Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators

<p>Abstract</p> <p>Background</p> <p>Independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. We developed U2transLUC, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method.</p> <p>Results</p> <p>The U2transLUC system is based on a stable cell line expressing a GFP-tagged FOXO transcription factor and a luciferase reporter gene under the control of human FOXO-responsive enhancers. The U2transLUC assay measures nuclear-cytoplasmic FOXO shuttling and FOXO-driven transcription, providing a means to analyze these two key features of FOXO regulation in the same experiment. We challenged the U2transLUC system with chemical probes with known biological activities and we were able to identify compounds with translocation and/or transactivation capacity.</p> <p>Conclusion</p> <p>Combining different biological read-outs in a single cell line offers significant advantages over conventional cell-based assays. The U2transLUC assay facilitates the maintenance and monitoring of homogeneous FOXO transcription factor expression and allows the reporter gene activity measured to be normalized with respect to cell viability. U2transLUC is suitable for high throughput screening and can identify small molecules that interfere with FOXO signaling at different levels.</p

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

Púrpura trombocitopénica trombótica asociada a lupus eritematoso sistémico y artritis reumatoidea. Reporte de un caso

Thrombotic thrombocytopenic purpura (TTP) was first described in the 1970-1980s, it is characterized by an intravascular aggregation process, produced by the deficiency of the metalloproteinase ADAMTS 13. It represents a rare hematological disorder, which has an incidence of 4 cases per million inhabitants per year. Treatment with plasmatic sparing has led to a fundamental change in the clinical course of adult patients with TTP. However, prolonged follow-up has revealed a progressively increasing rate of relapse.La púrpura trombótica trombocitopénica (PTT) fue descrita por primera vez en década de 1970–1980, esta se caracteriza por un proceso de agregación intravascular, producido por la deficiencia de la metaloproteinasa ADAMTS 13. Representa un trastorno hematológico raro, siendo la incidencia de la PTT idiopática de 4 casos por millón de habitantes al año. El tratamiento con recambios plasmáticos ha supuesto un cambio fundamental en el curso clínico de los pacientes adultos con PTT. Sin embargo, el seguimiento prolongado ha revelado una tasa de recaídas progresivamente creciente

A Dual-Color Fluorescence-Based Platform to Identify Selective Inhibitors of Akt Signaling

Background: Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells. Methodology/Principal Findings: In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway. BaFiso relies on the co-culture of isogenic cell lines that have been engineered to sustain interleukin-3 independent survival of the parental Ba/F3 cells, and that are individually tagged with different fluorescent proteins. Whilst in the first of these two lines cell survival in the absence of IL-3 is dependent on the expression of activated Akt, the cells expressing constitutively-activated Stat5 signaling display IL-3 independent growth and survival in an Akt-independent manner. Small molecules can then be screened in these lines to identify inhibitors that rescue IL-3 dependence. Conclusions/Significance: BaFiso measures differential cell survival using multiparametric live cell imaging and permits selective inhibitors of Akt signaling to be identified. BaFiso is a platform technology suitable for the identification of smal

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity

The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management. © 2021, The Author(s)

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P &lt; 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men