Rank Difference Analysis of Microarrays (RDAM), a novel approach to statistical analysis of microarray expression profiling data

BACKGROUND: A key step in the analysis of microarray expression profiling data is the identification of genes that display statistically significant changes in expression signals between two biological conditions. RESULTS: We describe a new method, Rank Difference Analysis of Microarrays (RDAM), which estimates the total number of truly varying genes and assigns a p-value to each signal variation. Information on a group of differentially expressed genes includes the sensitivity and the false discovery rate. We demonstrate the feasibility and efficiency of our approach by applying it to a large synthetic expression data set and to a biological data set obtained by comparing vegetatively-growing wild type and tor2-mutant yeast strains. In both cases we observed a significant improvement of the power of analysis when our method is compared to another popular nonparametric method. CONCLUSIONS: This study provided a valuable new statistical method to analyze microarray data. We conclude that the good quality of the results obtained by RDAM is mainly due to the quasi-perfect equalization of variation distribution, which is related to the standardization procedure used and to the measurement of variation by rank difference

Global DNA hypomethylation prevents consolidation of differentiation programs and allows reversion to the embryonic stem cell state.

DNA methylation patterns change dynamically during mammalian development and lineage specification, yet scarce information is available about how DNA methylation affects gene expression profiles upon differentiation. Here we determine genome-wide transcription profiles during undirected differentiation of severely hypomethylated (Dnmt1⁻/⁻) embryonic stem cells (ESCs) as well as ESCs completely devoid of DNA methylation (Dnmt1⁻/⁻;Dnmt3a⁻/⁻;Dnmt3b⁻/⁻ or TKO) and assay their potential to transit in and out of the ESC state. We find that the expression of only few genes mainly associated with germ line function and the X chromosome is affected in undifferentiated TKO ESCs. Upon initial differentiation as embryoid bodies (EBs) wild type, Dnmt1⁻/⁻ and TKO cells downregulate pluripotency associated genes and upregulate lineage specific genes, but their transcription profiles progressively diverge upon prolonged EB culture. While Oct4 protein levels are completely and homogeneously suppressed, transcription of Oct4 and Nanog is not completely silenced even at late stages in both Dnmt1⁻/⁻ and TKO EBs. Despite late wild type and Dnmt1⁻/⁻ EBs showing a much higher degree of concordant expression, after EB dissociation and replating under pluripotency promoting conditions both Dnmt1⁻/⁻ and TKO cells, but not wild type cells rapidly revert to expression profiles typical of undifferentiated ESCs. Thus, while DNA methylation seems not to be critical for initial activation of differentiation programs, it is crucial for permanent restriction of developmental fate during differentiation

A Conserved GA Element in TATA-Less RNA Polymerase II Promoters

Initiation of RNA polymerase (Pol) II transcription requires assembly of the pre-initiation complex (PIC) at the promoter. In the classical view, PIC assembly starts with binding of the TATA box-binding protein (TBP) to the TATA box. However, a TATA box occurs in only 15% of promoters in the yeast Saccharomyces cerevisiae, posing the question how most yeast promoters nucleate PIC assembly. Here we show that one third of all yeast promoters contain a novel conserved DNA element, the GA element (GAE), that generally does not co-occur with the TATA box. The distance of the GAE to the transcription start site (TSS) resembles the distance of the TATA box to the TSS. The TATA-less TMT1 core promoter contains a GAE, recruits TBP, and supports formation of a TBP-TFIIB-DNA-complex. Mutation of the promoter region surrounding the GAE abolishes transcription in vivo and in vitro. A 32-nucleotide promoter region containing the GAE can functionally substitute for the TATA box in a TATA-containing promoter. This identifies the GAE as a conserved promoter element in TATA-less promoters

Accelerated Tryptophan Degradation Predicts Poor Survival in Trauma and Sepsis Patients

Immune system activation and inflammation accompanies immune dysfunction in trauma and sepsis patients. Immunodeficiency may develop in such patients as one consequence of an activated chronic pro-inflammatory response. According to recent data, degradation of L-tryptophan (TRP) via the kynurenine (KYN) pathway by the cytokine-inducible enzyme indoleamine 2,3-dioxygenase (IDO) could represent an important contributor to the deficient responsiveness of immunocompetent cells. Compared to healthy controls, patients post trauma or with sepsis had increasing KYN concentrations and KYN to TRP ratios (KYN/TRP) whereas TRP concentrations decreased. Likewise, concentrations of cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and of immune activation marker neopterin increased in patients (all p < 0.001). Furthermore in patients KYN/TRP, KYN and neopterin concentrations were further increasing (all p < 0.001), whereas the changes of TRP, TNF-α and IL-6 concentrations were not significant. Compared to the survivors, the non-survivors had a higher concentration of KYN, neopterin, TNF-α and IL-6 as well as a higher KYN/TRP ratio. KYN/TRP correlated with neopterin (p < 0.001) and also with TNF-α (p < 0.01) and IL-6 concentrations (p < 0.05) and inversely with the in vitro response of stimulated monocytes. We conclude that increased TRP degradation in patients post trauma is closely associated with immune activation. Cytokines released during the pro-inflammatory response may induce the activity of IDO and thus accelerate TRP degradation. Thus, increased IDO activity most likely represents a result of host response to pro-inflammation in patients. Data support a possible role of inflammation-induced IDO in the diminished immunoresponsiveness in patients

Antifactor Xa activity in critically ill patients receiving antithrombotic prophylaxis with standard dosages of certoparin: a prospective, clinical study

INTRODUCTION: Deep venous thrombosis with subsequent pulmonary embolism or post-thrombotic syndrome is a feared complication in the intensive care unit. Therefore, routine prophylactic anticoagulation is widely recommended. Aside from unfractionated heparin, low molecular weight heparins, such as certoparin, have become increasingly used for prophylactic anticoagulation in critically ill patients. In this prospective study, we evaluated the potency of 3,000 IU certoparin administered once daily to reach antithrombotic antifactor Xa (aFXa) levels of 0.1 to 0.3 IU/ml in 62 critically ill patients. METHODS: AFXa levels were determined 4, 12 and 24 h after injection of certoparin. Prothrombin time, activated partial thromboplastin time, antithrombin, fibrinogen, hemoglobin, platelet count, serum urea and creatinine concentrations were documented before and 12 and 24 h after injection of certoparin. RESULTS: Four hours after certoparin injection (n = 32), 28% of patients were within the antithrombotic aFXa range. After 12 and 24 h, 6% achieved antithrombotic aFXa levels. Because of a severe pulmonary embolism in one study patient, an interim analysis was performed, and the dosage of certoparin was increased to 3,000 IU twice daily. This regime attained recommended antithrombotic aFXa levels in 47%, 27%, 40% and 30% of patients at 4, 12, 16 and 24 h, respectively, after twice daily certoparin injection (n = 30). Antithrombin and fibrinogen concentrations slightly increased during the observation period. Low antithrombin concentrations before certoparin were independently correlated with underdosing of certoparin. Patients with aFXa levels <0.1 IU/ml 4 h after certoparin injection required vasopressors more often and had lower serum concentrations of creatinine and urea than patients with antithrombotic aFXa levels. CONCLUSION: Standard dosages of certoparin of 3,000 IU given once or twice daily are ineffective for attaining the recommended aFXa levels of 0.1 to 0.3 IU/ml in critically ill patients. Low antithrombin levels before certoparin administration were independently associated with low aFXa levels. Renal function and vasopressor therapy may further influence the effectiveness of certoparin in ensuring adequate antithrombotic prophylaxis

Mid-Term Outcome after Extracorporeal Life Support in Postcardiotomy Cardiogenic Shock: Recovery and Quality of Life

Background: Extracorporeal life support (ECLS) therapy for refractory postcardiotomy cardiogenic shock (rPCS) is associated with high early mortality rates. This study aimed to identify negative predictors of mid-term survival and to assess health-related quality of life (HRQoL) and recovery of the survivors. Methods: Between 2017 and 2020, 142 consecutive patients received ECLS therapy following cardiac surgery. The median age was 66.0 [57.0–73.0] years, 67.6% were male and the median EuroSCORE II was 10.5% [4.2–21.3]. In 48 patients, HRQoL was examined using the 36-Item Short Form Survey (SF-36) and the modified Rankin-Scale (mRS) at a median follow-up time of 2.2 [1.9–3.2] years. Results: Estimated survival rates at 3, 12, 24 and 36 months were 47%, 46%, 43% and 43% (SE: 4%). Multivariable Cox Proportional Hazard regression analysis revealed preoperative EuroSCORE II (p = 0.013), impaired renal function (p = 0.010), cardiopulmonary bypass duration (p = 0.015) and pre-ECLS lactate levels (p = 0.004) as independent predictors of mid-term mortality. At the time of follow-up, 83.3% of the survivors were free of moderate to severe disability (mRS \u3c 3). SF-36 analysis showed a physical component summary of 45.5 ± 10.2 and a mental component summary of 50.6 ± 12.5. Conclusions: Considering the disease to be treated, ECLS for rPCS is associated with acceptable mid-term survival, health-related quality of life and functional status. Preoperative EuroSCORE II, impaired renal function, cardiopulmonary bypass duration and lactate levels prior to ECLS implantation were identified as negative predictors and should be included in the decision-making process

Different neuroinflammatory profile in amyotrophic lateral sclerosis and frontotemporal dementia is linked to the clinical phase

Objective To investigate the role of neuroinflammation in asymptomatic and symptomatic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mutation carriers. Methods The neuroinflammatory markers chitotriosidase 1 (CHIT1), YKL-40 and glial fibrillary acidic protein (GFAP) were measured in cerebrospinal fluid (CSF) and blood samples from asymptomatic and symptomatic ALS/FTD mutation carriers, sporadic cases and controls by ELISA. Results CSF levels of CHIT1, YKL-40 and GFAP were unaffected in asymptomatic mutation carriers (n=16). CHIT1 and YKL-40 were increased in gALS (p<0.001, n=65) whereas GFAP was not affected. Patients with ALS carrying a CHIT1 polymorphism had lower CHIT1 concentrations in CSF (-80%) whereas this polymorphism had no influence on disease severity. In gFTD (n=23), increased YKL-40 and GFAP were observed (p<0.05), whereas CHIT1 was nearly not affected. The same profile as in gALS and gFTD was observed in sALS (n=64/70) and sFTD (n=20/26). CSF and blood concentrations correlated moderately (CHIT1, r=0.51) to weak (YKL-40, r=0.30, GFAP, r=0.39). Blood concentrations of these three markers were not significantly altered in any of the groups except CHIT1 in gALS of the Ulm cohort (p<0.05). Conclusion Our data indicate that neuroinflammation is linked to the symptomatic phase of ALS/FTD and shows a similar pattern in sporadic and genetic cases. ALS and FTD are characterised by a different neuroinflammatory profile, which might be one driver of the diverse presentations of the ALS/FTD syndrome

Executive summary. Expert consensus statement on the diagnosis and treatment of paediatric pulmonary hypertension. The European Paediatric Pulmonary Vascular Disease Network, endorsed by ISHLT and DGPK

The European Paediatric Pulmonary Vascular Disease (PVD) Network is a registered, non-profit organisation that strives to define and develop effective, innovative diagnostic methods and treatment options in all forms of paediatric pulmonary hypertensive vascular disease, including specific forms such as pulmonary arterial hypertension (PAH)-congenital heart disease, pulmonary hypertension (PH) associated with bronchopulmonary dysplasia, persistent PH of the newborn, and related cardiac dysfunction. Methods The writing group members conducted searches of the PubMed/MEDLINE bibliographic database (1990-2015) and held five face-to-face meetings with votings. Clinical trials, guidelines, and reviews limited to paediatric data were searched using the terms 'pulmonary hypertension' and 5-10 other keywords, as outlined in the other nine articles of this special issue. Class of recommendation (COR) and level of evidence (LOE) were assigned based on European Society of Cardiology/American Heart Association definitions and on paediatric data only, or on adult studies that included >10% children. Results A total of 9 original consensus articles with graded recommendations (COR/LOE) were developed, and are summarised here. The topics included diagnosis/monitoring, genetics/biomarker, cardiac catheterisation, echocardiography, cardiac magnetic resonance/chest CT, associated forms of PH, intensive care unit/ventricular assist device/lung transplantation, and treatment of paediatric PAH. Conclusions The multipaper expert consensus statement of the European Paediatric PVD Network provides a specific, comprehensive, detailed but practical framework for the optimal clinical care of children with PH

Preclinical safety and efficacy of a therapeutic antibody that targets SARS-CoV-2 at the sotrovimab face but is escaped by Omicron

The recurrent emerging of novel viral variants of concern (VOCs) with evasion of preexisting antibody immunity upholds severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) case numbers and maintains a persistent demand for updated therapies. We selected the patient-derived antibody CV38-142 based on its potency and breadth against the VOCs Alpha, Beta, Gamma, and Delta for preclinical development into a therapeutic. CV38-142 showed in vivo efficacy in a Syrian hamster VOC infection model after post-exposure and therapeutic application and revealed a favorable safety profile in a human protein library screen and tissue cross-reactivity study. Although CV38-142 targets the same viral surface as sotrovimab, which maintains activity against Omicron, CV38-142 did not neutralize the Omicron lineages BA.1 and BA.2. These results highlight the contingencies of developing antibody therapeutics in the context of antigenic drift and reinforce the need to develop broadly neutralizing variant-proof antibodies against SARS-CoV-2