Global gene expression of histologically normal primary skin cells from BCNS subjects reveals "single-hit" effects that are influenced by rapamycin

Studies of dominantly heritable cancers enabled insights about tumor progression. BCNS is a dominantly inherited disorder that is characterized by developmental abnormalities and postnatal neoplasms, principally BCCs. We performed an exploratory gene expression profiling of primary cell cultures derived from clinically unaffected skin biopsies of BCNS gene-carriers (PTCH1 +/-) and normal individuals. PCA and HC of untreated keratinocytes or fibroblasts failed to clearly distinguish BCNS samples from controls. These results are presumably due to the common suppression of canonical HH signaling in vitro. We then used a relaxed threshold (p-value <0.05, no FDR cut-off; FC 1.3) that identified a total of 585 and 857 genes differentially expressed in BCNS keratinocytes and fibroblasts samples, respectively. A GSEA identified pancreatic β cell hallmark and mTOR signaling genes in BCNS keratinocytes, whereas analyses of BCNS fibroblasts identified gene signatures regulating pluripotency of stem cells, including WNT pathway. Significantly, rapamycin treatment (FDR<0.05), affected a total of 1411 and 4959 genes in BCNS keratinocytes and BCNS fibroblasts, respectively. In contrast, rapamycin treatment affected a total of 3214 and 4797 genes in normal keratinocytes and normal fibroblasts, respectively. The differential response of BCNS cells to rapamycin involved 599 and 1463 unique probe sets in keratinocytes and fibroblasts, respectively. An IPA of these genes in the presence of rapamycin pointed to hepatic fibrosis/stellate cell activation, and HIPPO signaling in BCNS keratinocytes, whereas mitochondrial dysfunction and AGRN expression were uniquely enriched in BCNS fibroblasts. The gene expression changes seen here are likely involved in the etiology of BCCs and they may represent biomarkers/targets for early intervention

Automated classification of early afterdepolarizations grades in flipr calcium assays

Présentation PosterInternational audienceThe detection and classification of proarrhythmic phenotypes like early afterdepolarizations (EADs) is fundamental in the safety pharmacology assessment of developing drugs. Calcium flux analysis enables sensitive and high throughput in vitro assays to assess potential cardiac liabilities of compounds of interest in cardiomyocytes, generating large amounts of multi-parametric data in a single experiment. However, instrument-agnostic, robust automated data analysis platforms to predict and characterize EADs are not readily available.We developed a new integrated approach able to automatically classify severity grades of EADs observed in high throughput calcium flux data. StemoniX microHeart screening plates promote cardiomyocyte alignment and enable greater resolution over the analysis of pro-arrhythmic phenotypes observed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs were maintained in standard or microengineered microHeart 384-well screening plates for seven days prior to compound treatment and calcium flux analysis. Compound responses were categorized into 7 phenotypes: normal rythmic calcium flux behavior, five severity grades of EADs, and cessation of beating activity (quiescence). Data generated in the microHeart platform was used by Cybernano to develop and validate a statistical learning algorithm in the statistical computing environment R to characterize transient patterns associated with different classes of EADs. Parametric modeling of the beating pattern was associated with a numeric degree of EADs with a value normalized between 0 (no beating) and 1 (no EAD). Finally, an multiclass classification is applied to discriminate the five severity grades of EADs. EAD classifications obtained from standard manual data plotting were compared to classifications obtained with Cybernano algorithms, and concordance between the datasets was analyzed for validation. Robust concordance was observed through this analysis, indicating that the algorithm has satisfactory fidelity to streamline data analysis for automated detection of EADs from high throughput calcium flux analysis in cardiomyocytes.In summary, the comparison of data analysis obtained have demonstrated the proof of concept and very promising perspectives for automatic detection and grading EADs in high throughput calcium flux assays. This comparative study will serve as basis for extending the analysis to a larger library of molecules for additional validation

p53 modulates Hsp90 ATPase activity and regulates aryl hydrocarbon receptor signaling

The aryl hydrocarbon receptor (AhR), a client protein of heat shock protein 90 (Hsp90), is a ligand-activated transcription factor that plays a role in polycyclic aromatic hydrocarbon (PAH)-induced carcinogenesis. Tobacco smoke activates AhR signaling leading to increased transcription of CYP1A1 and CYP1B1, which encode proteins that convert PAHs to mutagens. Recently, p53 was found to regulate Hsp90 ATPase activity via effects on activator of Hsp90 ATPase (Aha1). It is possible, therefore, that AhR-dependent expression of CYP1A1 and CYP1B1 might be affected by p53 status. The main objective of this study was to determine whether p53 modulated AhR-dependent gene expression and PAH metabolism. Here, we show that silencing p53 led to elevated Aha1 levels, increased Hsp90 ATPase activity, and enhanced CYP1A1 and CYP1B1 expression. Overexpression of wild-type p53 suppressed levels of CYP1A1 and CYP1B1. The significance of Aha1 in mediating these p53-dependent effects was determined. Silencing of Aha1 led to reduced Hsp90 ATPase activity and downregulation of CYP1A1 and CYP1B1. In contrast, overexpressing Aha1 was associated with increased Hsp90 ATPase activity and elevated levels of CYP1A1 and CYP1B1. Using p53 heterozygous mutant epithelial cells from patients with Li-Fraumeni syndrome, we show that monoallelic mutation of p53 was associated with elevated levels of CYP1A1 and CYP1B1 under both basal conditions and following treatment with benzo[a]pyrene. Treatment with CP-31398, a p53 rescue compound, suppressed benzo[a]pyrene-mediated induction of CYP1A1 and CYP1B1 and the formation of DNA adducts. Collectively, our results suggest that p53 affects AhR-dependent gene expression, PAH metabolism, and possibly carcinogenesis

The Impact of a 12-Hour Shift Compared to an 8-Hour Shift on Nurse Health and Safety

Abstract Background: An international healthcare concern is extensive shift length among registered nurses and the negative factors associated with nurse burnout. Cognitive impairment, medical errors, sleep deprivation, job dissatisfaction, and overall declined health and safety of nurses have been associated to long shift work hours among registered nurses globally. However, there is limited published evidence about the difference between 8-hour and 12-hour shift work. Objective: This systematic review summarized evidence on the impact that 12-hour shifts compared to 8-hour shifts has on the health and safety of registered nurses. Methods: PubMed, SCOPUS, EBSCOHost, NCBI, Google Scholar, and APHA’s Medical Care databases were searched, covering the period from 2010-2021. Studies were included if their focus concentrated on nurses working 12-hour shifts in comparison to 8-hour shifts. All were of observational design. Results: Upon analysis of 12 peer-reviewed studies, in which data was collected on roughly 36,709 nurses in 8 countries, the findings indicate that shift length is in fact a contributing factor in nurse health and safety internationally. Adverse outcomes that frequently occur with extensive shift length are exhaustion, fatigue, burnout, sleep deprivation/inefficiency, job dissatisfaction, cognitive impairment, and medical errors. These outcomes play a role in diminishing emotional, physical, and mental health and safety of registered nurses. Conclusions: Longer shift lengths for registered nurses is associated with greater health and safety decline for nurses. Policy interventions are needed to resolve the shift hours required of registered nurses

p53 protein regulates Hsp90 ATPase activity and thereby Wnt signaling by modulating Aha1 expression

The p53 tumor suppressor gene encodes a homotetrameric transcription factor which is activated in response to a variety of cellular stressors, including DNA damage and oncogene activation. p53 mutations occur in >50% of human cancers. Although p53 has been shown to regulate Wnt signaling, the underlying mechanisms are not well understood. Here we show that silencing p53 in colon cancer cells led to increased expression of Aha1, a co-chaperone of Hsp90. Heat shock factor-1 was important for mediating the changes in Aha1 levels. Increased Aha1 levels were associated with enhanced interactions with Hsp90, resulting in increased Hsp90 ATPase activity. Moreover, increased Hsp90 ATPase activity resulted in increased phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β), leading to enhanced expression of Wnt target genes. Significantly, levels of Aha1, Hsp90 ATPase activity, Akt, and GSK3β phosphorylation and expression of Wnt target genes were increased in the colons of p53-null as compared with p53 wild type mice. Using p53 heterozygous mutant epithelial cells from Li-Fraumeni syndrome patients, we show that a monoallelic mutation of p53 was sufficient to activate the Aha1/Hsp90 ATPase axis leading to stimulation of Wnt signaling and increased expression of Wnt target genes. Pharmacologic intervention with CP-31398, a p53 rescue agent, inhibited recruitment of Aha1 to Hsp90 and suppressed Wnt-mediated gene expression in colon cancer cells. Taken together, this study provides new insights into the mechanism by which p53 regulates Wnt signaling and raises the intriguing possibility that p53 status may affect the efficacy of anticancer therapies targeting Hsp90 ATPase

Correction: MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease.

[This corrects the article DOI: 10.1371/journal.ppat.1005032.]

Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported