Identification of glucocorticoid-related molecular signature by whole blood methylome analysis

Objective Cushing's syndrome represents a state of excessive glucocorticoids related to glucocorticoid treatments or to endogenous hypercortisolism. Cushing's syndrome is associated with high morbidity, with significant inter-individual variability. Likewise, adrenal insufficiency is a life-threatening condition of cortisol deprivation. Currently, hormone assays contribute to identify Cushing's syndrome or adrenal insufficiency. However, no biomarker directly quantifies the biological glucocorticoid action. The aim of this study was to identify such markers. Design We evaluated whole blood DNA methylome in 94 samples obtained from patients with different glucocorticoid states (Cushing's syndrome, eucortisolism, adrenal insufficiency). We used an independent cohort of 91 samples for validation. Methods Leukocyte DNA was obtained from whole blood samples. Methylome was determined using the Illumina methylation chip array (~850 000 CpG sites). Both unsupervised (principal component analysis) and supervised (Limma) methods were used to explore methylome profiles. A Lasso-penalized regression was used to select optimal discriminating features. Results Whole blood methylation profile was able to discriminate samples by their glucocorticoid status: glucocorticoid excess was associated with DNA hypomethylation, recovering within months after Cushing's syndrome correction. In Cushing's syndrome, an enrichment in hypomethylated CpG sites was observed in the region of FKBP5 gene locus. A methylation predictor of glucocorticoid excess was built on a training cohort and validated on two independent cohorts. Potential CpG sites associated with the risk for specific complications, such as glucocorticoid-related hypertension or osteoporosis, were identified, needing now to be confirmed on independent cohorts. Conclusions Whole blood DNA methylome is dynamically impacted by glucocorticoids. This biomarker could contribute to better assessment of glucocorticoid action beyond hormone assays

Whole blood methylome-derived features to discriminate endocrine hypertension

Background: Arterial hypertension represents a worldwide health burden and a major risk factor for cardiovascular morbidity and mortality. Hypertension can be primary (primary hypertension, PHT), or secondary to endocrine disorders (endocrine hypertension, EHT), such as Cushing's syndrome (CS), primary aldosteronism (PA), and pheochromocytoma/paraganglioma (PPGL). Diagnosis of EHT is currently based on hormone assays. Efficient detection remains challenging, but is crucial to properly orientate patients for diagnostic confirmation and specific treatment. More accurate biomarkers would help in the diagnostic pathway. We hypothesized that each type of endocrine hypertension could be associated with a specific blood DNA methylation signature, which could be used for disease discrimination. To identify such markers, we aimed at exploring the methylome profiles in a cohort of 255 patients with hypertension, either PHT (n = 42) or EHT (n = 213), and at identifying specific discriminating signatures using machine learning approaches. Results: Unsupervised classification of samples showed discrimination of PHT from EHT. CS patients clustered separately from all other patients, whereas PA and PPGL showed an overall overlap. Global methylation was decreased in the CS group compared to PHT. Supervised comparison with PHT identified differentially methylated CpG sites for each type of endocrine hypertension, showing a diffuse genomic location. Among the most differentially methylated genes, FKBP5 was identified in the CS group. Using four different machine learning methods—Lasso (Least Absolute Shrinkage and Selection Operator), Logistic Regression, Random Forest, and Support Vector Machine—predictive models for each type of endocrine hypertension were built on training cohorts (80% of samples for each hypertension type) and estimated on validation cohorts (20% of samples for each hypertension type). Balanced accuracies ranged from 0.55 to 0.74 for predicting EHT, 0.85 to 0.95 for predicting CS, 0.66 to 0.88 for predicting PA, and 0.70 to 0.83 for predicting PPGL. Conclusions: The blood DNA methylome can discriminate endocrine hypertension, with methylation signatures for each type of endocrine disorder

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Analyse de la méthylation d’ADN sur puces : étude comparative d’échantillons de tissus congelés et tissus FFPE

International audienceLes études de méthylome permettent de mesurer sur l’intégralité du génome les niveaux de méthylation des sites CpG qui conditionne l’expression des gènes dans chaque cellules et sont réalisées à partir de tissus cryo-préservés ou fixés. En clinique, la méthode de fixation dans le formol et d’inclusion dans la paraffine (FFPE) représente un moyen incontournable de conservation des biopsies. L’étude du méthylome à partir des ADNs extraits de tissus FFPE,souvent dégradés en raison des conditions de fixation et d’inclusion des tissus, reste un défi. Plusieurs techniques d’analyse de méthylation ont été développées dont la puce Infinium Methylation EPIC (850K) d’Illumina. Cette puce commercialisée depuis 2015, permet d’analyser 850 000 sites de méthylation répartis sur l’ensemble du génome humain. Afin d’améliorer la qualité des ADNs issus de matériel conservé par FFPE et par conséquent la reproductibilité de la méthode, une optimisation de protocole par une étape de « restauration » a été introduite. Dans une étude pilote, nous avons comparé par puce Illumina Infinium Methylation EPIC, le méthylome de tumeurs cérébrales ayant été conservées en parallèle par congélation et en paraffine dans le but d’optimiser l’analyse du méthylome sur tissus FFPE. Nous avons donc comparé des paires de biopsies tumorales : Cryo-préservées versus FFPE. Les résultats montrent une distribution des valeurs β des niveaux de méthylation similaire entre les échantillons FFPE et leurs paires congelées, bien que les intensités des sondes soient plus faibles dans le cas des tissus FFPE dégradés par rapport aux congelés ; elles restent néanmoins exploitables.La corrélation des paires FFPE-congelés reste élevée sur la base des valeurs β des sondes filtrées sur les SNP (r2 moyen = 0.92, intervalle entre 0.86 et 0.98). Une corrélation est aussi observée au niveau des intensités des sondes qui s’hybrident sur le chromosome Y et qui permettent de valider l’appartenance au même sexe des échantillons appariés. Cependant, sur la base des valeurs β de l’ensemble des sondes, la corrélation est faible entre certains échantillons appariés. Cela est probablement lié à la composition cellulaire entre la composante congelée et celle incluse en paraffine. Ces résultats suggèrent que les tissus FFPE peuvent être utilisés, après réalisation du protocole de restauration d’Illumina, pour l’analyse de la méthylation par puces EPIC. Ces analyses ont par ailleurs permis l’identification de différentes sous classes de tumeurs dans les tissus FFPE et congelés et devraient aider à la caractérisation encore en cours de potentiels marqueurs de diagnostic et de pronostic. Cette étude pilote sur tissus FFPE réalisée en collaboration avec le Dr Franck BIELLE (ICM), nous permet de proposer une nouvelle prestation sur notre plateforme, l’analyse de la méthylation sur les échantillons FFPE. Cette nouvelle offre complète le catalogue de puces à ADN de la plateforme P3S

Alternative splicing in normal and pathological human placentas is correlated to genetic variants

International audienceTwo major obstetric diseases, preeclampsia (PE), a pregnancy-induced endothelial dysfunction leading to hypertension and proteinuria, and intra-uterine growth-restriction (IUGR), a failure of the fetus to acquire its normal growth, are generally triggered by placental dysfunction. Many studies have evaluated gene expression deregulations in these diseases, but none has tackled systematically the role of alternative splicing. In the present study, we show that alternative splicing is an essential feature of placental diseases, affecting 1060 and 1409 genes in PE vs controls and IUGR vs controls, respectively, many of those involved in placental function. While in IUGR placentas, alternative splicing affects genes specifically related to pregnancy, in preeclamptic placentas, it impacts a mix of genes related to pregnancy and brain diseases. Also, alternative splicing variations can be detected at the individual level as sharp splicing differences between different placentas. We correlate these variations with genetic variants to define splicing Quantitative Trait Loci (sQTL) in the subset of the 48 genes the most strongly alternatively spliced in placental diseases. We show that alternative splicing is at least partly piloted by genetic variants located either in cis (52 QTL identified) or in trans (52 QTL identified). In particular, we found four chromosomal regions that impact the splicing of genes in the placenta. The present work provides a new vision of placental gene expression regulation that warrants further studies

Colopexia em ovinos da raça Dorper com prolapso retal Colopexy in Dorper lambs with rectal prolapse

Prolapso de reto é afecção comum em ovinos de cauda curta. Neste trabalho relata-se a técnica de colopexia para redução de prolapso retal em trinta ovinos da raça Dorper, dos quais, três vieram a óbito no período pós-operatório e três tiveram que ser sacrificados, pois além de apresentarem recidiva, um deles era idoso, e os outros três por se encontrarem bastante debilitados. Aos 15 dias após a cirurgia, cinco animais apresentaram recidiva do prolapso, sendo a colopexia refeita em três deles tendo bom resultado em apenas um, e os outros dois foram sacrificados. Aos 30 dias de pós-operatório um animal apresentou prolapso retal, os outros dezenove (63,3%) estavam em adequado estado físico. A realização de colopexia é uma alternativa para o tratamento de prolapso retal em ovelhas da raça Dorper, porém recidivas e complicações são comuns.<br>Rectal prolapse is a common affection in lambs of short tail. This study aimed at reporting the colopexy to reduce the rectal prolapse in Dorper lambs. Thirty animals were submitted to surgery and three of them died in the postoperative period. Three animals had to be sacrificed, because they have shown prolapse recurrence (one of them was old, and the other three were in a strong debilitated state). At 15 days after the surgery, five animals showed prolapse recurrence and the colopexy was performed again in three having good results in only one, the other two were sacrificed. At the 30 postoperative days, an animal showed rectal prolapse signals, the other nineteen animals (63,3%) were healthy. The colopexy use is an option to rectal prolapse treatment in Dorper lambs, although, recurrences and complications are expected

Immune Microenvironment and Lineage Tracing Help to Decipher Rosette-Forming Glioneuronal Tumors

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Pan-Genomic Regulation of Gene Expression in Normal and Pathological Human Placentas

In this study, we attempted to find genetic variants affecting gene expression (eQTL = expression Quantitative Trait Loci) in the human placenta in normal and pathological situations. The analysis of gene expression in placental diseases (Pre-eclampsia and Intra-Uterine Growth Restriction) is hindered by the fact that diseased placental tissue samples are generally taken at earlier gestations compared to control samples. The difference in gestational age is considered a major confounding factor in the transcriptome regulation of the placenta. To alleviate this significant problem, we propose here a novel approach to pinpoint disease-specific cis-eQTLs. By statistical correction for gestational age at sampling as well as other confounding/surrogate variables systematically searched and identified, we found 43 e-genes for which proximal SNPs influence expression level. Then, we performed the analysis again, removing the disease status from the covariates, and we identified 54 e-genes, 16 of which are identified de novo and, thus, possibly related to placental disease. We found a highly significant overlap with previous studies for the list of 43 e-genes, validating our methodology and findings. Among the 16 disease-specific e-genes, several are intrinsic to trophoblast biology and, therefore, constitute novel targets of interest to better characterize placental pathology and its varied clinical consequences. The approach that we used may also be applied to the study of other human diseases where confounding factors have hampered a better understanding of the pathology