Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

Linking electric field simulations and physiological measurements to reveal how TMS stimulates the human motor hand area

Knowledge of the type and position of the neural population stimulated by transcranial magnetic stimulation (TMS) is pivotal for a better understanding of the underlying physiological mechanisms and for advancing a systematic targeting and dosage approach. We will provide evidence that realistic field modeling informed by individual structural MRI, combined with measurements of the elicited motor evoked potentials (MEPs) can help to pinpoint the stimulated brain region and reveal the stimulation depth. We will further show that field modeling can contribute to the understanding of the neural origin of inter-individual differences in MEP latencies. In 9 healthy participants, we systematically varied the orientation of a standard figure-8 coil and compared the MEP threshold changes for monophasic TMS with the electric field changes in the motor cortex that were calculated using the Finite-Element Method (FEM). In addition, in another 9 participants, we used three figure-8 coils having different field decays to correlate the differences in the electrophysiological thresholds for current direction posterior-to-anterior (PA) with the differences in the calculated field distributions. These two experiments consistently showed that TMS stimulates the region of the crown and posterior lip of the precentral gyrus and that the maximal electric field strength in this region is significantly related to the MEP threshold. Finally, in 13 participants, we tested the correlation between the field in the motor cortex and the MEP onset delays for anterior-to-posterior (AP) current orientation. We demonstrated that the part of the motor cortex found in the two prior experiments also exhibited a significant negative correlation between the onset delays and the field strength. The results of our experiments validate the FEM-based field calculations by demonstrating a significant correlation between the electric field estimates and the physiological response to TMS. They further help to resolve uncertainties on the stimulation depth of TMS. They suggest that TMS at the optimal current orientation might mainly stimulate subarea BA 4a of the motor cortex at the transition from the posterior wall to the crown of the precentral gyrus. In addition, in those subjects in which lower field strengths are sufficient to induce a motor response for current orientation AP, later I-waves are recruited. This suggests that local inter-individual differences in cortical organization in the upper part of M1 might underlie the observed latency differences

Multinomial, ordinal and stereotype logistic regression - an introduction to the regression analysis of categorial outcome variables

For about three decades now a number of suitable regression models for categorical outcome variables have been described in the literature. These are also available as analysis tools in statistical software packages. This overview presents some of the models which are suitable for medicine and health sciences. The lesser-known stereotypical model in particular is emphasized since the ordinal and the multinomial models have been in use for some time.The article is aimed at statistics users: It covers types of categorical data, models and their implementation in statistical software. Estimation and inference theory is omitted. It demonstrates, firstly, model selection depending on the order structure of the outcome variables and, secondly, appropriate interpretation of the model parameters. This is illustrated by one example each for the multinomial and the stereotypical model. Notes to use the software are included.Seit rund drei Jahrzehnten gibt es in der Literatur eine Reihe geeigneter Regressionsmodelle zur Analyse kategorialer Outcome-Variablen. Diese sind inzwischen in den großen Statistik-Softwarepaketen implementiert. Diese Übersicht soll einige für Medizin und Gesundheitswissenschaften geeignete Modelle darstellen, wobei das Schwergewicht auf den weniger bekannten stereotypen Modellen liegt. Die ordinalen und die multinomialen Modelle sind seit längerem in Gebrauch.Der Artikel richtet sich an Anwender. Er beschreibt die Typen von kategorialen Daten, Modellen und deren Implementierungen, auf Schätz- und Inferenztheorie wird verzichtet. Es wird gezeigt, wie in Abhängigkeit von der Ordnungsstruktur der Zielvariablen Modelle ausgewählt und wie die Modellparameter interpretiert werden können. Dies wird durch je ein Beispiel zum multinomialen und zum stereotypen Modell illustriert, Erläuterungen zum Umgang mit der Software sind eingeschlossen

Genomic imprinting recapitulated in the human β-globin locus

A subset of genes in mammals are subject to genomic imprinting. The mouse H19 gene, for example, is active only when maternally inherited and the neighboring Igf2 gene is paternally expressed. This imprinted expression pattern is regulated by the imprinting control region (ICR) upstream of the H19 gene. A maternally inherited H19 ICR inhibits Igf2 gene activation by the downstream enhancer due to its insulator function while it suppresses H19 gene transcription by promoter DNA methylation when paternally inherited. These parent-of-origin specific functions depend on the allele-specific methylation of the ICR DNA, which is established during gametogenesis. Therefore, the ICR may also function as a landmark for epigenetic modifications. To examine whether the ICR confers these activities autonomously, we introduced a 2.9-kbp ICR-containing DNA fragment into a human β-globin yeast artificial chromosome at the 3′ end of the locus control region and established transgenic mouse lines. Expression of all of the β-like globin genes was higher when the transgene was paternally inherited. In accord with this result, transgenic ICR DNA from nucleated erythrocytes was more heavily methylated when paternally transmitted. Chromatin immunoprecipitation assays confirmed that CCCTC binding factor is preferentially recruited to the maternal transgenic ICR in vivo. Surprisingly however, the parent-of-origin specific methylation pattern was not observed in germ cell DNA in testis, demonstrating that methylation was established after fertilization. Thus, the ICR autonomously recapitulated imprinting within the normally nonimprinted transgenic β-globin gene locus, but the temporal establishment of imprinting methylation differs from that at the endogenous Igf2/H19 locus