity and survival through a zero-inflated negative binomial regression model and multivariable Cox proportional hazards model, respectively., Results: Between January 2000 and July 2015, 98 patients met inclusion, criteria (84% male, median age of 65, 90% adenocarcinoma). Median tumor length was 2.5 cm with each 1-cm increase in length increasing the odds of node positivity (odds ratio 3.55, 95% confidence interval 1.50 to 8.40, P = .004) and decreasing overall survival (hazards ratio 1.18, 95% confidence interval 1.06 to 1.32, P < .003)., Conclusion: This study suggests an association among tumor length, lymph node metastasis, as well as overall survival in esophageal cancer patients who have not received neoadjuvant chemoradiotherapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The mysteriously diverse phenotypes in mice lacking the p53 homolog p73 are recently unified by new analysis showing p73 is required for formation of multiciliated epithelia. p73 directly activates FoxJ1, the central transcriptional driver for multiciliation, and induces a host of genes critical for ciliogenesis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The primary cilium is an antenna-like cellular protrusion mediating sensory and neuroendocrine signaling. Its localization within tissue architecture and a growing list of cilia-localized receptors, in particular G-protein-coupled receptors, determine a host of crucial physiologies, which are disrupted in human ciliopathies. Here, we discuss recent advances in the identification and characterization of ciliary signaling components and pathways. Recent studies have highlighted the unique signaling environment of the primary cilium and we are just beginning to understand how this design allows for highly amplified and regulated signaling., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves β-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161-β-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment., (© 2016 Pal et al.)

Background: Surgical resection is the standard of care for unilateral adrenal adenomas and hyperplasia resulting in primary aldosteronism (PA). Resolution of PA following surgery is variable and some patients continue to require some or all of their antihypertensives. Prior studies have investigated factors contributing to "cure" of PA (defined as no hypertension [HTN] medications required postoperatively). These models are a tool in patient selection, yet fail to consider the benefit of some reduction in medications, resolution of hypokalemia, or reduction in blood pressure which may improve long-term cardiovascular and renal outcomes. We sought to investigate factors contributing to postoperative improvement or complete resolution following surgery., Methods: This is a retrospective review of prospectively collected data on 58 adrenalectomies performed for PA from December 1999 to April 2013 at a single center. Patient demographics, PA characteristics, labs, and imaging studies were evaluated, as well as operative characteristics. Mean systolic and diastolic blood pressures were calculated over several visits after discharge, and postoperative antihypertensive regimen was recorded. Patients were stratified by cured vs not cured and then again by improved vs not improved based on differences in pre- and postoperative values. Aldosteronoma Resolution Score was also calculated for each patient., Results: Median age was 52.6 years, with 44.8% women and an average duration of HTN of 13.5 years. Average body mass index (BMI) was 31.5 kg/m(2); 74% of the lesions were adenomas. Patients with complications had higher BMIs than those without (36.9 vs 28.7 kg/m(2), P = .02). In comparing improved (n = 42, 72%) vs not improved (n = 16, 28%) patients, preoperative systolic blood pressure (147.5 vs 159.7 mm Hg, P = .047) and serum creatinine (.94 vs 1.32 mg/dL, P = .016) were higher in the not improved group. Cured (n = 13) vs not cured (n = 45) patients differed in terms of BMI (27.4 vs 32.7, P = .009), duration of HTN (9.1 vs 14.9 years, P = .020), and number of preoperative antihypertensives (2.1 vs 3.7, P = .002). Aldosteronoma Resolution Score was significantly higher in cured patients (3.2 vs 1.0, P < .01)., Conclusions: A significant number of patients who are not cured by adrenalectomy for PA will still benefit from surgery. Obesity, duration of HTN, and number of medications may predict cure, yet fail to detect a population of patients with overall improvement. Attention to serum creatinine may help in distinguishing this population of patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Uveitis is a common complication of spondyloarthritis. The "phenotype" of the uveitis characteristic of ankylosing spondylitis (sudden onset, anterior, unilateral, recurrent, more often male) may differ from the phenotype often seen with either psoriatic arthritis or inflammatory bowel disease (insidious onset, anterior and intermediate, bilateral, chronic, and/or more often female). The frequency of uveitis is also much greater in association with ankylosing spondylitis than with either inflammatory bowel disease or psoriasis. Uveitis may affect the choice of therapy and can rarely be a complication of therapy. Uveitis and arthritis also co-exist in several animal models.

Diagnostic codes used in healthcare administration have been employed extensively in clinical research to identify target patient populations, including demonstration of important clinical outcomes among adults with congenital heart disease. However, little is known about the reliability of code-derived data in this context. We sought to determine the accuracy of International Classification of Disease-9th Revision (ICD-9) diagnoses and the reliability of retrieval algorithms in adults with congenital heart disease (ACHD). Pilot testing of a hierarchical algorithm to identify ACHD patients and determine their principle congenital diagnosis was performed. A revised algorithm was then applied retrospectively to a sample of all outpatients seen by providers who see general cardiology and ACHD patients. Using all ICD-9 codes available from any encounter, accuracy for detection and categorization of sub-types were compared to physician chart review. After initial testing on 334 patients, the revised algorithm was applied to 740 patients. The sensitivity and specificity for ACHD patient identification from this specialty clinic population were 99 and 88 %, respectively. Of 411 (56 %) non-ACHD patients, 49 were incorrectly categorized as ACHD by the algorithm. Of ACHD patients, 326 of 329 were correctly identified by diagnostic codes and categorization of ACHD defect sub-type was correct in 263 (80 %). Administrative data can be used for identification of ACHD patients based on ICD-9 codes with excellent sensitivity and reasonable specificity. Accurate categorization that would be utilized for quality indicators by ACHD defect type is less robust. Additional testing should be done using non-referral populations.

Short-rib polydactyly syndromes (SRPS) arise from mutations in genes involved in retrograde intraflagellar transport (IFT) and basal body homeostasis, which are critical for cilia assembly and function. Recently, mutations in WDR34 or WDR60 (candidate dynein intermediate chains) were identified in SRPS. We have identified and characterized Tctex1d2, which associates with Wdr34, Wdr60 and other dynein complex 1 and 2 subunits. Tctex1d2 and Wdr60 localize to the base of the cilium and their depletion causes defects in ciliogenesis. We propose that Tctex1d2 is a novel dynein light chain important for trafficking to the cilium and potentially retrograde IFT and is a new molecular link to understanding SRPS pathology.

Background: Age-related macular degeneration (AMD) is the leading cause of central vision loss in older adults. Anti-retinal autoantibodies (AAbs) have been found in individuals with AMD. The goal of the study was to determine the AAb specificity in different stages of AMD, and determine whether there is a prevalent AAb signature., Methods: Sera of 134 participants in the Age-related Eye Disease Study were analyzed for anti-retinal AAbs by western blotting. The subjects were classified by diagnostic subgroups based upon their clinical classification: No AMD, Intermediate AMD, and Late AMD - geographic atrophy (GA) and Late AMD - neovascular (NV)., Results: The presence of anti-retinal AAb was detected in 58% patients with Intermediate and Late AMD, and 54% of those with no AMD. AAbs bound to fifteen different retinal antigens. Most individuals had 1 specific AAbs (67%), with the remainder having 2 to 4 different AAbs. Over 40% of patients with Intermediate AMD, and 46% of those with GA had anti-enolase AAbs, compared with 29% of individuals with NV and 29% with no AMD. Different AAbs signatures related to NV as compared to GA and/or Intermediate AMD were distinguished. Anti-40-kDa (10%) and 42-kDa (16%) autoantibodies were associated with Intermediate AMD, while anti-30-kDa AAbs (23%) were primarily present in GA. Anti-32-kDa (12%), 35-kDa (21%), and 60-kDa (8%) AAbs were more frequent in NV AMD., Conclusions: A unique AAb pattern for each of the disease subgroups was present when AMD progressed from the intermediate to the late forms of severity. Differences in the frequency of specific AAbs between AMD subgroups suggested that they may participate in pathogenicity of AMD. Further studies are necessary to confirm these observations in the larger cohort and individual AMD patients over time.

We have developed a novel 3D cell culture model that uses mouse inner-medullary collecting duct (mIMCD3) cells to generate epithelial spheroids. This model is amenable to efficient siRNA knockdown and subsequent rescue with human patient-derived alleles. Spheroids develop apicobasal polarity and complete lumens, and they are consequently an ideal model for polarity defects seen in renal ciliopathies such as nephronophthisis. Briefly, mIMCD3 cells are transfected and subsequently passaged to a Matrigel mixture, which is seeded in chamber slides and covered in growth medium. Once the spheroids are formed, Matrigel is dissolved and immunocytochemistry is performed in the chamber slides. The technique is amenable to semiautomatic imaging analysis, and it can test multiple genes simultaneously, gene-dosing effects and a variety of therapeutic interventions. The spheroid technique is a unique and simple 6-d in vitro method of interrogating ex vivo tissue organization.

In this issue, two studies from Yue Xiong and colleagues (Li et al., 2014; Yan et al., 2014) show that CUL7, OBSL1, and CCDC8, all mutated in 3M short stature syndrome, form a centrosomal complex that regulates CUL9 and its substrate survivin to link mitosis to cell survival., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Early brain injury (EBI) during the first 72 h after subarachnoid hemorrhage (SAH) is an important determinant of clinical outcome. A hallmark of EBI, global cerebral ischemia, occurs within seconds of SAH and is thought to be related to increased intracranial pressure (ICP). We tested the hypothesis that ICP elevation and cortical hypoperfusion are the result of physical blockade of cerebrospinal fluid (CSF) flow pathways by cisternal microthrombi. In mice subjected to SAH, we measured cortical blood volume (CBV) using optical imaging, ICP using pressure transducers, and patency of CSF flow pathways using intracisternally injected tracer dye. We then assessed the effects of intracisternal injection of recombinant tissue plasminogen activator (tPA). ICP rose immediately after SAH and remained elevated for 24 h. This was accompanied by a decrease in CBV and impaired dye movement. Intracisternal administration of tPA immediately after SAH lowered ICP, increased CBV, and partially restored CSF flow at 24 h after SAH. Lowering ICP without tPA, by draining CSF, improved CBV at 1 h, but not 24 h after SAH. These findings suggest that blockade of CSF flow by microthrombi contributes to the early decline in cortical perfusion in an ICP-dependent and ICP-independent manner and that intracisternal tPA may reduce EBI and improve outcome after SAH.

Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the << molar tooth sign >>. JS is genetically heterogeneous, involving 20 genes identified to date, which are all required for cilia biogenesis and/or function. In a consanguineous family with JS associated with optic nerve coloboma, kidney hypoplasia, and polydactyly, combined exome sequencing and mapping identified a homozygous splice-site mutation in PDE6D, encoding a prenyl-binding protein. We found that pde6d depletion in zebrafish leads to renal and retinal developmental anomalies and wild-type but not mutant PDE6D is able to rescue this phenotype. Proteomic analysis identified INPP5E, whose mutations also lead to JS or mental retardation, obesity, congenital retinal dystrophy, and micropenis syndromes, as novel prenyl-dependent cargo of PDE6D. Mutant PDE6D shows reduced binding to INPP5E, which fails to localize to primary cilia in patient fibroblasts and tissues. Furthermore, mutant PDE6D is unable to bind to GTP-bound ARL3, which acts as a cargo-release factor for PDE6D-bound INPP5E. Altogether, these results indicate that PDE6D is required for INPP5E ciliary targeting and suggest a broader role for PDE6D in targeting other prenylated proteins to the cilia. This study identifies PDE6D as a novel JS disease gene and provides the first evidence of prenyl-binding-dependent trafficking in ciliopathies., (© 2013 WILEY PERIODICALS, INC.)

Activation of Checkpoint kinase 1 (Chk1) following DNA damage mediates cell cycle arrest to prevent cells with damaged DNA from entering mitosis. Here we provide a high-resolution analysis of cells as they undergo S- and G₂-checkpoint bypass in response to Chk1 inhibition with the selective Chk1 inhibitor GNE-783. Within 4-8 h of Chk1 inhibition following gemcitabine induced DNA damage, cells with both sub-4N and 4N DNA content prematurely enter mitosis. Coincident with premature transition into mitosis, levels of DNA damage dramatically increase and chromosomes condense and attempt to align along the metaphase plate. Despite an attempt to congress at the metaphase plate, chromosomes rapidly fragment and lose connection to the spindle microtubules. Gemcitabine mediated DNA damage promotes the formation of Rad51 foci; however, while Chk1 inhibition does not disrupt Rad51 foci that are formed in response to gemcitabine, these foci are lost as cells progress into mitosis. Premature entry into mitosis requires the Aurora, Cdk1/2 and Plk1 kinases and even though caspase-2 and -3 are activated upon mitotic exit, they are not required for cell death. Interestingly, p53, but not p21, deficiency enables checkpoint bypass and chemo-potentiation. Finally, we uncover a differential role for the Wee-1 checkpoint kinase in response to DNA damage, as Wee-1, but not Chk1, plays a more prominent role in the maintenance of S- and G₂-checkpoints in p53 proficient cells.

Human monogenic obesity syndromes, including Bardet-Biedl syndrome (BBS), implicate neuronal primary cilia in regulation of energy homeostasis. Cilia in hypothalamic neurons have been hypothesized to sense and regulate systemic energy status, but the molecular mechanism of this signaling remains unknown. Here, we report a comprehensive localization screen of 42 G-protein-coupled receptors (GPCR) revealing seven ciliary GPCRs, including the neuropeptide Y (NPY) receptors NPY2R and NPY5R. We show that mice modeling BBS disease or obese tubby mice fail to localize NPY2R to cilia in the hypothalamus and that BBS mutant mice fail to activate c-fos or decrease food intake in response to the NPY2R ligand PYY3-36. We find that cells with ciliary NPY2R show augmented PYY3-36-dependent cAMP signaling. Our data demonstrate that ciliary targeting of NPY receptors is important for controlling energy balance in mammals, revealing a physiologically defined ligand-receptor pathway signaling within neuronal cilia., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Here we report that GNE-783, a novel checkpoint kinase-1 (CHK1) inhibitor, enhances the activity of gemcitabine by disabling the S- and G2 cell-cycle checkpoints following DNA damage. Using a focused library of 51 DNA-damaging agents, we undertook a systematic screen using three different cell lines to determine which chemotherapeutics have their activity enhanced when combined with GNE-783. We found that GNE-783 was most effective at enhancing activity of antimetabolite-based DNA-damaging agents; however, there was a surprisingly wide range of activity within each class of agents. We, next, selected six different therapeutic agents and screened these in combination with GNE-783 across a panel of cell lines. This revealed a preference for enhanced chemopotentiation of select agents within tumor types, as, for instance, GNE-783 preferentially enhanced the activity of temozolomide only in melanoma cell lines. Additionally, although p53 mutant status was important for the overall response to combinations with some agents; our data indicate that this alone was insufficient to predict synergy. We finally compared the ability of a structurally related CHK1 inhibitor, GNE-900, to enhance the in vivo activity of gemcitabine, CPT-11, and temozolomide in xenograft models. GNE-900 significantly enhanced activity of only gemcitabine in vivo, suggesting that strong chemopotentiation in vitro can translate into chemopotentiation in vivo. In conclusion, our results show that selection of an appropriate agent to combine with a CHK1 inhibitor needs to be carefully evaluated in the context of the genetic background and tumor type in which it will be used., (©2013 AACR.)

Nicotinamide adenine dinucleotide (NAD) is a critical metabolite that is required for a range of cellular reactions. A key enzyme in the NAD salvage pathway is nicotinamide phosphoribosyl transferase (NAMPT), and here, we describe GNE-618, an NAMPT inhibitor that depletes NAD and induces cell death in vitro and in vivo. While cells proficient for nicotinic acid phosphoribosyl transferase (NAPRT1) can be protected from NAMPT inhibition as they convert nicotinic acid (NA) to NAD independent of the salvage pathway, this protection only occurs if NA is added before NAD depletion. We also demonstrate that tumor cells are unable to generate NAD by de novo synthesis as they lack expression of key enzymes in this pathway, thus providing a mechanistic rationale for the reliance of tumor cells on the NAD salvage pathway. Identifying tumors that are sensitive to NAMPT inhibition is one potential way to enhance the therapeutic effectiveness of an NAMPT inhibitor, and here, we show that NAMPT, but not NAPRT1, mRNA and protein levels inversely correlate with sensitivity to GNE-618 across a panel of 53 non-small cell lung carcinoma cell lines. Finally, we demonstrate that GNE-618 reduced tumor growth in a patient-derived model, which is thought to more closely represent heterogeneous primary patient tumors. Thus, we show that dependence of tumor cells on the NAD salvage pathway renders them sensitive to GNE-618 in vitro and in vivo, and our data support further evaluation of the use of NAMPT mRNA and protein levels as predictors of overall sensitivity.

Checkpoint kinase 1 (ChK1) is a serine/threonine kinase that functions as a central mediator of the intra-S and G2-M cell-cycle checkpoints. Following DNA damage or replication stress, ChK1-mediated phosphorylation of downstream effectors delays cell-cycle progression so that the damaged genome can be repaired. As a therapeutic strategy, inhibition of ChK1 should potentiate the antitumor effect of chemotherapeutic agents by inactivating the postreplication checkpoint, causing premature entry into mitosis with damaged DNA resulting in mitotic catastrophe. Here, we describe the characterization of GNE-900, an ATP-competitive, selective, and orally bioavailable ChK1 inhibitor. In combination with chemotherapeutic agents, GNE-900 sustains ATR/ATM signaling, enhances DNA damage, and induces apoptotic cell death. The kinetics of checkpoint abrogation seems to be more rapid in p53-mutant cells, resulting in premature mitotic entry and/or accelerated cell death. Importantly, we show that GNE-900 has little single-agent activity in the absence of chemotherapy and does not grossly potentiate the cytotoxicity of gemcitabine in normal bone marrow cells. In vivo scheduling studies show that optimal administration of the ChK1 inhibitor requires a defined lag between gemcitabine and GNE-900 administration. On the refined combination treatment schedule, gemcitabine's antitumor activity against chemotolerant xenografts is significantly enhanced and dose-dependent exacerbation of DNA damage correlates with extent of tumor growth inhibition. In summary, we show that in vivo potentiation of gemcitabine activity is mechanism based, with optimal efficacy observed when S-phase arrest and release is followed by checkpoint abrogation with a ChK1 inhibitor., (©2013 AACR.)

Background: Autoimmune retinopathy (AR) and Cancer-Associated Retinopathy (CAR) are associated with a diverse repertoire of anti-retinal autoantibodies (AAbs) but not all antigenic targets have been characterized. Identification of new AAbs may help with clinical diagnosis and prognosis of retinal dysfunction in AR. The goal was to identify frequently targeted retinal autoantigens within the 60-70-kDa molecular weight range., Methods: Human retinal proteins were separated by SDS-PAGE and 2D gel electrophoresis (2-DE) and sera from AR patients with and without cancer were used to identify immunoreactive proteins by Western blotting. Proteins were identified following separation by electrophoresis, Coomassie staining using in-gel trypsin digestion and mass spectrometric analysis. Circulating serum hsp60 and anti-hsp60 antibody levels were determined by quantitative ELISA., Results: Retrospective evaluation of 819 patients with anti-retinal AAbs showed that 29% patients had AAbs targeted proteins between 60-70-kDa. Shotgun mass spectrometry of human retinal proteins present in 1D-gel found 66 species within this range. To identify the immunoreactive proteins, we performed Western blots of 2-DE gels and showed a group of heat shock proteins (hsps), including hsp60 and CRMP proteins that were frequently recognized by AR patient AAbs, irrespective of cancer status. These results were validated by immunostaining of purified hsp60 and CRMP2 proteins. ELISA results revealed that patients with AR and CAR had significantly increased levels of serum anti-hsp60 antibodies compared to control healthy subjects (p < 0.0001). However, circulating hsp60 protein was not significantly elevated in sera of either patient group., Conclusions: Different anti-retinal antibodies frequently co-exist in a single patient, creating antibody-arrays related to the syndrome. Hsps and CRMP-2 are newly identified autoantigens in AR. A frequent co-association of anti-hsp antibodies with other anti-retinal AAbs may augment pathogenic processes, leading to retinal degeneration.

VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.

In this issue, Choi et al. (2013) discover a molecular link between the Nek8 kinase, mutated in the renal ciliopathy nephronophthisis, and DNA damage control by cyclin A/Cdk2 and ATR-Chk1, providing new ideas for targeted therapies limiting tissue degeneration., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The Institute of Medicine's report, To Err is Human, concluded that "medical errors are not a result of isolated individual actions but rather faulty systems, processes, and conditions that lead people to make mistakes." In situ simulation offers the unique opportunity to train the teams of people who deliver healthcare while enhancing policies, evaluating new technologies, and improving the systems that support the delivery of safe healthcare. For this reason, the Institute of Medicine, the Joint Commission, and the Agency for Healthcare Research and Quality recommend medical simulation as one of the most important safe practice interventions to reduce errors and risks associated with the process of care. This review builds on other reports in this issue and discusses the application of in situ simulation to identify, address, and test systems improvements., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Activation of a cellular senescence program is a common response to prolonged oncogene activation or tumor suppressor loss, providing a physiological mechanism for tumor suppression in premalignant cells. The link between senescence and tumor suppression supports the hypothesis that a loss-of-function screen measuring bona fide senescence marker activation should identify candidate tumor suppressors. Using a high-content siRNA screening assay for cell morphology and proliferation measures, we identify 12 senescence-regulating kinases and determine their senescence marker signatures, including elevation of senescence-associated β-galactosidase, DNA damage and p53 or p16 (INK4a) expression. Consistent with our hypothesis, SNP array CGH data supports loss of gene copy number of five senescence-suppressing genes across multiple tumor samples. One such candidate is the EPHA3 receptor tyrosine kinase, a gene commonly mutated in human cancer. We demonstrate that selected intracellular EPHA3 tumor-associated point mutations decrease receptor expression level and/or receptor tyrosine kinase (RTK) activity. Our study therefore describes a new strategy to mine for novel candidate tumor suppressors and provides compelling evidence that EPHA3 mutations may promote tumorigenesis only when key senescence-inducing pathways have been inactivated.

Valosine containing protein (VCP), also known as p97, is a member of AAA ATPase family that is involved in several biological processes and plays a central role in the ubiquitin-mediated degradation of misfolded proteins. VCP is an ubiquitously expressed, highly abundant protein and has been found overexpressed in many tumor types, sometimes associated with poor prognosis. In this respect, VCP has recently received a great deal of attention as a potential new target for cancer therapy. In this paper, the discovery and structure-activity relationships of alkylsulfanyl-1,2,4-triazoles, a new class of potent, allosteric VCP inhibitors, are described. Medicinal chemistry manipulation of compound 1, identified via HTS, led to the discovery of potent and selective inhibitors with submicromolar activity in cells and clear mechanism of action at consistent doses. This represents a first step toward a new class of potential anticancer agents.